Live cell assays: from research to health and regulatory applications

BIOMEDICAL ENGINEERING SERIES

Live Cell AssaysFrom Research to Health

and Regulatory Applications

Christophe Furger

http://www.wiley.com/go/eula

Live Cell Assays

Series Editor Marie-Christine Ho Ba Tho

Live Cell Assays

From Research to Health and Regulatory Applications

Christophe Furger

First published 2016 in Great Britain and the United States by ISTE Ltd and John Wiley & Sons, Inc.

Apart from any fair dealing for the purposes of research or private study, or criticism or review, as permitted under the Copyright, Designs and Patents Act 1988, this publication may only be reproduced, stored or transmitted, in any form or by any means, with the prior permission in writing of the publishers, or in the case of reprographic reproduction in accordance with the terms and licenses issued by the CLA. Enquiries concerning reproduction outside these terms should be sent to the publishers at the undermentioned address:

ISTE Ltd John Wiley & Sons, Inc. 27-37 St George’s Road 111 River Street London SW19 4EU Hoboken, NJ 07030 UK USA

www.iste.co.uk www.wiley.com

© ISTE Ltd 2016 The rights of Christophe Furger to be identified as the author of this work have been asserted by him in accordance with the Copyright, Designs and Patents Act 1988.

Library of Congress Control Number: 2016941698 British Library Cataloguing-in-Publication Data A CIP record for this book is available from the British Library ISBN 978-1-84821-858-1

Contents

Foreword . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xix

Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxi

List of Cell Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxiii

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxvii

Chapter 1. Principles and Position . . . . . . . . . . . . . . . . . . . . . 1

1.1. Live cell assay principles . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1.2. Application areas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1.3. Positioning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

1.3.1. Definition and typology of cell tests . . . . . . . . . . . . . . . . . 6 1.3.2. The regulatory and industrial dimension . . . . . . . . . . . . . . . 8

1.4. Market . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 1.5. Competitive advantages . . . . . . . . . . . . . . . . . . . . . . . . . . 12

1.5.1. Cells are live information models . . . . . . . . . . . . . . . . . . . 12 1.5.2. Development: high throughput . . . . . . . . . . . . . . . . . . . . 13 1.5.3. Development: multiplex analysis . . . . . . . . . . . . . . . . . . . 13 1.5.4. Development: miniaturization . . . . . . . . . . . . . . . . . . . . 14 1.5.5. Development: molecular engineering . . . . . . . . . . . . . . . . 14 1.5.6. Development: standardization . . . . . . . . . . . . . . . . . . . . 14

1.6. Can measurements of cells in culture be extrapolated to effects in the organism? . . . . . . . . . . . . . . . . . . . 15

1.6.1. Toxicokinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 1.6.2. Components of the immune system . . . . . . . . . . . . . . . . . 16

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1.6.3. Biotransformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 1.6.4. The macrocellular environment . . . . . . . . . . . . . . . . . . . . 16

1.7. Limits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 1.7.1. Importance of cellular microenvironment . . . . . . . . . . . . . . 17 1.7.2. Other limits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Chapter 2. History and State of the Art . . . . . . . . . . . . . . . . . . 21

2.1. Origins of cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 2.1.1. Pioneering studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 2.1.2. Alexis Carrel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 2.1.3. Were Dr Carrel’s cells immortal? . . . . . . . . . . . . . . . . . . 25

2.2. The HeLa line and the first applications of cell culture . . . . . . . . . 27 2.2.1. A vaccine against poliomyelitis . . . . . . . . . . . . . . . . . . . . 29 2.2.2. Cells in space . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 2.2.3. Cell cloning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

2.3. New cell lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 2.3.1. The CHO line . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 2.3.2. An increasing number of cell lines . . . . . . . . . . . . . . . . . . 31

2.4. Cross-contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 2.5. Cell lines, an ethical issue . . . . . . . . . . . . . . . . . . . . . . . . . . 35 2.6. The first generation of cell assays (1969–1983) . . . . . . . . . . . . 37

2.6.1. The karyotype test . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 2.6.2. The MTT assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 2.6.3. The NRU test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

2.7. The first target of regulatory assays: genotoxicity (1983–1986) . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

2.7.1. Ames test (OECD guideline 471) . . . . . . . . . . . . . . . . . . 43 2.7.2. In vitro mammalian chromosome aberration test (OECD guideline 473) . . . . . . . . . . . . . . . . . . . 44 2.7.3. In vitro mammalian cell gene mutation test (OECD guideline 476) . . . . . . . . . . . . . . . . . . . . . . . . . 45 2.7.4. In vitro sister chromatid exchange assay in mammalian cells (OECD guideline no. 479) . . . . . . . . . . . . . . 46 2.7.5. DNA damage and repair, unscheduled DNA synthesis in mammalian cells (OECD guideline 482) . . . . . . . . . . 47

Chapter 3. Cell Models and Technologies . . . . . . . . . . . . . . . . 49

3.1. Fluorescence and bioluminescence . . . . . . . . . . . . . . . . . . . . . 50 3.1.1. Green fluorescent protein . . . . . . . . . . . . . . . . . . . . . . . 51 3.1.2. BRET . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 3.1.3. FRET . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

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3.1.4. Other applications of GFP . . . . . . . . . . . . . . . . . . . . . . . 57 3.1.5. The reporter gene approach . . . . . . . . . . . . . . . . . . . . . . 58

3.2. Impedance variation in cell population . . . . . . . . . . . . . . . . . . 60 3.3. Optical signals modified by state of cells . . . . . . . . . . . . . . . . . 62 3.4. Cellular autofluorescence . . . . . . . . . . . . . . . . . . . . . . . . . 65

3.4.1. The case of chlorophyll . . . . . . . . . . . . . . . . . . . . . . . . 66 3.5. The different cell models and culture modes available . . . . . . . . . 67

3.5.1. Immortalized lines . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 3.5.2. Primary cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 3.5.3. Three-dimensional cell culture . . . . . . . . . . . . . . . . . . . . 69

Chapter 4. Loss of Cell Homeostasis: Applications in Toxicity Measurement . . . . . . . . . . . . . . . . . . . 71

4.1. What relevant information to use in the living cell? . . . . . . . . . . 71 4.2. Lysosomal activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 4.3. Redox balance and oxidative stress . . . . . . . . . . . . . . . . . . . . 76 4.4. Integrity of the plasma membrane . . . . . . . . . . . . . . . . . . . . . 80 4.5. Cellular efflux . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 4.6. Homeostasis of ion exchanges . . . . . . . . . . . . . . . . . . . . . . . 89

4.6.1. The calcium ion . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 4.6.2. Maintenance of membrane potential . . . . . . . . . . . . . . . . . 91

4.7. Metabolism and cell respiratory activity . . . . . . . . . . . . . . . . . 92 4.8. Genotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 4.9. Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

Chapter 5. The Replacement of Animal Testing: A Driving Force in Live Cell Assay Development . . . . . . . . . . . . 103

5.1. On the pertinence of in vitro assays . . . . . . . . . . . . . . . . . . . . 104 5.2. On the pertinence of animal tests . . . . . . . . . . . . . . . . . . . . . 105 5.3. The problem with extrapolation . . . . . . . . . . . . . . . . . . . . . . 106

5.3.1. The interspecies barrier . . . . . . . . . . . . . . . . . . . . . . . . 106 5.3.2. The striking example of TGN1412 . . . . . . . . . . . . . . . . . . 107

5.4. Toxicological assessment of substances . . . . . . . . . . . . . . . . . 109 5.5. Irritation and eye corrosion: the long (ongoing) quest for an alternative to the Draize test . . . . . . . . . . . . . . . . . . . 111

5.5.1. The CM test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 5.5.2. Ex vivo approaches . . . . . . . . . . . . . . . . . . . . . . . . . . 113 5.5.3. 3D culture models . . . . . . . . . . . . . . . . . . . . . . . . . . . 114 5.5.4. Recent attempts and validations . . . . . . . . . . . . . . . . . . . 115

5.6. Measurement alternatives for skin absorption, corrosion and irritation (2004–2010) . . . . . . . . . . . . . . . . . . . . . 116

viii Live Cell Assays

5.6.1. Skin absorption: in vitro method (OECD guideline no. 428) . . . . . . . . . . . . . . . . . . . . . . . . . 117 5.6.2. Reconstituted skin models for corrosion and irritation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 5.6.3. In vitro skin corrosion: human skin model test (OECD guideline no. 431) . . . . . . . . . . . . . . . . . . . 118 5.6.4. In vitro membrane barrier test method for skin corrosion (OECD guideline 435) . . . . . . . . . . . . . . . . . 121 5.6.5. In vitro skin irritation: reconstructed human epidermis test method (OECD guideline no. 439) . . . . . . . . 121

5.7. The live cell test for phototoxicity measurement (2004) . . . . . . . . 122 5.8. Assays for endocrine disruptor tracking (2009–2011) . . . . . . . . . 123

5.8.1. Detection of estrogenic agonist-activity of chemicals (OECD guideline 455) . . . . . . . . . . . . . . . . . . . . 124 5.8.2. H295R steroidogenesis assay (OECD guideline 456) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124

5.9. The four last live cell assays to be validated (2012–2015) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

5.9.1. Eye corrosion: fluorescein leakage test method (OECD guideline 460) . . . . . . . . . . . . . . . . . . . . . . . 125 5.9.2. Mammalian cell micronucleus test (OECD guideline 487) . . . . . . . . . . . . . . . . . . . . . . . . . 126 5.9.3. ARE-Nrf2 luciferase test method for in vitro skin sensitization (OECD guideline no 442D) . . . . . . . . . . . 127 5.9.4. Short-time exposure in vitro test method for identifying (1) chemicals inducing serious eye damage and (2) chemicals not requiring classification for eye irritation or serious eye damage (OECD guideline 491) . . . . . . . . . . . . . . . 127

Chapter 6. Regulatory Applications and Validation . . . . . . . . . . 129

6.1. Brief history of the validation process in Europe . . . . . . . . . . . . . 129 6.2. The validation process of a live cell assay . . . . . . . . . . . . . . . . . 130 6.3. Live cell assays adopted by the OECD . . . . . . . . . . . . . . . . . . 132 6.4. The future of regulatory cell tests: the TOX21 and SEURAT programs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134

6.4.1. TOX21, a new paradigm in the assessment of health and environmental risks . . . . . . . . . . . . . . . . . . . . . . 134 6.4.2. The SEURAT-1 program (2011–2016) . . . . . . . . . . . . . . . 138

6.5. The REACH regulatory context . . . . . . . . . . . . . . . . . . . . . . 139 6.5.1. Assessment approach by weight of evidence (WoE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140

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6.5.2. Up-date on the use of live cell assays under REACH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140 6.5.3. Acute toxicity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141 6.5.4. Skin corrosion and irritation . . . . . . . . . . . . . . . . . . . . . 142 6.5.5. Eye irritation and severe damage . . . . . . . . . . . . . . . . . . . 142 6.5.6. Skin sensitization . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142 6.5.7. Repeated doses (long-term effects) . . . . . . . . . . . . . . . . . 142 6.5.8. Genotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 6.5.9. Reproductive toxicity (reprotoxicity) . . . . . . . . . . . . . . . . 143 6.5.10. Carcinogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 6.5.11. Bioaccumulation and toxicity in fish . . . . . . . . . . . . . . . . 144 6.5.12. Long-term toxicity and reprotoxicity in birds . . . . . . . . . . . 144

6.6. Implementation of the 7th amendment to the Cosmetics Directive . . . . . . . . . . . . . . . . . . . . . . . . . . . 144

6.6.1. Acute toxicity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 6.6.2. Eye corrosion and irritation . . . . . . . . . . . . . . . . . . . . . . 145 6.6.3. Skin irritation and corrosion . . . . . . . . . . . . . . . . . . . . . 146 6.6.4. Skin sensitization . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146 6.6.5. Genotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147 6.6.6. Skin absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147

6.7. Food safety and biocides directive . . . . . . . . . . . . . . . . . . . . 147 6.7.1. Food safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147 6.7.2. The biocides directive . . . . . . . . . . . . . . . . . . . . . . . . . 148

Chapter 7. Cell Signaling: At the Heart of Functional Assays for Industrial Purposes . . . . . . . . . . . . . . 149

7.1. Membrane receptors, the primary target of drugs . . . . . . . . . . . . 149 7.1.1. Development of the therapeutic target/receptor concept . . . . . . 150 7.1.2. Purification, sequencing and heterologous expression . . . . . . . 151 7.1.3. The therapeutic importance of seven transmembrane domain receptors . . . . . . . . . . . . . . . . . . . . . . 152

7.2. Second messenger, base unit of the functional live cell assay . . . . . 153 7.2.1. The second messenger concept . . . . . . . . . . . . . . . . . . . . 153 7.2.2. Adenylyl cyclase and phosphodiesterase regulate the concentration of cyclic AMP . . . . . . . . . . . . . . . . . . 155

7.3. The concept of cell transduction . . . . . . . . . . . . . . . . . . . . . . 156 7.3.1. The protein kinase A, the (near) universal target of cyclic AMP . . . . . . . . . . . . . . . . . . . . . . . . 157 7.3.2. Decrypting the transduction pathways . . . . . . . . . . . . . . . . 158 7.3.4. G proteins, the missing link in cell transduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160

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7.3.5. Connection between transduction and genic expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161

7.4. The transduction pathways used in the context of live cell assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162

7.4.1. First level of regulation – activation of the transduction pathway . . . . . . . . . . . . . . . . . . . . . . . . . . 163 7.4.2. Second level of regulation – desensitization and recycling . . . . . . . . . . . . . . . . . . . . . . . . . 164 7.4.3. Third level of regulation – allosteric modulation . . . . . . . . . . 165

Chapter 8. Applications in New Drug Discovery . . . . . . . . . . . . 167

8.1. High-throughput screening, the leading market sector for cell assays . . . . . . . . . . . . . . . . . . . . . . . . . . . 167

8.1.1. The role of cell assays in screening programs . . . . . . . . . . . . 169 8.1.2. The contribution of functional cell assays . . . . . . . . . . . . . . 171 8.1.3. Exploitation of transduction pathways . . . . . . . . . . . . . . . . 171

8.2. Measurements in the immediate environment of receptors . . . . . . . 173 8.2.1. Assays on receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . 173 8.2.2. β-arrestin activity assays . . . . . . . . . . . . . . . . . . . . . . . . 174

8.3. Measuring cyclic AMP . . . . . . . . . . . . . . . . . . . . . . . . . . . 177 8.3.1. Classic cyclic AMP assays on cellular lysates . . . . . . . . . . . . 177 8.3.2. Cyclic AMP assays on live culture cells . . . . . . . . . . . . . . . 180

8.4. Measurement of the PKC pathway and discrimination of the PKA/PKC pathways . . . . . . . . . . . . . . . . . . . 183

8.4.1. IP3 measurement tests . . . . . . . . . . . . . . . . . . . . . . . . . . 183 8.4.2. Assays for the measurement of Ca2+ . . . . . . . . . . . . . . . . . 183 8.4.3. Discrimination between the cyclic AMP and IP3/Ca2+ pathways by label-free methods . . . . . . . . . . . . . . . . 184

8.5. Measurement of distal signals . . . . . . . . . . . . . . . . . . . . . . . 185 8.6. Cell assays concerning other therapeutic targets . . . . . . . . . . . . . 186

8.6.1. Measurement on ion channels . . . . . . . . . . . . . . . . . . . . . 186 8.6.2. Measurements on receptor tyrosine kinases (RTK) . . . . . . . . 188

8.7. Pharmacokinetics (ADME) in vitro . . . . . . . . . . . . . . . . . . . . 191 8.7.1. M for metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192 8.7.2. A for absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193 8.7.3. T for toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195

Chapter 9. Impact on Health and the Environment . . . . . . . . . . . 197

9.1. Patient diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197 9.1.1. Cytogenetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198 9.1.2. Diagnosis of tuberculosis . . . . . . . . . . . . . . . . . . . . . . . . 200

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9.1.3. Cell assay for the detection of pyrogenic substances . . . . . . . . . . . . . . . . . . . . . . . . . . . 201 9.1.4. Cell assays for predicting efficacy of chemotherapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203

9.2. Military programs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204 9.2.1. Detection and screening of botulinum toxin inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205 9.2.2. Antibody-based toxin neutralization assays (TNA): application on anthrax and ricin . . . . . . . . . . . . . . 208 9.2.3. Field measurement of water potability . . . . . . . . . . . . . . . . 209

9.3. Pollution and quality of environment . . . . . . . . . . . . . . . . . . . 211 9.3.1. The MicroTox assay . . . . . . . . . . . . . . . . . . . . . . . . . . 211 9.3.2. Mobility of the Daphnia test . . . . . . . . . . . . . . . . . . . . . 212 9.3.3. Fish embryo acute toxicity (FET) test (OECD guideline no. 236) . . . . . . . . . . . . . . . . . . . . . . . . . 213 9.3.4. The DR CALUX assay . . . . . . . . . . . . . . . . . . . . . . . . 214 9.3.5. Biomonitoring and field issues . . . . . . . . . . . . . . . . . . . . 215

Chapter 10. Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219

10.1. Stem cells, an opportunity for the future of cell assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219 10.2. Organs-on-a-chip . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222

10.2.1. Homo chippiens . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224 10.2.2. The contribution of PBPK models . . . . . . . . . . . . . . . . . 225

10.3. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226

Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247

Foreword

A book discussing live cell assays, a subject at the heart of scientific, technical, sanitary and economic development, should be made accessible to the global citizen. To underscore the importance of this book, I have decided to use my own expertise as a researcher and public consultant to provide the reader with some perspective while bringing useful clarifications to the text of this work.

A researcher’s view

As a researcher in Biology of my generation, this subject underpins my entire career not only at the university but also throughout my work as an authority evaluating health risks connected with exposure to chemical substances. I should mention that the discovery of the double helix of DNA won Watson and Crick the Nobel Prize in 1962 while I was in my senior year of high school, and that doctors Lwoff, Monod and Jacob received the Nobel Prize for their discovery of messenger RNA in 1965, which was my first year in the Toulouse Faculty of Science. These events were to ignite my passion for biochemistry and ever since guide my career as a researcher. Essentially, the work of these Nobel Laureates touched on the workings of life by chemical reactions and biological interactions, continuing on to the molecular level. I should add that at the time of my own initiation to research during my Master’s in 1969, all our experiments in physiology were performed on anesthetized living animals, while in vitro studies were performed on isolated organs (stomach, intestine, heart, etc.) or on tissue sections (brain, liver, kidney, etc.) kept alive in glass ampoules containing nutritive physiological liquid, oxygenated and maintained at 37°C.

xiv Live Cell Assays

Passing from physiology to biochemistry, we began to reproduce vital phenomena at a molecular level by using what are known as acellular systems, or machines (mitochondria, endoplasmic reticulum, etc.) and parts (transfer or messenger RNA, amino-acids, energetic cofactors, enzymes, etc.). However, in studying the various machines that make cells work (energy production, endogenous or exogenous molecule synthesis or degradation, etc.), biologists were removed not only from the workings of the animal as a whole (as our physiological education taught us), but also from the cell as a biological entity. Indeed, only those geneticists, microbiologists or algae biologists working with unicellular species, for example with bacteria or yeast, would readily cultivate cells.

While working on my thesis treating the regulation of protein synthesis in fish, an increasing number of articles were being published on cell culture, albeit, essentially on immortalized cancer cells, which have the drawback of a transformed metabolism compared with tissue cells. Techniques making use of cells isolated from their tissue and kept alive in a culture medium were also developed, but cell condition would degrade rapidly, limiting viability to several days.

On my arrival in Bordeaux, I established my own biochemical toxicology team. Through the first few years of the 1980s, we tried to acquire these culture techniques, but this required fitting the laboratory with specialized equipment and retraining the technical personnel. After several placements in hospital laboratories, we decided to abandon our efforts. In France, we could not readily adapt an existing laboratory to new techniques due to problems in mobilizing the requisite budget, to the corporatism of researchers who dislike multidisciplinary work and to the difficulty in retraining personnel (an animal specialist does not become a specialist in cell culture overnight).

Nonetheless, concerning the subject of chemical carcinogenesis, it had become expedient to evaluate the genotoxic and mutagenic potential of the molecules studied. In response to this, I brought a new team to my laboratory and set up Dr. Ames’s mutagenicity test, which uses the modified Salmonella thyphimurium bacteria, provided to us by an UCLA researcher. I required a specialist in bacterial culture as an assistant, and indeed, succeeded in recruiting a microbiology assistant who had been to the Institut du Cancer in Villejuif to learn this same technique. We were then able to work on the same molecule both in vivo for carcinogenesis and in vitro for genotoxicity. Thanks to these complementary techniques, we were able to collaborate internationally and to publish in reviews of repute.

Foreword xv

The second development of my research group took place almost 20 years later on the subject of endocrine disruptors. As before, the subject required a new team to join the laboratory, including a senior lecturer in endocrinology together with the technical acquisition of the modified cell with a reporter gene and fluorescent sensors. We were then able to progress to human and mammalian cells. Additionally, as these assays were suited for the purposes of microplates, a specific plate reader was required. Of course, the technical personnel had to undergo training with the organization that commercializes these assays, particularly those concerning the detection of dioxin-related substances or estrogen-related endocrine disruptors. Compared to the first extension of our group, there was one major difference: everything had become monetized (cell lines, training, royalties). Furthermore, as available public funds had dwindled, our operations were possible only with the help of a large private company.

The third development affected the miniaturization of ecotoxicological tests with the arrival in 2010 of a young manager to the head of my team. This allowed us to progress from studies in vivo on mollusks and fish in an aquarium to techniques in vitro, including on microplates, not only using cells but also the larvae or embryos of aquatic animals. With these new techniques, we were then able to multiply the number of assays and measure new parameters of toxicity in connection with behavior or development.

Simultaneously, the introduction of health regulations required us to repeatedly increase levels of investment to keep our animal facilities within norms. Indeed, the laboratory facility housing rats was closed around 20 years ago. In effect, the increasing use of cell models and in vitro techniques has led to a corresponding reduction in animal tests despite the persistent difficulties in financing both material assets and personnel training for cell assays due to the reduced allocations of public funding over the past 30 years.

A consultant’s view

Since 1988, in my duties as a public consultant evaluating health risks in connection to chemical substance exposure at both national and international levels, I was well placed to follow the apparition of cell assays in the regulatory context. From its beginnings, toxicology (the study of poisons) has been concerned with using certain substances (medical toxicology) or defining exposure levels to certain substances (nutritional, environmental or professional toxicology) that do not provoke illness in the long or medium

xvi Live Cell Assays

term. Since pathologies result in clinical signs observable in the individual, the accepted practice (set in stone by the OECD) was essentially based on the clinical observation of animals subjected to a series of toxicological tests over the short, medium and long term, allowing for the setting of an acceptable daily intake (ADI) for human beings. Advances in molecular biology have introduced the notion of mechanism of action into this process, which would allow for a better understanding of the cascading events that connect the presence of an active chemical entity to pathology.

OECD protocols have integrated live cell assays primarily in the area of carcinogenesis. In fact, these assays apply to bacterial cells (Ames test) or blood cells (lymphocytes) and show genotoxic effects (capable of altering genetic material). It is, therefore, a means of detecting the first stage of multistage carcinogenic process, which, in vivo, manifests as the apparition of malignant tumors. Even so, the connotations of “carcinogenic” and “genotoxic” are different since exposure to a genotoxin does not systematically induce the apparition of a cancer, and inversely, some compounds can induce cancers without being genotoxic.

The same problem applies to endocrine disruptors since the term applies to a mechanism of action and not to pathology. An endocrine disruptor’s characteristic is determined in large part by the response to several specific cell assays together with the application of the QSAR models. In fact, this mechanism is implicated in numerous pathologies (hormone-dependent cancers, impairment of reproductive function, diabetes, obesity, etc.) without there being a systematically causal link: expression of the pathology is dependent on conditions of exposure and susceptibility.

At present, the debate continues within health agencies and regulatory bodies concerning the integration of the cell approach into the risk assessment process, and ever more so now that the in vivo approach has demonstrated its numerous technical, economic, temporal and ethical limits, which will necessarily require profound changes in the methods of evaluating dangers and risks in which cell assays will have a significant role.

The author

For a book to be written, the subject must find its author. The work’s quality then depends on the quality of the author’s thought and, for technical subjects, on the author’s experience. It so happens that Christophe Furger was closely associated with the developments in cell assays from their point

Foreword xvii

of conception, through their development, technical adaptations, applications, validation and even commercialization. His extensive culture, his ability to integrate and his multidisciplinary openness provide him with a sense of perspective and vision going forward which, combined with his technical and scientific ability, assure him a clear understanding of the scientific importance, the possibilities and the limits in the use of live cell assays.

The clarity of wording will help the reader come to terms with this highly technical subject in a comprehensive approach. This book will become indispensable to students, specialists, engineers, doctors and medical professionals, journalists, environmental activists, animal rights campaigners and more generally to the informed citizen of the world. And I must say that I am particularly pleased to preface this work in the knowledge that the author’s home is Toulouse, the stamping ground of so many great scientists, authors, poets and musicians, and also of visionaries and activists. I sense the same fervor in the genesis of this work.

Jean-François NARBONNE Toxicologist and Professor at the University of Bordeaux 1,

France May 2016

Acknowledgments

I would especially like to thank Cécile Dufour, Camille Gironde, Sylvain Derick and Olivier Nosjean for their comments and critical reading of the manuscript, and of course Jean-François Narbonne for his Foreword, a very generous gesture for me.

Abbreviations

ABC: ATP Binding Cassette

ADME: Absorption, Distribution, Metabolism and Excretion (also known as pharmacokinetics or DMPK)

AMP: Adenosine Mono Phosphate

ATCC: American Type Culture Collection

ATP: Adenosine Triphosphate

BRET: Bioluminescence Resonance Energy Transfer

CHO: Chinese Hamster Ovary

DNA: Deoxyribo Nucleic Acid

EC50: 50% Effective Concentration

ECVAM: European Center for Validation of Alternative Methods or EURL ECVAM

FDA: Food and Drug Administration

FISH: Fluorescence In Situ Hybridization

FRET: Förster Resonance Energy Transfer

GFP: Green Fluorescent Protein

GHS: Globally Harmonized System of Classification and Labeling of Chemicals

GLP: Good Laboratory Practice

xxii Live Cell Assays

HCS: High Content Screening

HTS: High Throughput Screening

IP: Inositol Phosphate

iPSC: Induced Pluripotent Stem Cell

LED: Light-Emitting Diode

NADP: Nicotinamide Adenine Dinucleotide Phosphate

NOAEL: No Observable Adverse Effect Level

OECD: Organization for Economic Co-operation and Development

PBPK: Physiologically Based Pharmaco Kinetic (mathematical models)

QSAR: Quantitative Structure–Activity Relationship

REACH: Registration, Evaluation, Authorization and Restriction of Chemicals (European Regulations)

RET: Resonance Energy Transfer

ROS: Reactive Oxygen Species

RTK: Receptor Tyrosine Kinase

WoE: Weight of Evidence

List of Cell Assays

A-B: Cell permeability by ADME transporters

AK: Adenylate kinase, membrane permeability

AKT: RTK activity by AKT translocation

Alamar Blue: Metabolism by reductase activity

Alpha Screen: cAMP, IP or RTK activity on lysates

Ames: Mutagenesis assay on bacteria

Annexin V: Apoptosis after phosphatydylserines presentation

ARE-NRF2: Skin sensitization by reporter gene

ATPlite: ATP quantitative analysis by bioluminescence

Bind: Cell biomass by optical measurement

BrdU: Sister chromatid exchange

Brilliant Black: Membrane potential by quenching

C11-Bodipy: Lipid peroxidation

Ca++-Aequorin: Intracellular calcium ion by BRET

Cameleon: Intracellular calcium ion by FRET

cAMP Glosensor: Bioluminescence cAMP measurement on co-cultures

Calcein-AM: Cell efflux by ABC transporters

xxiv Live Cell Assays

Candles: Cell cAMP by bioluminescence

Caspase-3: Apoptosis

CAT: Lysosomal activity by amphiphilic cationic tracer

CellKey: Cell activity by impedance measurement

CellRox: Redox activity

Cell Titer GLO: ATP quantitative analysis by bioluminescence

CM: Metabolism by potentiometry (pH)

CM-H2DCFDA: Redox activity

Comet: DNA fragmentation on electrophoresis gel

CRE: AMPc pathway activation by reporter gene

cytokine release: detection of cytokine release syndromes

ΔΨ potential: Markers of mitochondrial membrane potential

DIBAC: Ion channels by fluorescence quenching

DR CALUX: Ah receptor activation by reporter gene

ECIS: Cell activity by impedance measurement

EPIC: Cell biomass by optical measurement

EpiDERM: Skin irritation on 3D epidermis model

EpiOcular: Eye irritation on 3D epithelium model

EpiSkin: Skin irritation on 3D epidermis model

EST-100: Skin irritation on 3D epidermis model

FLUO-4: Intracellular calcium ion evaluation

fluorescein leakage: Loss of cell layer sealing function

Gα15/16: 7 domain receptor activation by IP3/Ca++ pathway

FMP: FLIPR membrane potential, ion channel activity

GAPDH: G-3-P dehydrogenase, membrane permeability

GF-AFC: Intracellular protease activity

H295R: Endocrine disruption by steroidogenesis

h-CLAT: Skin sensitization by cytometry

List of Cell Assays xxv

hERG: hERG receptor function in cardiotoxicity

HitHunter: cAMP evaluation on lysates

Hoechst 33342: Cell efflux by ABC transporters

H3-Thymidine: Unscheduled DNA synthesis

HTRF: Transduction pathway analysis by time-resolved FRET

Karyotype: Chromosomal abnormalities

Lance: cAMP evaluation on lysates

LDH: Lactate dehydrogenase, membrane permeability

LUCS: ABC transporter efflux alteration by photosensitization

Skin sensitization by reporter gene

Pyrogenic substance by Interleukin 1β production

Monochlorobimane, glutathione level evaluation

Aneugenic and clastogenic genotoxic effects

Toxin evaluation by fluorescent bacteria

Presence of superoxide ion

Methyl-thiazolyl-tetrazolium, metabolic activity

Skin sensitivity by cytometry

Metabolic activity by cell auto-fluorescence

Intracellular calcium ion by reporter gene

Neutral red uptake, lysosomal activity (pH)

Cytochrome P450 activity for ADME

Chlorophyll photosynthetic activity

Signaling by enzyme fragment complementation

UVA effects on cell by NRU assay

Membrane permeability by protease

LuSens:

MAT:

MCB:

Micro Nucleus:

MicroTox:

MitoSOX Red:

MTT:

MUSST:

NAD(P)H:

NFAT-RE:

NRU:

P450-Glo:

PAM:

PathHunter:

Phototoxicity:

Protease:

QFT-GIT: Medical diagnosis by interferon γ release

xxvi Live Cell Assays

Rhodamine 123: Cell efflux by ABC transporters

SkinEthic: Eye irritation on 3D model of cornea

SkinEthic RHE: Skin irritation on 3D model of epidermis

SNAP25: Botulinum toxin activity by ELISA

SNARE: Botulinum toxin activity by FRET

SRE: Rho pathway by reporter gene

STE: Eye damage by short-term exposure

STTA: Endocrine disruption by estrogen activity

TA ERBG1Luc: Endocrine disruption by reporter gene

TagLite: Dimerization of seven domain receptors

Tango: β-arrestin activity by reporter gene

T-BARS: Lipid peroxidation

TK: Mutagenesis assay on mammal cells

TNA: Neutralization of toxins such as anthrax or ricin

Transfluor: β-arrestin plasma membrane recruitment

T-SPOT: Medical diagnosis by interferon γ release

TUNEL: DNA fragmentation

VIPR: Membrane potential by FRET

xCELLigence: Cell activity by impedance measurement

Introduction

“The word cell makes us think not of a monk or a prisoner but of a bee… Who knows if the human mind, consciously borrowing the term cell from the beehive in order to designate the element of the living organism, did not also borrow, almost unconsciously, the notion of the cooperative work that produces the honeycomb?”

Georges CANGUILHEM [CAN 09]

The term “live cell assay” refers to all of the approaches that use the cell as an information medium for measuring purposes. This very broad definition covers a wide range of experimental contexts in which the levels of information, flow or standardization are particularly diverse and numerous. Clearly, it would be tedious to describe them all here.

For the purposes of this work, the definition will be narrowed: a live cell assay shall be defined as an approach or a technology in cell biology, which, due to its high levels of standardization, may be used by the wider scientific and industrial community for purposes of measurement and comparison. Conversely, the term “application” will be taken in its broader sense, covering areas as diverse as fundamental research, industrial R&D, regulatory contexts, the environment and patient diagnostics.

These applications owe their development to our capacity of manipulating the living cell and, moreover, in conserving its integrity outside of the organism. Indeed, much time was required (1910–1950) to succeed in separating (tearing would be just as appropriate) the cell from the human or animal specimen to which it belonged, and then to recognize that the isolated cell could individualize and live its own existence. From the 1960s, these

xxviii Live Cell Assays

developments in cell culture allowed for the emergence of the first standardized cell assays, namely, the karyotype and the Ames test.

Live cell assays are held in relatively high esteem by today’s society. We will see that this was not always the case. In so far as the models used lead to a reduction in the use of animals, cell assays are at present considered to be the more ethical choice, whether it is for research, for the discovery of new medicine or for assessing health risks.

Due to the extreme diversity of the living world, live cell assays are naturally polyvalent. They can be conceived to measure extremely specific cell activity or on the other hand, very generic activity. They can be performed on a wide array of models, from bacteria to human cells but also plants, fungi or all varieties of microorganisms. The two most common functions targeted by live cell assays are homeostasis in cases of toxicity measurements and the modulation of potential therapeutic targets in cases of pharmaceutic research. While the former represents a growing market since the introduction of international agreements such as REACH in Europe, the latter has been, for around 15 years now, a market worth several billion dollars, based in large part on the measuring of cell signaling pathways. Cyclic AMP assays remain the foremost commercial assay in terms of volume.

No reference book of which I am aware has addressed this issue in any comprehensive manner. The specialist literature remains compartmentalized, shutting itself off in various sectors such as the search for knowledge, industry, diagnostics, the environment or regulations. However, the same technologies are (or should be) at work in these different sectors. On investigation, it is clear that some of these disciplines using cell assays have not learned to communicate with each other. The objective of this work is to shed light on the contributions of these various schools of thought so as to improve the ease of exchange and, perhaps, to promote the spread of cell technology.

The development of cell assays requires knowledge of the intimate workings of the cell. Acquiring this knowledge has been particularly laborious and drawn out over several decades. A historical perspective could help to understand the importance of these works, which are seldom cited today. Indeed, historical perspective shows us the value of time. Often 20 to 30 years go by between understanding a biological mechanism and the emergence of applications that use it.

Introduction xxix

Measuring toxicity seems to have been the first application of live cell assays. In 1995, the combined contributions of molecular biology, fluorescence and genomics led to an explosion in our understanding of cellular biology, which itself led to the emergence of new generations of more varied and more informative assays in response to new requirements, particularly in the area of new drug research. These new approaches greatly benefitted from contemporary advances in miniaturization and robotization. We will see how, due to differences in schools of thought, these advances were not adopted with equal rapidity across all the sectors.

One of the major revolutions that took place in 1994 was due to the ability to substitute proteins for chemically-based fluorescent compounds. These fluorescent proteins, or GFPs, present in several species of marine animals, are coded by genes, the heterologous expression of which has been mastered. This advance unlocked vast possibilities of investigation, which naturally pushed the cell assay developers to engage without delay.

Finally, it seems appropriate to attempt this first reference book of live cell assays insofar as today we can acknowledge that the approach has acquired a certain maturity and that the applications considered are sufficiently numerous and recognized by academic, industrial and regulatory actors.

1

Principles and Position

1.1. Live cell assay principles

Cell culture aims to isolate cells from organisms then to keep them alive for experimental uses. Cell models vary widely. For practical reasons, the available human cells are, for the most part, tumorous in origin, having been immortalized so as to remain living for numerous generations. Culture cells can also be natural, which means that cells are collected in tissue or in organs for the purposes of an experiment. These are known as primary cells. Additionally, cells can be modified by a bioengineer so as to express genes that they did not originally possess. These are known as transgenic models.

In any case, cells must adapt to their new way of existing in vitro, a world in which they can no longer benefit from the multiple opportunities of complex exchange and communication inherent to their natural environment. Consequently, their behavior in culture is typically remote from the role they fulfilled as part of the organism.

Cell culture has been understood for over half a century. This long history provides it with a backlog of numerous applications spanning more than just cell assays (Figure 1.1).

Historically, cell cultures have acted as models for fundamental research and knowledge acquisition, particularly in cellular and molecular biology. Transgenic cultures, primarily based on the Chinese Hamster Ovary model (see section 2.3), have since been used as “factories” for the mass production of biopharmaceutics such as hormones or antibodies. More recently, cells have been in the limelight due to the first developments in cell therapy, a sector with great potential though very much still in development.

Live Cell Assays: From Research to Health and Regulatory Applications, First Edition. Christophe Furger. © ISTE Ltd 2016. Published by ISTE Ltd and John Wiley & Sons, Inc.

2 Live Cell Assays

Figure 1.1. Position of cell assays within the various application areas of cell culture

And finally, cell cultures have been used to perform evaluations and measurements. This is the area of cell assays.

The principle of cell assays is founded on the evaluation of an experimental condition, a cell model and a means of measurement. The choice of cell model is essential. Unlike other industrial or clinical applications, cell assays use the cell only to produce information. Accordingly, the cell model is chosen for its faithful representation of the biological context in which the information is being sought. The matching of the cell model to the experimental objective is clearly the key to evaluating if a proposed cell assay is fit for purpose. Any discussion about measurement quality will be dependent on the demonstration of this match.

This difficulty can be eased by considering the cell as representing a certain level of information to be reached. For example, the information in the living model is capable of integrating the effect of the experimental condition in the form of a global response. This is often the case in studies of cytotoxicity where the signals of interest are limited to global effects such as proliferation, apoptosis, alteration in DNA or membrane integrity. In such cases, the choice of the model ultimately counts for little. The response measured is shared by the vast majority of cell types. Ultimately, numerous

Principles and Position 3

assays work in this way, utilizing the living cell by default, as a simple demonstration of the effect sought on a living model.

However, some specific properties can be used for application purposes. These properties are dependent on the level of differentiation that the cell managed to retain in culture. These levels increase the pertinence of the cell assay’s information level. For example, neurons or cardiac cells can be used to measure signals of electrical excitability, liver cells can be used to metabolize and thereby activate or deactivate a compound’s toxicity.

To study the expression of a specific signal typically requires genome modification by transgenesis, which is the preferred method of orientating a cell toward a particular phenotype. Cell models developed in this way will have acquired a truly specific response. This strategy is widely employed in the pharmaceutical industry to create models that coexpress the therapeutic target of interest and the measured signal, based for the most part on fluorescent or luminescent proteins.

Notwithstanding, the question of the measurement method is more readily resolved. These methods are numerous and benefitted greatly from advances in molecular biology through the decade 1985–1995. Over the last 20 years, these advances have been consolidated while providing demonstrations of their viability.

1.2. Application areas

Live cell assays can be broadly categorized according to three areas of application (Table 1.1):

– cytotoxicity measurement;

– discovery of new medicines;

– diagnostics (pathological, military and environmental).

Cytotoxicity measurement represents a driving force in the development of live cell assays. Indeed, in a certain way, this is their natural application. There are two reasons for this: measuring cytotoxicity is above all a major issue in public health and increasingly so due to the modern preoccupation for pollution. However, cytotoxicity is difficult to evaluate without engaging

4 Live Cell Assays

living models as toxicity must be expressed. Then the cell becomes an essential target for toxicity. In the first instance, this typically manifests by a loss of homeostasis (reactive oxygen species generation, increase in ATP consumption, loss of membrane integrity, mitochondrial changes, DNA changes). The living cell in culture has proved itself to be an attractive model for such assessments. Homeostasis measurement methods are both reliable and numerous. Today, they cover the entirety of intimate, inner cell functions (see Chapter 4). Furthermore the cell is rendered fragile by being maintained in culture, often presenting high susceptibility to the effect of exogenous compounds.

Live cell tests are widely employed at various stages in the discovery of new medicines, from identifying therapeutic targets to validating compounds of interest. The essential area of application, in volume at least, is molecular screening. The strategy here consists of creating a cell model expressing the therapeutic target, and then employing it to select compounds of interest from chemical libraries according to both their capacity to bind themselves to the target in question and obtaining the expected response. Screening can be at high or ultrahigh throughput (with libraries of several thousands or hundreds of thousands of compounds) or high content (multiplex analysis of different cell parameters by image analysis). This vast area of application will be treated in more depth in Chapter 8.

Diagnostics represent the third main area of application for cell assays. The three main subsets of this area are public health, military programs and the environment. In public health, diagnostics consist of putting cells into cultures that have only recently been extracted from patients (see section 9.1). The signals observed will typically be genomic (karyotype), infectious (presence of antibodies) or therapeutic (efficacy in chemotherapy). Applications in diagnostics have a long history, with the first assays (see section 2.2) being perfected in the 1950s within the context of programs studying poliomyelitis. Military programs use assays to protect soldiers’ health in the theater of operations (see section 9.2). The principle is to ensure the extemporaneous identification of toxins in the event of bioterrorist acts. The environmental issue joins the military one but on a far more vast panel of polluting compounds (see section 9.3). The measuring technologies employed here are the same as other applications, albeit with cell models approaching those used in ecology (fish, bacteria, algae, etc.).


Cytotoxicity measurement Regulatory (health checks on chemical or cosmetic products)

Evaluation of drug candidates (pharmaceutic industry)

Diagnostics Pathological

Military (bioterrorism, theater of operations)

Environmental (pollution)

Drug Discovery High content or throughput screening

Pharmacokinetics (ADME)

Table 1.1. Main applications of live cell assays

1.3. Positioning

Cell assays are positioned at the half-way point between physicochemical tests, which measure the presence of substances or specific activities in abiotic systems, and animal tests, which are of a functional nature and provide answers at the organism level. Indeed, both of these varieties of tests are historically well-established. In the current industrial and regulatory landscape, cell tests are still considered as something of an alternative strategy with both advantages and disadvantages.

Physicochemical tests are mono-informative and quantitative by their very nature. While they measure the presence of molecular species in a clear, precise and standardizable way, they do not supply any indication on the effect or impact of this presence on the living being. Furthermore, they are often bonded to specific molecular species. By and large, they find only what they look for. Ultimately, these tests give rise to throughput problems and often require support from more onerous and expensive technologies.

On the other hand, animal tests are qualitative. The main interest of these tests is their capacity for evaluating the effect or the impact of a chemical species or mixture on an organism. With regards to effects on humankind, the extrapolation of these tests is dubious. Furthermore, they are very poorly adapted to high throughput, very hard to standardize and extremely expensive. They also give rise to major ethical problems that will be addressed in depth later.

6 Live Cell Assays

The final goal of live cell assays is to surpass the limitations that competing tests are subject to, in terms of the predictability of effects in human beings, throughput, cost, standardization and ethical considerations, all of which may be significant for the increasingly stringent quality requirements of industrial and regulatory applications.

1.3.1. Definition and typology of cell tests

The matter of definition is fundamental. Cell biology abounds with a great many measurement methods, which have been developed in response to various issues raised over the decades. Where can a line be drawn between the cell assay and method? On what criteria should we base an assessment of the relative importance of each method? The outcome from a regulatory standpoint can be considered initially. Indeed, all tests that have followed through in implementing the organization for economic co-operation and development (OECD) guidelines or, on occasion, an ISO norm, have necessarily succeeded along the whole value chain. Nonetheless, we will see that regulatory bodies are extremely conservative and the happy few that are chosen for their list, 15 at most, are too restrictive and do not represent the diverse needs of applications.

A more reasonable criterion then is to consider the capacity of an approach to be standardized. This idea takes into account the numerous tests validated by use and not by a regulatory body. The criterion of access to high throughput will automatically permit a test to be taken into the applicative dimension and can also be retained. Several approaches that are widely practiced by the scientific community though without being standardized due to reasons of the complex process or a lack of industrial interest may also be considered as cell assays. And lastly, several approaches inspired by recently acquired developments in cell biology that are considered as fertile ground for the future of cell assays will also be brought into consideration.

The issue of cell assay typology has never truly been broached either. And this gives rise to a question: how can we rank the highly varied approaches whose only commonality is their foundation on cells in culture? The most straightforward way is to proceed according to the type of application in line with the three main areas mentioned above.

A second way to proceed is by reference to the technologies employed, which for the most part are the same in all three types of application. These


technologies can be categorized into four main classes: colorimetry, fluorescence, bioluminescence and label-free methods (see Chapter 3).

A third way to address this issue is to consider the information level delivered by the approach. An assay in which the end measurement is read directly in the live cell in culture, by image analysis, for example, may be considered as more informative and pertinent than an ELISA-type test in which cells have been lysed to make the medium more homogeneous. Although found in various publications, this point of view is of debatable value since the best test is above all the one that provides the information corresponding to the question in consideration.

Finally, a last way of address the issue is to consider the status of the cell under analysis. Here we may note the following propensities, divided according to their level of complexity:

– The first consists of employing non-modified cells, or at least modified no more than least required (immortalization) for culture. In this way, cells are as close as possible to the physiological reality and may be considered to be in homeostasis. The analysis will then consist of measuring the disturbance levels of this homeostasis under the effects of a physical agent or chemical disruptor. This process finds many applications in questions of toxicity (Chapter 4) or pollution (section 9.3). In general, this approach employs colorimetric, fluorescent or bioluminescent agents, which can nonetheless disturb the signals under analysis. This problem may be avoided by means of label-free approaches that make use of a cell’s autofluorescence or of certain noninvasive electrical or optical properties (from section 3.2).

– The second consists of modifying the cell’s genome so as to transform the physical or chemical agent’s effect into a fluorescent or bioluminescent intracellular signal produced directly by the cell. Green fluorescent protein (GFP) and reporter gene strategies are typically considered to belong to this category (section 3.1).

– The third practice, and most significant in terms of activity volume, consists of verifying an independent cell function or homeostatic function, often enzymatic activity or the signaling pathway associated with a target, particularly pathological targets. Often this process requires the addition of a second genomic modification so as to create a model that independently co-expresses the luminescent signal and the target of interest. This is quite naturally put to work on the part of the pharmaceutical industry in researching new medicines. The practice will be described in Chapter 8.

8 Live Cell Assays

All of these typologies are admissible and any preference for one or another depends only on the standpoint that actors may take within their sector of activity. The cell assays will be described here in accordance with their area of application: routine toxicity measurements (Chapter 4), regulatory toxicity measurements (end of Chapter 2, Chapter 5), researching new medicines (Chapter 8) and diagnostics (Chapter 9). The major technologies that are common to all of these various applications will be preliminarily introduced in Chapter 3.

1.3.2. The regulatory and industrial dimension

In regards to the market and access to the market, segmentation between industrial and regulatory applications becomes a necessity. Although both are engaged in live cell assays, each has developed in complete independence from the other, albeit in a parallel way. These two schools of thought were launched (or initiated) from opposite reasoning (or logic).

The main client of cell assays, the pharmaceutical industry, standardized numerous, and often highly sophisticated, cell approaches over 20 years ago in response to questions concerning the validation of therapeutic targets, the identification and validation of new compounds or toxicity measurements. More cell approaches have come to light in recent years in the area of pharmacokinetics (absorption, distribution, metabolism and excretion [ADME]), the stage preceding clinical tests. This is a significant market covering all therapeutic areas.

Regulatory authorities, on the other hand, are committed to protecting citizens from the potential dangers brought about by the industrial and agri-food industries, which every day invent and produce new substances that must be tested for their innocuousness to public health and the environment. For decades, the means employed by regulatory authorities have relied on the exclusive use of animals for their measurements. The progression of live cell assays into the market is still ongoing, and policy toward alternative approaches has remained, until recently, hesitant. Since the implementation of the 7th Amendment of the European Cosmetics Directive in Europe, banning all studies of toxicity performed on animals since 2013, the nature of the game has slowly changed. This has also been the way in the whole of the industrial sector due to the Registration, Evaluation, Authorization and Restriction of Chemicals (REACH) European Regulations. This applies to 125,000 substances, produced by the industry, whose toxicity must be tested by 2018. While the REACH regulations allow for a large part of these tests to


be performed on animals, there is pressure being exerted on the official bodies that approve alternative tests to accelerate the legal availability of new approaches, in particular that of live cell assays. This pressure is even more pronounced considering that in vivo tests, aside from the ethical considerations that they raise, are adapted to neither the societal nor economic stakes in terms of both attainable throughput and cost.

It is regrettable that regulatory and industrial bodies remain so closed-minded. It appears that regulatory bodies have not fully understood the advances that have been made by both the pharmaceutical industry and in academic research over these past 20 years. Or perhaps this understanding has indeed arrived, albeit very late. In any case, the lethargic pace of decision making on the part of public authorities has left legislators wishing to fulfill their obligations concerning Directives and Regulations with a relatively short list of cell assays and, more widely speaking, in vitro tests, for the most part developed in the 1970s and 1980s.

It is worth noting that the term in vitro may at times be employed to designate live cell assays. As far as regulatory organizations are concerned, the term covers all alternative methods to animal tests. In fact, most of the in vitro methods accepted as such by the official organizations are live cell assays. Some approved in vitro tests do nonetheless use extracts of human skin tissue, poultry eyes, bovine cornea or acellular biological membranes. It should also be noted that the term in silico has been accepted in reference to certain alternative methods, which, by means of software, describe the structure–function relationships (SAR) or quantitative SAR (QSAR) of compounds. These last approaches have not been validated by regulations but instead enter into certain tiered processes.

1.4. Market

Market studies constitute a burgeoning sector of activity and the global market for live cell assays is no different. Any Internet search engine will provide dozens of results on the subject at the click of the mouse.

To introduce the subject, it is worth recalling that the global market for cell assays was worth $300 million in 2002 according to a study [FRO 02] by Front Line. At that time, cell assays were considered as emergent and divided in technological terms between fluorescence (75%), bioluminescence (20%) and the remainder (nanoparticles, quantum dots, which represented <5%). Live cell tests were at the time defined as:

10 Live Cell Assays

The tools used in analyzing the biological response of cells, specifically studies that establish the response to potential drug compounds.

It would seem that the market was directed only at the pharmaceutical industry. Their use at this time was primarily in the validation stage of targets following human genome sequencing. The massive shift toward high throughput screening had yet to take place. At that time, the factors that were associated with market growth were in connection with the discovery of new medicines, and particularly, the possibility of acquiring patents on proteins or target genes. A norm had already been established on culture in 384 well plates together with a trend toward outsourcing screening due to the cost involved and at times, due to the expertise required.

The situation is somewhat different today. A dozen studies, for the most part titled “Global Cell-based Assay Market”, which, according to their market evaluation in terms of volume, fall into three groups. Group 1 (low bracket) includes studies by Global Industry Analysis and Transparency Market Research, which evaluates the market at around $1.5 billion in 2017–2018. Group 2 (The Market Publisher, Companies & Markets, VisionGain, Research & Market) estimates the same market between $3.9 and 4.7 billion for 2015–2016. VisionGain expects a progression to (or: up to) $5.5 billion for 2018. The studies in group 3 (high bracket), particularly BCC Research, Markets & Markets [MAR 14], IQ4I (Intelligence Quotient for Innovation), Research & Consultancy and Market Research have announced values between $14.8 and 27 billion to be reached by 2018–2020. The significant discrepancies between these evaluations can cast doubt on their worth here, despite the studies each costing between €3,000 and 5,000. The estimate of $4.7 billion (VisionGain) is often accepted in reference articles.

According to publically released data [CLI XX], live cell assays are still an essential aspect of the pharmaceutical industry. In the sector of high throughput screening alone, live cell assays now constitute 50% of total use. This is considerable. Their versatility is appreciated. One aspect of this versatility is in allowing for an early assessment of toxicological and metabolic aspects, thereby avoiding problematic compounds before beginning highly expensive clinical trials. This is a highly desirable attribute in the eyes of industrial actors who are obsessed with knowing which candidate medicines will be defective in terms of cytotoxicity as soon as


possible. The identification of such candidate medicines is invariably too late. Far too often, it arises during ADME/Tox (40%), animal testing (10%) or even during the assessment of harmful effects and side effects in humans (10%).

In terms of technologies, a study by Research & Market [RES 15] found that FRET represents the largest part of the market, the other main parts being fluorescence in situ hybridization (FISH), high content screening (HCS) and radiometric analysis. Nonetheless, a significant technological development has occurred since the 2002 study cited above in the arrival of label-free methods, which now represent a considerable part of the market.

All of the studies agree that industrial demand for live cell tests will develop according to an annual growth rate of 8–12%. Another significant development in recent years concerns the emancipation of functional cell assays and their marketing in the phenotypic screening sector (see section 8.1), which is focused on the cell’s expression of pathology, independent of any knowledge of the therapeutic target. This is now the largest part of the market in the United States, also presenting the strongest growth.

The study performed by BCC Research singles out the emergence of live cell assays in the ADME/Tox sector. In 2018, these assays will represent 21% of the market in terms of volume (against 70% for screening and target validation), and thereby constitutes the most dynamic sector [BCC 14].

According to Frost and Sullivan, the market trend is towards customized solutions [FRO 08]. Indeed, personalized medicine harbors vast potential for growth, particularly with regards to cell assays establishing the biomarker profiles of individual patients.

VisionGain addresses the British market in its analysis, which represents around 15% of the European market [VIS 15]. The British market is projected to grow from $98 million in 2014, to $164 million in 2018, to $251 million in 2022 and to reach $328 million in 2025. Overall, the study expects that the trend of outsourcing will increase from 24.5% in 2011 to 30.2% in 2023, and market growth to be supported by technological advances in sensitivity, precision with regards to cytotoxicity, miniaturization and multiplex analysis. Finally, control over stem cells, particularly induced pluripotent stem cells (iPSCs; see section 10.1), has the potential to provide more pertinent pathological models that may also help support market growth.

12 Live Cell Assays

It should be noted that the 2010 report by Global Industry Analysts mentions the emergence of an in vitro toxicity market superseding animal models [GIA 14]. In this way, live cell assays are presented as essential prediction tools, whether used in batteries or in tiered testing. However, the study mentions that very few assays have been able to reach any market due to regulatory limitations.

1.5. Competitive advantages

The underlying idea of live cell assays is to appropriate the advantageous attributes of physicochemical and animal approach types by making use of advances in molecular engineering together with the wide variety of available cell models.

By nature, live cell assays are reductionist. They simplify the natural environment to limit the number of variables and to quantify the achieved effect. This facet of assays is both an advantage and a disadvantage. On the one hand, the signal is not drowned out by environmental interference, on the other, it is less (some would say hardly or not) representative of reality.

1.5.1. Cells are live information models

Cell assays possess a decisive advantage in using live models. In a way, this likens them to animal tests, since they retain the capacity to integrate an observed response. Furthermore, this information level is obtained according to a favorable cost/throughput basis and without ethical issues, with, for human cells at least, the consent of the patient or healthy donor (a propos see the debate on HeLa cells in section 2.5).

The success of cell assays, as demonstrated in the market studies presented above, comes primarily from the ability of numerous models, in particular human models, to adhere to plastic supports and, in conditions providing nutrition and growth factors, to reorganize themselves to survive in a group as long as needed for manipulation.

As a living unit, the cell acts autonomously. The cell’s responses to an external stimulus are coordinated and, above all, integrated. At a molecular level, this is manifested by the convergence of complex signaling pathways resulting in the expression of specific genes, cytoskeleton mobilization, the production of biomolecules, etc.


1.5.2. Development: high throughput

Increasing throughput is one of the major advantages of live cell assays. In the mid-1990s, the pharmaceutical industry constructed large chemical libraries as part of their race to identify new medicines. This practice was later continued by certain academic laboratories or institute groups. Today, some libraries contain several million compounds. Meanwhile, the emergence of the consumer society since the 1960s, followed by the globalization of economic exchange since the 1980s, has contributed to the commercialization of over 130,000 chemical compounds, according to a survey carried out for the REACH European Directive (see section 6.5), all of which must now be tested for innocuousness. This global context requires high throughput measuring technologies. Today, these technologies constitute the main body of live cell assays in terms of volume.

Over the course of the last two decades, live cell assays have successfully adapted to the standards required by the ever-increasing size of chemical libraries used in testing. For example, assays have moved on from 96 well plates to the new formats of the same size containing 384 or 1536 wells [MAY 09]. This experimental configuration has been accompanied by a parallel development in automation and robotic tools. Indeed, robotic tools ensure that the information level and quality provided by live cell assays remains consistent. In particular, robotization has contributed to improve kinetic evaluation and measurements of dose–response with an EC50 quantitative evaluation measurement capability [CON XX].

A further advantage of high throughput is the ability to perform parallel assays on different cell lines with different characteristics, for both tissue cells (epithelial, endothelial, excitable, circulating) and organ cells (liver, kidney, nervous system, heart).

1.5.3. Development: multiplex analysis

Multiplex analysis is a sector in which live cell assays possess near exclusivity. This approach was brought about in the 1990s following the development of image analysis systems combined with new molecular devices, fluorescence in particular. The American company Cellomics, which was later purchased by the Thermo Fischer group for $49 million in 2005, led research in the sector to create high-throughput screening platforms that would allow for the measurement of multiple simultaneous parameters at cell level, with the molecular components being marked by fluorescent tags [ABR 08].

14 Live Cell Assays

1.5.4. Development: miniaturization

At the same time, the development of throughput standards underwent a process of miniaturization, or even of ultraminiaturization. Live cell assays have also adapted well in these terms. Considering that plate size has remained constant at 12.8 × 8.5 cm, working on plates with 384 or 1,536 wells allows us to significantly reduce reaction volume (from 200 µl in 96 well plates to 5 µl in 1,536 well plates), the quantities of substances to test and to streamline costs simultaneously. Waste products have similarly been significantly reduced.

1.5.5. Development: molecular engineering

Be it human or of another species, the cell genome can easily be modified and controlled. Revolutions in molecular biology (through the 1970s and 1980s) followed by those in the genome (through the 1990s and 2000s) have provided the tools necessary for cell engineering, the most prominent of which are gene knock-outs or knock-ins, introduction of mutations or deletions, and the construction of chimeric genes. Numerous cell assays use models of transgenic and/or clonal origin. We will see from section 3.1 how the fluorescent properties expressed by different animal or vegetable species can be transferred for measurement purposes to human or other mammal cells. Or moreover, how cells can be durably equipped with therapeutic targets and confronted with compounds of therapeutic interest.

1.5.6. Development: standardization

One of the objectives of live cell assays is to simplify the experimental implementation and decrease the number of variables so as to quantify them individually in a controlled environment. Of course, this choice draws cell assays away from the physiological context but provides access to a high level of standardization. It follows that variability between experiments is minimized. Variability can be quantified by means of experimental derivative measurement tools like factor Zʹ, which is widely employed by the pharmaceutical industry. This is based on a particularly challenging statistical analysis:

Zʹ = 1 − 3(σp + σn) |µp − µn|


where σp and σn are the standard deviations of the, respectively, positive and negative controls and µp and µn the means of these same controls. The ideal cell assay gives the value Zʹ = 1, with a value of Zʹ between 0.5 and 1 indicating an excellent test [ZHA 99]. This factor can be applied at intraplate level (to establish the variability between the wells of the same plate), or more generally for high-throughput screening problems at interplate level (to establish the variability in responses between one plate and another).

The application of good laboratory practice (GLP) is an important part of standardization. This consists of following a collection of rules or simple recommendations, depending on the context, as defined by the OECD and made law by a European directive in 2004. GLP ensures the quality and integrity of results. Finally, standardization is greatly facilitated by the commercial availability of cell models, disposable kits and cell engineering methods, all produced in controlled conditions.

1.6. Can measurements of cells in culture be extrapolated to effects in the organism?

Aside from the clear advantages described above, one question remains unaddressed concerning the classical applications of cell assays such as toxicity measurements and the discovery of new medicines. Is the cell functionally representative of the organism from which it originated? The response is clearly negative. The key stages that result in the expression of toxicity or in cell response at organism level are absent in approaches based on cell culture. The cell is indeed the living unit, however no living being is simply the sum total of its cells.

1.6.1. Toxicokinetics

The key data that are absent in live cell assays are above all toxicokinetics, which refer to the measurements of the relationship between exposure and the toxic dose actually exerted within an organism. The outcome of an exogenous agent in the organism depends to a great extent on its absorption, its distribution, its metabolism and its excretion. However, toxicokinetic data are only accessible by means of extrapolation according to mathematical prediction models known as physiologically based pharmaco-kinetics (PBPK), which were developed from animal data. These sophisticated models include numerous variables such as blood flow,

16 Live Cell Assays

respiration, biodistribution, etc. They are considered robust and independent of (or at least adjustable to) the animal species in consideration.

Nonetheless, various studies have been published which address PBPK models based on in vitro/in vivo correlations. These studies have demonstrated that toxicokinetics could be evaluated based on cell assays, and in particular on organotypic culture models (see section 10.2).

1.6.2. Components of the immune system

Any exogenic agent entering into contact with an organism will inevitably find itself confronted by a response from the immune system. Neutralization antibodies (see section 9.2) are produced in case of intrusion by proteins for example, resulting in their elimination from the organism. Inflammation also represents a possible response by the organism to the presence of a xenobiotic or candidate medicine. At the level of a cell integrated in the organism, this is manifested by damage, leading to the recruitment and activation of specialized cells and the secretion of inflammation molecules such as cytokines at the area. These events are absent in assays performed on cells in culture.

1.6.3. Biotransformation

Numerous substances must be metabolized prior to activation or deactivation. In the living world, this activity is regulated by enzymes specialized in intracellular decontamination such as P450 cytochromes, which are particularly present in the liver of vertebrates. A cell model cannot recapitulate this capacity for metabolization by the organism. However, certain models, in particular those developed of liver cancers, can express certain P450 cytochromes and manifest biotransformation activity. At times, the use of microsomes containing these same cytochromes can be integrated to experimental protocols.

1.6.4. The macrocellular environment

On the macroscopic level, an organism’s cells are surrounded by an environment that provides them with nutriments, dioxygen and growth factors among others, which ensure their growth, their function, and their


homeostasis. These aspects are relatively well controlled in culture and adequate media are commercially available.

However, the bioavailability of the compound with respect to its target cells within the organism is less well controlled in culture. Indeed, with the particularly high levels of plasma proteins at the interior of an organism, a number of these proteins bind the circulating compounds and thereby reduce their solubilized fraction. It follows that compounds are less available to exert their effects on cells. Plasma operates like a reservoir, capturing compounds, maintaining them in an inactive state in pharmacological terms and redistributing them later on, thereby significantly altering their life expectancy inside the organism. Accordingly, a compound’s bioavailability is a function of time and its concentration can be decorrelated in reality from the quantity distributed initially. In terms of cell culture, a standardized proportion of serum is conventionally added to the culture medium to partially compensate for this problem.

Moreover, numerous feedbacks, exerted by means of hormones for example, which regulate homeostasis at organism level, are not taken into consideration by cell assays. The same can be said for systemic boundaries such as the blood brain barrier or the placenta. We will see in section 10.2 how in this respect organotypic cell culture allows for potential solutions.

Finally, the organism is subject to the harmful effect of compounds that act at a low dose but over long periods. This is also a difficult subject to address by cellular biology even if, here too, significant developments are in progress (see the Seurat program, section 6.4).

1.7. Limits

1.7.1. Importance of cellular microenvironment

Setting aside the difficulties of extrapolating systemic effects mentioned above, the cell approach in its conventional form also presents intrinsic limitations of which the leading two are dissociation, which isolates the cell from its counterparts and the loss of the extracellular matrix. In section 10.2, we will see the workaround strategies currently in development.

The following arguments are treated more extensively in the excellent reference article by Anna Astashkina et al. [AST 12] published in 2012 by Pharmacology & Therapeutics. The cellular microenvironment comprises the

18 Live Cell Assays

relations from cell to cell, interactions between cells and the extracellular matrix and the role of tissue architecture. These aspects are essential for the maintenance of the cell in physiological conditions and are often not addressed by interpretations of in vitro results.

Intercellular exchanges ensure that cells function in an integrated manner within a tissue via their capacity to transmit messages to a whole population. They ensure a sort of social cohesion, which is manifested in cellular terms by a shared response such as, in case of aggression, adhering to a mass movement of apoptosis or, inversely, expressing the genes implied in the upkeep of homeostasis. In fact, this type of behavior is naturally lost in cells that are dissociated from their natural environment to be put into culture.

The issue of the extracellular matrix and the adhesion proteins, which attach cells to their support, is certainly the weak link in in vitro assays performed on models of dissociated cells. These adhesion proteins form a large family, collectively referred to as cell-membrane adhesion molecules (CAM). The cadherins are represented by more than 350 members. They complex with catenins as a means of transferring adhesion messages to the cytoskeleton. In this way, they operate as mechanical transmitters. The gap junctions form hemi-channels that by associating themselves with their alter-egos found in neighboring cells form direct intracytoplasmic contacts connecting numerous cells between one another in structures called syncytia, which are capable of synchronizing populations and ensuring group homeostasis. Other proteins such as ICAMs or selectins are also involved in intercellular relations concerning inflammation.

Indeed, the nature of the in vitro culture’s adhesion surface, plastic more often than not, is minimalist. In any case, it is insufficient to allow cells to retain a normal phenotype, even if the support is coated in collagen or with a combination of matrix proteins, and even issued by real contexts such as Matrigel. The relationship between the cell and the matrix is fundamental, based on mechanical (forces conveyed to the cytoskeleton) and chemical (signaling molecules, growth factors, inflammation modulators) information and highly sensitive to events in the microenvironment. Proteins such as catenins and integrins play a significant part in these exchanges.

Furthermore, a compound’s toxicity inside the body can be expressed by the destruction of certain intercellular communications or certain adhesion proteins, initiating a cascade of events leading to tissue necrosis and cellular apoptosis concomitant with an inflammatory reaction. As the initial event is not considered in vitro, the consequences raised here escape analysis. In fact,


it is not that the adhesion proteins are not expressed by in vitro models, but that they are expressed though only at levels of expression far removed from physiological conditions.

These critiques of cell assays only apply to conventional live cell assays performed on dissociated cell models and adhering to a plastic support, also referred to as 2D culture. Nonetheless, this analysis does limit the range of in vitro methods such as they are generally practiced today. This does not mean that live cell assays do not have any value but merely that they cannot be obliged to say more than they are capable of saying, and indeed, this book intends to prove that they are capable of saying a lot.

Simply put, the choice of a live cell assay must be an accurate reflection of the question being posed [AST 12].

1.7.2. Other limits

A classic criticism of live cell assays is that they are, for the most part, attached to cancerous lineages. Indeed, this reality is often detrimental and considerably limits the diversity of available cell phenotypes. Stem cells, the primary cells and models of other cells issued from other classes of the living world provide a limited workaround to this problem.

Ultimately, assays using human or mammal cells remain quite complex to implement since they require a well-equipped laboratory environment (dedicated room, sterile enclosure, incubator at 37°C, CO2 controlled atmosphere). They also represent a potential hazard, since human or primate models are possible hosts for a transmittable virus. Consequently, certain models of bacteria, yeast or other insect cells can be safer to manipulate.

2

History and State of the Art

2.1. Origins of cell culture

The history of live cell assays is closely intertwined with that of cell culture. From the turn of the 20th Century to the 1930s, popular imagination culminated at the notion of cultivating cells in vitro. All kinds of applications were envisaged, the most ambitious of which sought to obtain models allowing for the potential regeneration of the organs or tissues from which cells had been collected. Certain renowned researchers, such as Alexis Carrel, thought that transplantations would soon be a viable option. And while cell culture was indeed relatively well understood by 1920 to 1930, its applicable projects were to fail utterly. Cells survived being put into culture and even succeeding in dividing, but only with a very short life expectancy of no more than several days at most. When researchers finally succeeded in maintaining cells over several generations, the cells tended to dedifferentiate rapidly. It was not before the 1950s that the first lines of immortalized cells emerged. From then on, the first applications began to appear. Cells in culture became a medium in the development of biological material such as viruses required for vaccine production. They were also used in the diagnosis of certain infectious pathologies. With the development of cryopreservation, cells could be transported by plane, and before long would be found in laboratories around the world. The first live cell assays were then to appear along with standardized protocols. Above all, these assays were concerned with toxicity measurements.


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2.1.1. Pioneering studies

The accepted practice [FRE 14] is to trace the history of cell culture in vitro to the works of the American embryologist Ross Harrison (1870–1959). It was Harrison who established the first culture protocol to exert long-term influence on our understanding of cell culture through the years. Several researchers had attempted this before Harrison, some successfully cultivating animal and vegetable cells. It appears that the doctor and physiologist Alfred Vulpain (1826–1887) was the first to have made successful attempts as early as 1859. He conserved tadpole tails in culture for several months, albeit without observing any signals of cell growth.

The honor of the first long-term animal cell culture doubtless goes to Parisian doctor and hematologist Justin Jolly (1870–1953), professor at the Collège de France. In attempting to describe the structure of chromatin during the stages of cell division, he discovered in 1901 that triton erythrocytes (which are nucleated in amphibians) presented cellular division in vitro. Following this discovery, in 1903, he demonstrated that erythrocytes and red blood corpuscles can survive for several months outside of the organism. Moreover, he observed that erythrocytes continued to proliferate during this time. Indeed, in a letter dated February 24, 1937, Harrison recognized “the major role that the aforementioned played in the history of our understanding of cells extracted from the organism and that he must be considered one of the pioneers in cell culture” [COU 53].

The major contribution that Harrison was to bring 4 years later was to demonstrate that conserved fragments of amphibian embryonic neuronal tissue not only survived for several months in their lymph droplet, but transformed, developing classical neuronal attributes [HAR 10a] (Figure 2.1). This observation would lead to the numerous technical developments of the first few decades of the 20th Century.

While the Jolly’s works went unnoticed, Harrison’s efforts were greeted with skepticism by his contemporaries, who quite simply did not believe him. Essentially, the scientific world was doubtful when presented with the expression of autonomous life in vitro. Indeed, these were the results that would revolutionize the very notion of life. What becomes of the idea of individual life if each of its constituent parts is capable of living independently?

Transcending the domain of biological research, this question would fascinate both intellectuals and the general public through the 1930s, even

History and State of the Art 23

influencing philosophical thought and literature. Indeed, life had finally escaped from the milieu intérieur as envisioned by Claude Bernard. Thereby, a central pillar of 19th Century biology collapsed.

Figure 2.1. Neuronal growth in culture obtained from a tissue fragment. Harrison’s experiment of April 28 and 29, 1908. Drawings

after microscopic observation. Reprint from [HAR 10a]

2.1.2. Alexis Carrel

The Frenchman Alexis Carrel was a central figure of this new vision of the living world. As a doctor and researcher living in New York, Carrel spent his entire career at the Rockefeller Institute, igniting the public imagination with his work on in vitro cell culture between the 1910s and 1940s. In 1908, Ross Harrison gave a talk about his amphibian neurons in culture at the Harvey Society of New York. Carrel was in attendance. A colleague of Carrel, Montrose Burrows contacted Harrison on March 22, 1910, later spending several months at Yale in Harrison’s laboratory. Burrows learned about cell culture technique during this time, having already planned to apply it to the calls of warm-blooded animals. Once back at the Rockefeller Institute, Burrows set to work. While cultivating poultry heart cells, Burrows observed that the fragment cultivated in plasma continued to beat. Moreover, as the cells began to individualize by migration from the explant, they

24 Live Cell Assays

continued to beat individually and in a synchronous manner [LAN 07, p. 49]. He concluded that cell functions are autonomous, since the sample was isolated from any contact with the nervous system or other capable of regulating the beat.

Carrel and Burrows published their first results between October 22 and December 12 in a series of seven articles published simultaneously in French and English in the Comptes Rendus de la Société de Biologie (Paris) and the Journal of the American Medical Association (JAMA). In these articles, they presented their ability to cultivate and maintain cells unchanged irrespective of their origin in the kidney, bone marrow, the spleen or in cancerous tissue. Indeed, their enthusiasm surpassed the reality of their observations somewhat. During a conference that November in Paris, Justin Jolly unleashed a scathing critique of Carrel’s affirmations, pointing out that while the cells were indeed alive after several days, they had lost all of the distinctive features that linked them to their organ of origin. The cells had become dedifferentiated. Moreover, there were no signs of division. The exchanges were animated between the two men, with Jolly maintaining his position on the requirement of the undivided body as a means of maintaining life. Carrel was shaken: he had no response for the critiques presented by Jolly.

Nonetheless, Carrel undertook to demonstrate the immortal nature of cells in culture. He chose chicken embryo cells to this end. The culture began officially on January 17, 1912, working with experiment number 720. The project was taken over in June of that year by the laboratory technician Albert Ebeling. After 104 days in culture, the cells ceased their pulsations but continued to grow [LAN 07, p. 79]. By February 3, 1913, the cells had been the subject of 138 passages (transfers to a fresh nutritive medium) and by May 1914, 358 passages had been reached. Before long, the culture had become famous around the world. In 1921 the 1500th passage [COR 64] was celebrated in grand style for, as Carrel’s assistant remarked, “the most remarkable career ever enjoyed by a chick”. In any case, the immortal line dating from 1912 boosted Carrel’s media profile, already burgeoning since receiving the Nobel Prize awarded the same year for his works in surgery [TRA]. The New York Times published a long article October 13, 1912, titled “Dr Carrel’s Miracles in Surgery”, extolling his advances in the development of poultry cell lines.

The social scientist Hannah Landecker [LAN 07] recently studied this period and drew some valuable conclusions on Carrel’s motivations. She explains that Carrel sought, above all, a philosophical explanation of life and that he intended to use his in vitro results to this end. In any case, the heart


that continued to beat in vitro had a resounding media impact. This was an image that left no doubts as to the question of life in vitro. Moreover, Carrel conveyed a mystical insight with which he could readily extrapolate the behavior of cells observed in culture. He believed that he had succeeded in extracting life from temporality [LAN 07, p. 71]. In 1913, in Paris, Jean Comandon directed the first film depiction of a cell culture with the support of Carrel. Landecker notes that it was at this time that the status of the cell changed, moving out of the realm of pure science and into the spheres of economics, technology and philosophy.

2.1.3. Were Dr Carrel’s cells immortal?

By now it was 1921. What happened after the famous culture of poultry heart cells? As the years went by, they never stopped growing. Carrel argued that in ideal cell culture conditions, “cells do not record time qualitatively; in fact, they are, immortal” [CAR 31]. Carrel was retired by the Rockefeller Institute in 1939. He then returned to France in July and openly expressed his willingness to work with the collaborationist Vichy government, which named him Directeur de la Fondation Française pour l’Etude des Problèmes Humains (Director of the French Foundation for the Study of Human Problems), working to propagate the eugenicist ideas presented in Man, the Unknown, published in 1935 [WEK 04]. He died in Paris in November 1944. Though this time, the peaceful existence of his immortal culture continued undisturbed. Albert Ebeling penned an article in 1942 in the Scientific American commemorating the 30th anniversary of the line’s birth. In the meantime, he had transferred the culture to the Lederle Laboratory of the American Cyanamide Company where it was put to use in testing germicide toxicity. In this respect, Carrel’s immortal culture represents one of the first examples in history of cell assays for toxicity. The culture would finally be abandoned April 26, 1946, 2 years after the death of Carrel.

However, one question remains unanswered. How was it that no other team managed to create a similar immortal line between the two wars? Some researchers thought they had succeeded, though ultimately their cells ended up dying. Such cases were explained as accidents of technique. It was not before the 1960s that Carrel’s works were openly unraveled by experimentation. In 1961, Hayflick and Moorhead demonstrated very clearly that normal cells have a limited life in culture and cannot surpass 50±10 divisions in vitro [HAY 61]. Co-culture experiments revealed that this life expectancy was inherent to the type of cell observed. This is the phenomenon of cell senescence, which is widely accepted today.

26 Live Cell Assays

How then to explain Carrel’s results? The 1980 study lead by Jan Witkowski established three possible explanations [WIT 80].

The first explanation is based on the possibility that the line spontaneously transformed, as has sometimes been observed to happen. This hypothesis is improbable, since cells in culture have, in all observed cases, changed in morphology and behavior, starting to grow on top of one another, for example. Yet, in all the reports of the study by Eberling and Carrel, cells strictly maintain their unchanged appearance.

The second hypothesis relies on the possibility of contamination by fresh cells that could have been transferred with the poultry embryo extract, which was added to the plasma by Carrel to stimulate cell growth. However, a centrifugal step was systematically included in the extraction protocol, which theoretically eliminated all the parasite cells. In any case, if this did indeed occur, such contaminations were minimal and insufficient to explain the cells’ survival for 34 years.

The third hypothesis, retained as the most likely, is that of the regular restocking of the line with fresh cells. In this respect, Witkowski gives credence to Rockefeller Institute’s historian George Corner. According to Corner, Carrel did not discuss his work with colleagues and upheld the utmost confidentiality concerning his work. Rare were the lucky ones to actually glimpse the line, and visitors, in general, were not welcome at all. Carrel rather conveniently argued that the risk of infection was far too high to approach the culture. This was the experience of Dr Ralph Buchsbaum during his visit to Carrel’s laboratory in the summer of 1930. Fifty years later, he spoke in detail about the visit to Witkowski. Denied access by Parker, Carrel’s laboratory assistant, then again by Ebeling, who showed him several other experiments as a conciliatory gesture, Buchsbaum was reluctant to leave New York without seeing the famous culture and waited for the laboratory to empty before begging a young technician to let him in. She refused, afraid of Carrel or Parker’s reaction, but ultimately ceded. “When I looked at the cells”, explained Buchsbaum, “and said that they were full of fat globules” and obviously on the way out, she said slyly, ‘Well, Dr Carrel would be so upset if we lost the strain, we just add a few embryo cells now and then…’”.

It was advances in cell culture that propelled Carrel to the heights of popularity, but it was Carrel himself who succeeded in staying there. And without going into too much extraneous detail, let us recall that cell culture outside of the human organism was the subject of great popular excitement in the 1920s and 1930s. The general public was alarmed by the new control


exerted by scientists over the biological material. This was reflected on the literary front by the 1926 publication of a novel by the renowned cellular biologist Julian Huxley, titled The Tissue Culture King, presented as a parable about the dangers of modern science. This first fictional novel on the theme of biotechnology presents the monstrous consequences of a British researcher providing an ill-intentioned African tribal king with cell culture techniques. It is a cautionary tale for modern society illustrating the paradox presented by control over cell culture, which presents both a major and imminent danger and an extraordinary opportunity to improve the human condition. Incidentally, Julian Huxley, father of the transhumanism movement, arrived at a eugenist position from his ideas on cell culture.

These thoughts ultimately found their expression in the 1932 publication of Brave New World, whose author Aldous Huxley was Julian’s brother. This best-seller also depicts a utopia that becomes a nightmare, although the author warned readers about the nefarious consequences of germ cell culture development.

2.2. The HeLa line and the first applications of cell culture

Henrietta Lacks (Figure 2.2) was born into a modest African American family on August 18, 1920. Her mother died giving birth to her 10th child. After the war, Henrietta lived with her husband David and their four children in a rented five-room house on New Pittsburgh Street, Baltimore. In January 1951, Henrietta was admitted to John Hopkins Hospital suffering from intermenstrual bleeding. The doctor discovered a red object of approximately one inch in diameter on her left side. This was strange since tumors found in this location are typically clear. The gynecologist suspected a lesion due to syphilis but the anatomopathologic exam concluded that it was an unusual form of cervical cancer. Henrietta underwent a first gamma-ray treatment eight days later. The surgeon took the precaution of extracting a sample beforehand, which was sent to George and Margaret Gey, the team of cell biology researchers at John Hopkins. Both had been attempting desperately to keep tumorous human cells in culture for the long term. They were interested in the typology of cervical cancers and hoped that the behavior of tumorous cells in culture would provide useful information in understanding their action on the organism. On February 9, 1951, around noon, a laboratory technician, Mary Kubicek, accepted the delivery of the biopsy. She had already launched a dozen cultures based on cervical cancers, but none with any success. The cells would invariably survive for several days before dying. According to the details collected by Michael Gold in his work A Conspiracy of Cells, which relates the

28 Live Cell Assays

episode [GOL 91], she casually finished eating her sandwich, named the box HeLa and set to work. Four days later, she extracted from the culture the shoots of flesh from the biopsy, transferring the remaining cells into a new tube. From that day, the cells have not ceased to grow. Months passed, and the cells were healthy. In the words of the researchers, the cells grew “luxuriously”, doubling in number each day. The first immortal line of human cells was born.

Figure 2.2. Henrietta Lacks around 1950

It was not common practice at that time to inform patients of experiments performed on tissue samples from their body, and indeed Henrietta never learned of the success or even about the existence of this research program. The doctors had been reassuring with regards to her health. The treatment had progressed well, and most patients with this type of cancer and this treatment were still alive after 5 years. At the end of July, the doctors discovered new tumors, now in the pelvis and in the lymph gland. The cancer had spread at a breathtaking rate, utterly unexpected, and the patient was inoperable. On August 8, she was admitted to hospital, 10 days before her 31st birthday. She died on November 4, shortly before midnight, without ever knowing that her cells were already eight months old and would fuel the entirety of the John Hopkins cancer program.


Soon later, George Gey went on television, presenting a tube of HeLa cells to the general public. News of this success spread quickly in the scientific community and by 1955, 600,000 boxes of culture were exchanged around the world. Commercial distribution was picked up by the company Microbial Associates.

2.2.1. A vaccine against poliomyelitis

It was at the beginning of the 1950s that Enders updated an idea as old as cell culture itself: using cells to produce viruses and thereby accelerate vaccine development. Cultures of human cells were rare up to this point. As early as 1913, Constanin Levaditi (1874–1953), a Romanian doctor and microbiologist working at the Institut Pasteur in Paris, had used cells to produce the poliomyelitis and rabies viruses. Carrel had followed suit in 1928 and at the end of the 1930s, the first vaccine against yellow fever had been developed by Max Theiler (1899–1972), a South African virologist, who had used a hundred cell subcultures in successfully attenuating the original, particularly the viral strain of the West African virus. The availability of the HeLa line as a truly industrial means of production was game-changing and accelerated research. Gey committed himself to the project. He had known Enders since 1950 and, along with Syverton and Scherer, launched himself into the culture of the poliomyelitis virus in 1953. Their success was startling, allowing a young virologist named Jonas Salk to begin development on the vaccine against poliomyelitis the very next year.

These spectacular results, along with Ender’s works, represent the first large-scale application of cells in culture. But Enders and his associates set their sights further. They had also observed that between 12 and 78 hours following infection by the polio virus, cells in culture change their morphology in a dramatic manner. This visual signal allowed for the rapid diagnosis of polio from then on, providing a real alternative to the infection method practiced until then on monkeys.

2.2.2. Cells in space

Studies on HeLa had soon colonized the planet, but did not stop there. The Soviet Union was first to send HeLa cells into space as part of their second satellite mission on August 19, 1960. They gathered data on growth, morphology and the viability of cells in space from this experiment. Frustrated at falling behind, the United States jumped into the race. On

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November 12, 1960, the Discoverer XVII mission sent synovial and conjunctive cells into orbit. These cells attained an altitude of 991 kilometers. HeLa cells were sent up three weeks later with the Discoverer XVIII mission. According to the study’s findings, they survived in space for 3 days and 48 orbits despite being subjected to a significant solar flare [DIC 91].

2.2.3. Cell cloning

Cell cloning, which involved obtaining a sustainable culture based on a unique cell, remained a failure until the 1940s. Was this due to a poor implementation of the technique or to biological impossibility? Be as it may, in 1948 Katherine Sanford, Wilton Earle and Gwendoline Likely demonstrated that an isolated cell could be cultivated, divide and engender a cloned population [SAN 48]. The first line, called L, was derived from a capillary. The isolated cell had been placed in culture in a medium in which other cells had grown. It was thus demonstrated that somatic cells are separate entities, both independent and capable of self-replication. Their existence was no longer, as had been believed, strictly bound to the integral body of the organism [LAN 07, p. 143 and suivantes]. By obtaining these cloned somatic lines, the path to studying genetics had been opened.

With cloning understood, the geneticist Theodore Puck (1916–2005), who was to be the first to clone a cell derived from the HeLa line in 1955, from then on considered the mammal cell to be equivalent to that of a microorganism. As a side note, the biologist Leigh Van Valen later returned to this standpoint and, by analyzing HeLa cells as a potential new species in accordance with the criteria typically reserved for studies on evolution or biodiversity, concluded in 1991 that HeLa cells ought to be considered as a completely separate microorganism due to their independence, their ability to compete for and conquer new areas and their genetic disparity to humans. He gave this new species the name Helacyton gartleri, although this initiative was hardly to be seized upon by the scientific community.

2.3. New cell lines

2.3.1. The CHO line

The CHO model is the first post-HeLa line to have achieved international notoriety. It was Theodore Puck who had the idea in 1957 to isolate dwarf Chinese hamster (Cricetulus griseus) ovary cells and place them in culture.


By cloning a unique cell, he obtained a robust line that manifested rapid growth. One of the daughter lines, CHO-K1, was transformed by mutagenesis in 1980 to provide a strain deficient in dihydro folate reductase (DHFR), the growth of which required glycine, hypoxanthine and thymidine (a mixture called GHT). This strain allowed for control over the stable transfection by associating the expression of the gene of interest with that of the gene DHFR, which ensures the growth of positive strains by selection in a GHT-depleted medium. This schema became the standard method to establish stable lines expressing the recombinant proteins of therapeutic interest (biopharmaceuticals).

The first lines made available around 1984 by the company Genentech produced interferon and tissue-type plasminogen activator (tPA). This tPA went on to become the first recombinant therapeutic agent to be approved in the United States by the Food and Drug Administration (FDA). More than 70% of cells currently used in the industry for the production of recombinant proteins in bioreactors are CHO subclones. In response to what has become a global demand, the size of individual culture containers has now reached volumes of 20,000 liters, with the global capacity for CHO cell growth in industrial bioreactors now close to 500,000 liters [DEJ 11]. Typically, production will last 21 days with maximal cell density from 10 to 15 million cells/ml and producing in the range of 50 to 60 pg of recombinant compounds per cell per day. The current trend is for disposable bioreactors, which have been available on the market for around 15 years. In such cases, cells are cultivated in plastic bags of 500 liters, which are mounted on a rocker.

2.3.2. An increasing number of cell lines

A noteworthy example in terms of application is the WI-38 line, developed by Leonard Hayflick and Paul Moorehead [HAY 61] in 1961. Composed of fibroblasts, this line was isolated from pulmonary tissue extracted from a human fetus following an interrupted pregnancy. Indeed, this was the first human diploid line, with the other lines at the time all presenting altered chromosomic characteristics due to their cancerous origin. The WI-38 line would be of particular use in the production of vaccines against zona, varicella, adenovirus or the triple measles-mumps-rubella (MMR).

An increasing number of lines were released through the 1960s. The Vero cells, for example, which were created in Japan in 1962, derived from the epithelial cells of the African green monkey. These cells were quickly put to

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work, like HeLa, in the production of viruses for vaccines or of protozoa-like trypanosomes to help in the fight against various tropical pathologies. During the 1990s, they found a new role in the clinical domain as part of in vitro fertilization protocols (medically assisted procreation) whereby they are co-cultivated with human embryos to stimulate the growth of the latter up to the blastocyst stage prior to reimplantation in the patient.

In terms of human lines, the new ones were mainly of cancerous origin. Human lines derived from normal cells continued to resist being placed in culture for long periods. One step to their immortalization was achieved thanks to the work of Renato Dulbecco (1914–2012, Nobel Prize in 1975) and Marguerite Vogt (1913–2007), who demonstrated in 1960 that normal cells (fibroblasts) recently extracted from mice could be transformed to neoplastic cells by infection with polyoma-type viruses [DUL 60]. Many lines were immortalized during this period due to other oncogenic viruses like the simian virus SV40.

In 1964, the Harvard cell biologist Howard Green created the line 3T3, derived from mouse fibroblasts and immortalized this time without the help of a virus. Unlike the polyomavirus or SV40 transformed lines, the 3T3 cells do not trigger tumor development after injection in mice. They also possess a completely new property: they stop their growth abruptly at low density and behave as quasi-normal cells even though their genome presents several chromosomic alterations. Still more surprising, rather than progressively dedifferentiating themselves as so many other culture cells do, they display a natural accumulation of lipids and can easily be differentiated in adipocytes. 3T3 cells went on to be widely used for research and regulatory reasons (see section 5.7).

By the 1970s, hundreds of cell models had been made available.

2.4. Cross-contamination

Concerning the HeLa cells, their incredible story continued. Or at least it did until around 1966. Laboratory exchanges had developed rapidly in large part due to the success of HeLa cells, but also with help from a lucky discovery by the British researcher Christopher Polge in 1949. On that day, this reproduction biology specialist accidentally placed the wrong bottle in the freezer, by mistake freezing sperm samples in glycerol. He was surprised to note that once defrosted, the sperm cells had retained all of their fertilization capabilities. From 1954 William Scherer and Alicia Hoogusian


transposed the experiment on somatic cells in culture. Since then, regardless of their origin, cells have been frozen with glycerol and maintained at –70°C until placed again in culture. This method was quickly generalized. Far from a banal development, in one swoop, this process eliminated time and space, two key aspects that slowed culture development. This represented an essential development that was to radically transform laboratory exchanges, and indeed, the future of cell biology.

However, this new means for exchanging biological material and experimental protocols between laboratories was the beginning of a scientific crisis without precedent. There were ever-increasing numbers of lines, but it was very difficult to recognize them with certainty. Due to the presence of HeLa cells in all the laboratories, some of the more informed minds began to consider the potential danger of cross-contamination. They recognized that HeLa easily occupies new territories. In a way, HeLa acts like a sort of invasive species. Since they grow faster on average than most other cells, even a very small contamination of just a few cells, could result in the complete replacement of the original culture by the invader. The decision was reached to create a reference center that could conserve the original of each publicly available line. This was the American Type Culture Collection (ATCC), a nonprofit, private company established in Rockville, Maryland, which, after cell identification, assumed the task of stocking frozen samples. The center was operational in 1962. HeLa cells are registered there as sample number 2.

But this was only the beginning of the problem. In 1966, the geneticist Stanley Gartler presented his results to a conference in Bedford, Pennsylvania. He had studied the genome of 18 cell lines, comparing them to the famous HeLa. Of the 19 lines, six come directly from the ATCC. His comparison technique relied on the electrophoretic analysis of glucose-6-phosphate dehydrogenase (G6PD) and phospho-glucomutase (PGM) polymorphism. Without exception, all of the lines possessed the same type A G6PD and type 1 PGM phenotypes. The G6PD variant is connected to gender and only present in the African American population (at a 30% frequency). The PGM gene is autosomic, possessing three variants. The probability of systematically finding type A G6PD and type 1 PGM is slim. Moreover, unlike HeLa, most lines were derived from patients of Caucasian origin. For Gartler, there was only one possibility: all of the lines had been contaminated and replaced by HeLa. It was suggested that the G6PD variant could have changed following its many divisions in culture. Gartler, however, as a true geneticist, explained that this was highly unlikely with the

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chances being infinitesimal for mutation from one variant to another. Michael Gold took the minutes of the conference [GOL 86]. In attendance was one of the founders of cell culture and co-organizer of the conference, Hayflick. He was particularly worried by this, having created the WISH line that had been used by Gartler in his study. Hayflick rose and asked to address the room. He pointed out that the WISH line was derived from cells of the amniotic sac that had surrounded his own daughter in the womb. Since he and his wife were both Caucasians, to loosen the rather tense attendees, he added that “I have just telephoned my wife who assured me that my worst fears are unfounded…”. However, the evidence was clear; all of the lines studied were one and the same – the HeLa line.

Other cell markers such as the karyotype were soon added to the G6PG phenotype analysis to help in characterizing the lines. By the 1970s, three markers had been definitively retained: the presence of type A G6PD, absence of the Y chromosome and the identification of a certain chromosomal motif. The works of Nelson-Rees, director of the Berkeley cell culture, would be referenced from this period. In May 1972, after the most troubling period of the Cold War, Nixon and Brezhnev decided on a rapprochement. They settled on a scientific cooperation agreement. As a manifestation of this agreement, it was decided that each would exchange cells to be studied by the other. The United States sent their cells to the Soviet Union, which, likewise, sent the Americans six human lines developed in Russia independently, and supposedly infected by human viruses [GOL 86, p. 40 and suivantes]. Nelson-Rees’s analysis was clear: all of the Russian lines were type A G6PD, with complementary analyses demonstrating that all were patently HeLa. Moreover, the human viruses identified were all of simian origin!

Through his studies, Nelson-Rees demonstrated that contamination by HeLa cells was widespread and global. Other intra- and interspecies cross-contaminations regularly emerged, further blackening the record. In 1981, Nature published an article cosigned by seven teams including that of Nelson-Rees. They had all participated in blind, parallel analyses of four cell cultures. All were supposedly derived from Hodgkin’s lymphoma patients. The good news was that none were contaminated by HeLa cells. The bad news was that none presented any connection with Hodgkin’s lymphoma. Three were unidentified human lines and a fourth was of animal origin, presenting a karyotype identical to that of a douroucouli, an owl monkey living in northern Colombia.


Between 1974 and 1981, Nelson-Rees published an alarming list of cell contaminations [NEL 81]. In total, he identified 279 contaminated lines derived from 45 different laboratories. Around 40 of these lines had been replaced by HeLa cells, with numerous others by cells of other species. As it was often the case that several years went by between the cell model studies and their identification as false lines, the situation soon became tense and inextricable. Through these results, Nelson-Rees had cast doubt on years of research, investment and hasty conclusions reached in hundreds of articles. With the pressure rising and emotions running high, researchers feared losing their credibility, and some even sought to challenge the conclusions reached by Nelson-Rees via editorials in such prestigious journals such as Nature. These repeated attacks ultimately succeeded, with Nelson-Rees withdrawing from the cause in 1981. Work weary, he left science and opened an art gallery in San Francisco.

Yet the problem remained despite the introduction of new stringent checks performed at national cell libraries, such as the ATCC in the United States. A 1999 study carried out for the German cell library (DSMZ, Braunschweig) demonstrated that 18% of the 252 lines provided to the library were still incorrectly identified or contaminated. In 2003, a joint study between the DSMZ and the Fujisaki Cell Center (Okayama, Japan) revealed that among the 550 lymphoma and leukemia lines, 15% were contaminated. And in a 2006 article published in Nature, they set the rate of cross contamination at 29% across all human tumor lines provided to the DSMZ [CHA 07a, NAR 07].

Today, the issue of traceability has been settled. Line certification tests can be performed at very little expense by means of new standardized DNA fingerprinting methods.

Without a doubt, this cellular imbroglio resulted in major delays of several years for line application, particularly in terms of measurements, which rely above on all the certainty that model in use is reliable and stable.

2.5. Cell lines, an ethical issue

Before long, another issue would ravage the world of cell lines. Yet the fears arising in the 1930s had come to nothing. Several decades had gone by without any conglomerates spawning a monster from cell culture. But researchers and doctors had another ethical problem in mind. After all, human cells were derived from human patients. Thereby, cells constitute an

36 Live Cell Assays

integral part of their being. Hannah Landecker remarks that by the simple name HeLa, the cell is personified, becoming a mirror image of the patient from whom it was derived [LAN 07, p. 174]. According to Landecker, HeLa represents a new way of being for human material.

At the time, the societal image projected by the line was, at least to begin with, positive. Of course, it was associated with a cruel death, but by means of its application in the fight against poliomyelitis, contributed above all to saving human lives. In this role, the line recalled an angelic and universal figure. This idealized role was accentuated by the film A New Approach, produced in John Enders’s laboratory, which presented the notion of replacing monkeys with HeLa cells for diagnostic applications.

But journalists soon began to question the real position occupied by the HeLa line in the domains of industry and research. They noted before long that the biomass of HeLa cells now greatly exceeded the body mass of Henrietta Lacks herself. HeLa cells were now seen in a different light. The media began to present them as a mass of flesh, voracious, aggressive, malicious, a sort of monstrosity that would cover all things in its passage by means of its incredible capacity for growth.

The ethical dimension first emerged through the 1980s. To whom did the HeLa cells belong or any of the other human lines for that matter? The problem was still more apparent given that several commercial enterprises now marketed the lines for profit. In The Immortal Life of Henrietta Lacks, a best-seller published in February 2010 in the United States and since translated into 25 languages, Rebecca Skloot gives an account of the parallel destinies of Henrietta, her family and the HeLa cells. It was Babette Lacks, the step-daughter of Henrietta, who in the spring of 1973 first discovered the existence of HeLa cells sold by the ATCC thanks to a care assistant acquaintance [SKL 12, LEL 12]. Soon, Lacks family members were contacted by doctors from John Hopkins for blood tests. They panicked. They did not understand why they were being asked to do these tests or the connection with Henrietta, their mother and wife. These tests were supposed to help in establishing a genetic map of the line following the contamination problems, but the Lacks family had not been informed and expected the worst. Rebecca Skloot explains [SKL 01] that the understanding of informed patient consent only came about much later.

The Lacks did not engage legal proceedings concerning the use of HeLa; however others did, like John Moore from Seattle. In October 1976, John Moore consulted the oncology department at UCLA Medical Center in Los


Angeles having been diagnosed with a rare form of cancer, hairy cell leukemia, a chronic disease of the blood characterized by an increase in spleen and liver size. His surgeon David Gold offered to remove his spleen in order to slow the progression of illness. The operation proved useful, being followed by a complete remission. Moore continued to return to the department for various tests until 1983. It was then that Gold asked him to sign a document in which he was requested to relinquish all possible rights over the cell line named Mo derived from his T-lymphocytes. Moore refused to sign the document and called a lawyer who discovered that a patent had been placed on the line in 1979. From then on, he has been continuously engaged in this cause.

In fact, David Gold had observed that John Moore’s cells produced proteins stimulating the growth of lymphocytes, which could be useful in fighting infections [MCL 01]. A 1991 Supreme Court of California ruling brought the discussion to an end. It ruled that Moore had no property right to the cells, nor was he entitled royalties due to their commercialization. John Moore died at the age of 56 in 2001.

2.6. The first generation of cell assays (1969–1983)

The interest in culture cells for evaluating toxicity began in earnest during the Second World War in relation to infected wounds. A study was published in 1941 demonstrating the effect of antibacterial sulfonamides (sulfanilamide, sulfathiazole and sulfapyridine) on the condition of poultry macrophages in adherent culture [JAC 41]. Thereafter, other studies were released in 1945 and 1946. The first showed the absence of any cytotoxic effect by DTT on primary cells of chicken (heart and intestine) or of rats (brain and spleen). The second studied the cytotoxicity of two other sulfonamides (marfanyl and V187), also on chicken macrophages.

In 1951, Robert Schrek demonstrated on lymphocytes that the discoloration of an oxidoreduction indicator, the DCPIP (currently used to measure photosynthetic activity), allows for a large range of toxicity agents to be measured, including cortisone, mustard gas or X-rays [SCH 51]. However, it was not until the 1960s that cell assays were first standardized to forms ready for use throughout the community.

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2.6.1. The karyotype test

One of the first assays to be performed on human cells was the collection of information about the chromosome structure of live cells in metaphase. The cytologist and geneticist Torbjörn Caspersson (1910–1997), together with his team at the Swedish Karolisnka Institute, noticed in 1969 that the coloration of chromosomes blocked in metaphase and colored with quinacrine of mustard distinguishes itself by contrasted dark-light bands (known by the name Q bands, for quinacrine). These patterns allow each chromosome pair to be individually identified. The initial observation made on vegetable cells was soon transposed to humans by the same team (1971). The more practical Gielsa colorant (G bands) soon replaced quinacrine (Figure 2.3). The first application in human health for this assay concerned Down syndrome screening. Modern karyotype technique as described in section 9.1 is very close to this original assay in principle, but has benefitted from later advances in multispectral fluorescence.

The first live cell assay addressing toxicity, more specifically genotoxicity, known as the Ames test, was published in the 1970s. It will be discussed in detail in section 2.7.

Figure 2.3. Karyotype assay. Colchicine treatment brings an end to cell division in metaphase. A photograph is taken of the sample under

microscope after coloration. The karyotype represented here is masculine


2.6.2. The MTT assay

The second live cell assay addressing toxicity was developed in 1983 by Mosmann, a researcher with the Californian biotechnology company DNAX Research Institute of Molecular and Cellular Biology. The main challenge facing them at that time was to circumvent the use of radioactive products in measuring cell proliferation or toxicity. The reduction of tetrazolium salts, typically colorless solutions, to formazans…, insoluble colored crystals has been understood since the works of German scientists at the end of the 19th Century. The formazan/tetrazolium redox combination was used from 1945 to verify the viability of cereal grains. In 1948, it was demonstrated that neoplastic tissues produced more formazan than normal tissues. One of these salts, the MTT (3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyl tetrazolium bromide) came to be used in histology as routine from the 1960s.

Mosmann’s major contribution was to adapt the MTT approach to cells in culture. He simplified the procedure and envisaged its transfer to a means of higher throughput [MOS 83]. To this end, he had the idea of combining the use of 96 well plates with an optical density reading in an ELISA spectrophotometer. The basic idea was that the quantity of formazan produced would be proportional to the number of viable cells contained in the sample. The MTT assay soon showed its value in the 1980s. Nonetheless, its mechanistic explanation remained subject to debate until the turn of the 21st Century.

Today we know that the MTT assay is the integrated measurement of a set of enzymatic activities that are more or less connected to cellular metabolism. Consequently, it can be said that this reflects, to a certain extent, cell viability. The understanding of the formazan production topology has been unveiled through the 1990s. In 1997, Liu and his colleagues proposed that the MTT transfer within the cell occurred by endocytosis, a hypothesis that was later refuted. Indeed, MTT has a positive charge, facilitating its intracellular transfer across the plasma membrane (of which the membrane potential is –30 to –60 mV), and, if it has not been reduced in the cytosol, to the mitochondrion (membrane potential of –150 to –170 mV).

It was long thought that the metabolic activity engaged in the reduction of MTT was closely related to mitochondrial respiration, particularly at the reduction sites of complex II of the respiratory chain implicating succinate dehydrogenase (SDH) (also known as succinate-coenzyme Q reductase,

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SQR). The production of formazan would then be mitochondrial and thereby associated with the oxidation of succinate in fumarate that intervenes during the Krebs cycle. However, it was clearly demonstrated from 1991 that the reduction of MTT is largely distributed in the cell and that the mitochondrial contribution seldom exceeds 10%. The debate appears to be over since the works of Berridge in 2005, which concluded experimentally that MTT is principally reduced by extramitochondrial enzymes of the NAD(P)H-dependent oxidoreductases variety like the P450 cytochromes [BER 05]. The MTT assay is ultimately, strictly speaking, a measure of NAD(P)H flow. Indeed, 20 years were required to specify the exact nature of this measurement (Figure 2.4).

Figure 2.4. Principle of MTT assay. As much as cell metabolism is active, NAD(P)H production supports activity of cytosolic oxydoreductases

(cytochromes P450) which reduce MTT to insoluble, colored formazan trapped in cell

As it is necessary to solubilize the formazan crystals in a homogeneous medium in order to read the MTT assay result, implementation is quite problematic. This problem was resolved thanks to the use of other tetrazolium salts such as XTT, MTS or WST-1, whose formazans are soluble in water. Moreover, the use of MTS has the advantage of eliminating the solubilization stage. On the other hand, it requires the addition of an electron


acceptor to facilitate the reduction stage. Finally, the introduction of WST-1 to the market has allowed for the further improvement of the protocol by removing the requirement of an electron acceptor.

2.6.3. The NRU test

Neutral red (3-amino-7-dimethylamino-2-methylphenazine hydrochloride) is a vital colorant, known since the 19th Century and used for the first time in 1911 by the diabetes specialist Robert Bentley (1867–1956) in order to identify the clusters characteristic of pancreatic cells known as islets of Langerhans. In 1937, Belkin and Shear had the idea of using the different marking topologies (diffuse vs. clearly identifiable) of neutral red to measure the viability of live murine tumor cells. It would not be before the works of Kull and Cuatrecasas (1983) and of Borenfreund and Puerner (1984) that an assay would become available for toxicity measuring applications on 96 well plates [KUL 83, BOR 84].

It is by no mere coincidence that the first two live cell assays in history emerged at exactly the same time, but due to a common will at that time to develop alternative methods to animal tests (see Chapter 3). This precise topic is addressed in an article by Borenfreund and Puerner who consider it a positive lesson.

Neutral red is a weakly cationic colorant that takes a yellow-orange hue in an alkali environment, turning red in an acid environment. This makes it an excellent marker for lysosomes or other intracellular organelles presenting acidity following the continued maintenance of a proton gradient. This connection between the neutral red and lysosomes has been known for a very long time. It is now established that for a cell in good health, the colorant penetrates the plasma membrane by passive diffusion, then concentrates in the lysosomes where it charges, remaining trapped so as to ultimately be associated by electrostatic hydrophobic bonds to phosphate or anionic groups of the lysosomal matrix (Figure 2.5). The intralysosomal acidic pH is maintained by means of the ATP activity, dependent on a proton pump. Technically, the maximal coloration is reached after 2 to 3 hours and the relation between absorption at 540 nm and the number of viable cells is well established. A differential coloration of lysosomes means then that the observed cell is in good health (the proton pump works), a diffuse coloration, that the cell has lost its homeostasis (see also section 4.2).

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Figure 2.5. Principle of NRU assay: (1) as long as cell metabolism is active, ATP is available to maintain the pH gradient between the lysosomes and the

cytosol: neutral red (NR) marks the lysosomes; (2) loss of metabolism abolishes pH gradient; NR labeling is then diffused within the cytosol

It is of note that the MTT and NRU assays only measure cytotoxicity indirectly by deducing the proportion of cells that remain in good health. It follows that, fundamentally, these assays measure cell viability.

2.7. The first target of regulatory assays: genotoxicity (1983–1986)

Damage to the genome constitutes by far one of the most fundamental forms of cellular aggression due to the importance of DNA for the future of both the individual cell and that of the organism on which it depends. If the damage is not repaired attentively, then it can put the cell’s homeostasis in peril. However, most genomic alterations remain invisible in both functional and phenotypic terms. Nonetheless, they are retained in the cell’s genetic heritage and later transmitted to its descendants. The accumulation of these sorts of alterations can ultimately provoke a major phenotypic modification and lead to the desocialization of the cell, which can for example lead to the development of a cancer.


Genetic damage can be due to environmental factors or be inherent in the cell’s metabolic process. Genomic stress factors of environmental origin are varied. These could be, for example UV waves, ionizing radiation, various toxins of vegetable or fungal origin like aflatoxins and other molecules containing aromatic chemical rings, such as certain polycyclic hydrocarbons like benzoapyrene. The production by the metabolism of highly reactive intermediary cell products like reactive oxygen species (ROS) represent another key source of genomic damage. Numerous cell assays have been devised to measure these aggressions, and are grouped together under the generic term genotoxicity assays. Some observe global disorders such as the presence (or the deletion) of chromosomes or parts of chromosomes, others are employed in the measurement of breaks, both simple and double, that can occur along the DNA strand. And other approaches measure the rate of mutation provoked by mutagenic agents. Research for diagnostic purposes concerning point mutations or deletions in genes implied in pathologies fall outside of this analysis.

Anomalies at the chromosomal level are rare but easy to observe. They can intervene during mitosis and lead to a loss of all or part of the chromosomes, or, inversely, generate the presence of extra chromosomes or segments of them that form characteristic micronuclei.

Along with those associated with mutagenic agents, these effects were the subject of the first genotoxicity cell assays adopted by the Organization for Economic Co-operation and Development (OECD) in the form of the applicable regulatory guidelines. The first assays adopted by the OECD in 1981 for the measurement of genotoxicity (series 4 guidelines) all used animal models. Two years later, the international body introduced the 470 subseries relating to genotoxicity in which live cell assays and in vivo tests both figured. Guidelines 471 to 473, published on May 26, 1983, for the first time proposed live cell assays for regulatory applications. Two of these assays (471 and 472) used prokaryotic models, and the third (473) used mammal cells. All three were dedicated to the measurement of genotoxicity.

2.7.1. Ames test (OECD guideline 471)

This was developed by Bruce Ames [AME 75], biochemist at the University of California-Berkeley. He had the privilege of developing the first live cell assay with applications that have been validated at the international level (1983). The Ames test was conceived at the beginning of the 1970s. It measures the capacity of a chemical compound to induce

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mutations in the genome of Salmonella tymphimurium bacteria. It has its foundations in a prokaryotic model, far removed from the human cells used in the cases of MTT or NRU. It follows that extrapolating the results to genotoxic effects on human beings remains relatively uncertain, particularly since there are compounds known to act almost exclusively on bacteria (antibiotics for example) or inversely, some act in a specific way on eukaryotic cells. This distance between species (or even between kingdoms) has been effectively reduced by a workaround, consisting in coincubating the cells with a microsomal fraction of human liver cells, which metabolize the substances tested in a similar way as would happen in the human body. The fact remains that the Ames test extrapolates to mammals the mutagenic effects observed on a bacterial model, which can generate false positive or false negative results.

In mechanistic terms, this is, in fact, an inverse mutation test, which relies on the properties of genotoxic agents to generate mutations that restore the bacteria’s capacity to synthesize an amino acid necessary for their survival (described as loss of auxotrophy). Bacteria under a genotoxic effect are detected by their ability to grow in the absence of the amino acid in question. In this case, the strain of Salmonella typhimurium used is auxotrophic to histidine amino acid. In fact, it possesses a mutation in the histidine synthesis gene and requires the addition of this amino acid into its medium in order to grow. This strain, referred to as His–, can indeed spontaneously return to the normal phenotype His+, although this event is very rare. The Ames test measures the capacity of genotoxic agents to accelerate the shift between His– and His+ by evaluating the number of colonies produced in the absence of histidine (Figure 2.6, top).

This approach had the advantage of providing evidence of the dangers of pesticide use very rapidly. It has received global recognition and remained for some time the star of in vitro tests. It should be noted that guideline 472, published on the same day, describes an equivalent test to Ames, but performed on the Escherichia coli bacteria. The two tests would be combined into one guideline (471) in 1997.

2.7.2. In vitro mammalian chromosome aberration test (OECD guideline 473)

Published in 1983, this assay proposed to evaluate chromatid and chromosome structural aberrations together with variations in chromosome number (ploidy). There are no restrictions concerning the choice of cell


models that can be primary or established lines. The cells are treated with the test substance, and then blocked in metaphase by colchicine. Indeed, this is manifestly no more than the karyotype protocol adapted for the purposes of measuring genotoxicity. Test 473 was the subject of an OECD amendment adopted in 2014.

2.7.3. In vitro mammalian cell gene mutation test (OECD guideline 476)

Unlike the Ames test, line 476 tries to address the mutagenic and potentially carcinogenic effects observed in human beings through the use of cell lines of animal (L5178Y mouse lymph cells, CHO cells) or human (TK6 lymphoblastic cells) origin. The idea is to use cells whose growth is dependent on the expression of a gene (or locus) that can be rendered nonfunctional by mutation. The target gene is selected in such a way that its functional absence renders the cell susceptible to a given genotoxic agent, known as the selective agent. The most common target is the TK gene, mutation of which renders cells deficient in thymidine kinase (TK). Deficiency in TK leads to resistance to trifluorothymidine (TFT) effects, which, in the absence of mutation, exerts an antiproliferative effect. Consequently, mutant cells proliferate in the presence of TFT, normal cells do not.

These same properties can be observed in hypoxanthine-guanine phosphoribosyl transferase (HPRT) or xanthine-guanine phosphoribosyl transferase (XPRT) coding genes, in association with the antiproliferative effects of thio-6 guanine (TG) or of aza-8 guanine (AG), respectively. Mutation frequency is determined by seeding cells in a medium that contains the selective agent or not. It is then extrapolated according to the number of cells to have manifested mutations at the locus in question (TK, HPRT, XPRT). The test is based on the hypothesis that mutations at a discrete locus of DNA reflect a generalizable genotoxic action on the rest of the genome. Mutation frequency is estimated by comparing the number of growth colonies obtained in the different conditions. The time required for expression of the phenotype can vary. For TK mutants, this can be 2 days, or 8 days for the other two mutants. Note that different genotoxic effects can be detected depending on the choice of mutant line and the autosomal location of the locus presenting the potential mutagen.

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Figure 2.6. Principle of the Ames test and its equivalent on mammal cells

In 1986, the OECD published a new series of four cell assays, again concerning genotoxicity (479–482). Two of these assays use mutations of Saccharomyces cerevisiae (baker’s yeast) as cell models. They aim to measure genetic mutations and mitotic crossing-overs. The two others use mammal cells, measuring the rate of exchange between sister chromatids (479) and DNA reparation capacity (482). However, the OECD retired these four assay guidelines from the regulatory framework April 2, 2014, judging them obsolete. In light of this, only assays 479 and 482 will be presented here, with emphasis placed on the exchange between sister chromatids, which, at least in terms of medical diagnosis, is still relevant.

2.7.4. In vitro sister chromatid exchange assay in mammalian cells (OECD guideline no. 479)

This test evaluates the mutagenic potential of a substance by measuring the exchanges that arise at the moment of DNA duplication (chromatid doubling). These exchanges are formed following the stalling of replication


forks in phase S. DNA is then repaired, replication resumes, although errors may occur. The test constitutes a measurement of the frequency of these exchanges by exposition of culture cells to bromo-deoxyuridine (BrdU). This synthetic thymidine analogue is integrated into the DNA during duplication. The cells (human lymphocytes, CHO) are conserved over two cell cycles. With DNA replication being semiconservative, each new formed chromatid is comprised of two DNA molecules, of which only one incorporates the BrdU. During the second incorporation cycle, each chromosome has a chromatid with both strands containing BrdU and one with only one strand of BrdU. The result is observed on metaphase chromosomes following the accumulation of metaphase cells after the treatment with a spindle inhibitor like colchicine (see karyotype in section 2.6).

After Giemsa marking and, if there have been exchanges, the genetic material between chromatid sisters appears as an alternately light and dark region. It is understood that the frequency of exchanges between sister chromatids increases under the effect of ultraviolet light, ionizing radiation or certain mutagenic agents. Indeed, this property is used to characterize Bloom syndrome.

2.7.5. DNA damage and repair, unscheduled DNA synthesis in mammalian cells (OECD guideline 482)

This radioactive test uses primary hepatocytes from rats, human lymphocytes or established lines (human diploid fibroblasts, for example). It is based on the addition of tritiated thymidine whose DNA incorporation attests to the resynthesis of DNA regions after deletion induced by a physical or chemical agent. The measurement is performed by autoradiography (counting marked cells) and by scintillation detector reading.

At this point, it is of note that this marked push by the OECD at the turn of the 1980s toward cell assay methods as an alternative to animal tests, in fact, remained without any follow-through for a very long time. At least 20 years went by before cell approaches would again be held in consideration. These approaches are detailed in section 5.5. In terms of genotoxicity, 27 years went by (2010) until a new test, the now classic micronuclei assay, which measures aneugenic and clastogenic effects, was finally adopted by the OECD. This is presented in section 5.9, along with the latest releases to have been validated in regulatory terms.

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Approach Cell function targeted Reading method Pertinence

Karyotype Presence of extra chromosomes

Colorimetry or fluorescence

Low throughput image analysis

MTT Measure of mitochondrial and cytosolic NAD(P)H dependent enzyme activity

Colorimetry Validated for cell viability measurement, integrated in several OECD lines

NRU Maintaining of pH-intralysosomal differential

Colorimetry Validated for cell viability measurement, integrated in several OECD lines

Ames DNA mutagenesis on bacterial model

Colorimetry OECD adopted assay Adapted at high throughput

In vitro genic mutation assay

DNA mutagenesis on mammal cells

Colorimetry OECD adopted assay

BrdU Sister chromatid exchange

Colorimetry OECD assay retired in 2014

Tritiated thymidine

Unscheduled DNA synthesis

Radioactivity OECD assay retired in 2014

Table 2.1. First cell assays made available in 1970s and 1980s

3

Cell Models and Technologies

Historically, colorimetric methods were the first to be developed for delving into the living cell (or the cell fixed by alcohol or formaldehyde) and retrieving information. Other studies in cell biology consisted of extrapolating cell functions by analyzing biochemical pathways or the interactions between molecules using preparations with fractioned and isolated cell compartments (plasma membrane, nucleus, cytoplasm, mitochondria, lysosomes, etc.). An indirect observation of cell functions during this period nonetheless bore its fruits, as in the decoding of metabolic cycles or cell signaling pathways, the principles of which are still widely employed today in major industrial programs.

The acquisition of new knowledge in cell biology over the last 20 years is in large part connected to the emergence of new technologies, without which the intimate dynamics of the cell would have remained almost invisible to the microscope. The technological watershed came at the beginning of the 1990s with the advent of fluorescence and bioluminescence combined with energy transfer methods. Of course, this technological leap would not have been achieved without advances toward high throughput in genomics and robotization/miniaturization, which occurred concomitantly.

The technological revolution that took place in the 1990s first made its presence felt in the heterologous expression of fluorescence proteins. This development resulted in access to a new dimension: dynamic perspectives of the cell. From this point, numerous elements and cell events such as molecular movements, biochemical reactions, protein interactions, cytoskeletal components, messenger RNA or even gene expression could all be analyzed in real time.


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Certain living beings emit light. This property can by summarily divided into two subcategories, fluorescence and bioluminescence, often combined in the wild. In both, photons are created following energy transitions between excited atomic states and their relaxation toward ground state. In fluorescence, the energy of atomic excitation comes from the absorption of another photon or in a nonradioactive way by resonance energy transfer. In bioluminescence, atomic excitation results from exothermic intracellular biochemical reactions, whose energy source is normally ATP.

The technologies associated with these properties both lent themselves readily to application uses. In fluorescence, incident photons are provided by instrumentation. The light signal is quite satisfactory but inevitably produces parasitic background photons. In bioluminescence, the photons are created by biochemical energy. Since they are more scarce, the light produced is more tenuous and difficult to detect, however, without incident photons, detection is undisturbed by any parasitic background light.

It should be noted that during this time a non-photonic means of analysis called label-free, based on the measurement of electric fields or on a selection of reflected optical waves, has also become a promising alternative to light-based approaches. All of these technologies have resulted in the commercialization of very competitive high- or ultrahigh-throughput dispositives (Figure 3.1).

3.1. Fluorescence and bioluminescence

The use of fluorescence in cell biology is particularly long-established. It has been used as a cell tracer since the first fluorescence microscope was conceived by Heimstaedt and Lehman of 1911. Intracellular labelling using fluorescent compounds first consisted of “tagging” the intracellular compounds to be followed, in particular the nucleic acids such as DNA [KRI 09]. The fluorescent tag can be covalently bonded to the target, or to an intermediate molecule itself bonded to the target or in a noncovalent fashion as in the case of intercalating agents or of compounds presenting an affinity for the DNA folds. Tags have numerous means of interaction. Some fluorescent compounds are structurally close to the constitutive bases of DNA and can replace them, thereby making the DNA fluorescent.

Cell Models and Technologies 51

Figure 3.1. Different technologies at work in live cell assays

These strategies have been applied to all cell compartments. For a long time they were in competition with radioactive sensors whose quantitative analysis has for a long time been considered more reliable and better adapted to the cell fractionation methods required for biochemical analysis. Today, fluorescent sensors have superseded them with an impressive range of fluorescent compounds being made available by suppliers. The main ones used in toxicity measurement will be described in Chapter 4.

3.1.1. Green fluorescent protein

No book on cell biology would be complete without covering green fluorescent protein (GFP), which has contributed immensely to both knowledge acquisition concerning the dynamic processes of the living cell

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and the ensuing industrial applications. GFP has one fundamental characteristic: it is genetically coded and as a consequence can be produced by cellular machinery in a functional manner. The other essential property of GFP is that it emits fluorescence. Excited between 395 and 475 nm, it emits photons of approximately 504 nm in wavelength. In 2008, Osamu Shimomura, Martin Chalfie and Roger Tsien were awarded the Nobel Prize in recognition for their work in the discovery, cloning and genetic engineering of GFP, which led to its use on human and mammal cells [ZIM 09].

Discovered in the Pacific by Shimomura as early as 1962, GFP is part of a protein family present in the cells of various species of jellyfish, such as Aequorea victoria, and several varieties of reef coral. Its genome was cloned by Douglas Prasher in 1992. In anticipation of these works, Martin Chalfie was the first to have suggested as early as 1988 the use of GFP as a reporter gene for a targeted cell function. The concept of reporter gene is quite simple. Since GFP is fluorescent, by attaching its gene to a controlled promoter of expression, controlled cell fluorescence can be induced. It remained to be confirmed whether species such as Homo sapiens, relatively distant from Aequorea victoria in phylogenetic terms, were capable of producing GFP in a functional way. Chalfie asked Prasher for the GFP clone, and in 1993 experimented on the Escherichia coli bacteria, and then on a worm of the nematode family, Caenorhabditis elegans, which was a study model that his laboratory worked on. Soon he obtained a fluorescent worm, demonstrating both GFP’s capacity for heterologous expression and its use as a reporter gene [CHA 94]. The result was published in Science on February 11, 1994.

These works were further advanced by those of Roger Tsien on the selection of mutant GFPs. Several mutants demonstrated new spectral properties, allowing access to a whole new palette of fluorescently emitted colors [DAY 09]. At the turn of the 21st Century, GFP became one of the main biomarkers of intracellular activity. The development of enhanced GFP (or eGFP), which retains the fluorescence emission peak of normal GFP even through its expression on cells of thermoregulated organisms at 37 °C, such as mammals, opened up a new field of GFP applications on human cells. Today, there are more than a hundred genetically modified fluorescent biosensors [PAL 11].


3.1.2. BRET

Bioluminescence is a biological system that uses enzymatic activities acquired through the species’ evolution to stimulate the creation of photons using biochemical energy. This ability to create light is common across a wide variety of species as diverse as insects, coelenterates, echinoderms and mushrooms. While some species use their own bioluminescent proteins to emit photons, others transfer the excitation energy from the bioluminescent protein by resonance energy transfer (RET) to a second protein (GFP), which, in turn, emits a photon of a different wavelength. Aequorea victoria, a species of pelagic jellyfish, expresses GFP through its magnificent crown without the need to receive excitation energy from sunlight, instead relying on the RET process initiated by a closely neighboring bioluminescent protein, aequorin (Figure 3.2).

RET is a non-radiative relaxation mechanism established between energy donor/acceptor partners in which the spectra of emission (donor) and absorption (acceptor) overlap. There are two essential properties in this means of energy transfer. First, it is optimal at distances in the order of a dozen nanometers (Förster radius). Second, its efficiency diminishes inversely to the power of six to the distance between the two molecules. In other words, RET efficiency very rapidly drops to zero as soon as the distance between the relevant partners varies slightest to the Förster radius. Applied to live cell assays, RET is an exceptional tool in the measurement of dynamic events transpiring at a molecular level, such as the interactions between proteins, through to conformational changes at inframolecular level.

In the case of Aequorea victoria, the aequorin–coelenterazine complex is the energy donor and GFP the acceptor. This is known as bioluminescence resonance energy transfer, or BRET. Figure 3.2(1) illustrates the BRET mechanism in Aequorea. The cells in the crown of the jellyfish express a protein, apoaequorin, which presents the chromophore coelenterazine and three calcium ion fixation sites. Occupation of these sites is regulated by the intracellular calcium flow and, with the presence of dioxygen, leads to chromophoric oxidation of excited coelenteramide. This then has two options for relaxation. Either it emits a blue photon (469 nm) (bioluminescence mode) or it transfers its energy via RET to the GFP (BRET mode).

This BRET mechanism has been widely employed in biotechnology. Nonetheless, industrial activities tend to use the system of bioluminescence that is present in the sea pansy, Renilla renimorphis. This cnidarian uses similar properties in the same scenario with one slight difference: here, the

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role of calcium ion is held by an enzyme, 2-mono-oxygenase, known as luciferase. It is this protein of the oxidoreductase family that catalyzes both the oxidation of coelenterazine and dioxygen to produce the excited coelenteramide, used as the BRET donor (Figure 3.2(2)).

Numerous cell approaches are based on BRET, including the β-arrestine (section 8.2) or Epac (section 8.3) assays. In such cases, cells are modified to coexpress two chimeric proteins, one composed of the association between luciferase and the first protein of interest, the second composed of a GFP (YFP for example) associated with the second protein of interest (Figure 3.2(3)). Coelenterazine is used as a substrate and provides energy for the transfer. A variant is provided by the calcium assay (section 4.6) in which BRET operates within the same aequorin–GFP fusion molecule. In addition, luciferase is widely employed in the reporter gene approach described in the next section.

Figure 3.2. Natural occurrence of BRET (1, 2) and application in live cell assays (3)


Another widely used approach is with a modified form of coelenterazine, called DeepBlueC, which leads to an emission of 400 nm when oxidized. This system known as BRET2 benefits from a better spectral separation between donor and acceptor, accompanied with an optimized BRET result [PFL 06].

3.1.3. FRET

In terms of application use, aequorin can be replaced by a fluorescent protein of the GFP family [FRO 09]. If the fluorescent protein couple is chosen in such a way that the emission spectrum of the first (the energy donor) overlaps the excitation of the second (the energy acceptor), then both GFPs can exchange energy by Förster resonance energy transfer (FRET) if they adhere to the terms of the Förster distance. There are many applications for FRET technology. For example, it is possible to measure the interaction or the colocalization of two proteins by fusioning each of them to a member of a FRET optimized GFP couple (Figure 3.3(1)). A classic example of application use is in cyclic AMP measurement by dissociation of the protein kinase A regulatory and catalytic subunit pair each fused to a GFP from a donor–acceptor couple (see section 8.3).

Using FRET, it is also possible to detect intramolecular conformation modifications. This is the case of the Cameleon system (Figure 3.3(2)), see also section 4.6), which is intended for measuring intracellular calcium or the activity of proteases such as caspase-3 intervening in apoptosis (see section 4.9). It is performed in the latter case by flanking the gene of interest (or merely the sequence coding the protein cleavage zone) with a fret optimized GFP couple. In this case, the protease activity results in the chimeric protein cleavage and the distancing of the two FRET partners (Figure 3.3(3)). Despite seeming complex a priori, these strategies have nonetheless found high throughput cell applications in screening campaigns in the pharmaceutics industry.

A FRET system allowing for the measurement of protein translocation between intracellular compartments has also been proposed for high throughput applications, particularly in the pharmaceutics industry [FUR 09]. This is the CYME-FRET (Figure 3.3(4)). The FRET couple is not optimized between two GFPs but between one mobile GFP (energy donor), whose location is functionally distributed either in the cytoplasm or the plasma membrane, and a membrane phospholipid (energy acceptor) such as the DiC-18. The interaction between the two FRET partners is not specific of an

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interaction but of dynamic colocalization in an action range approximately that of the Förster distance.

Figure 3.3. Different FRET methods applied to cell assays: (1) protein-protein interaction; (2, 3) intramolecular FRET with (2) trans-conformation of one protein flanked by two FRET partners (example of the chameleon approach) and (3) protein cleavage by protease; (4) translocation of protein between cell compartments


Measuring of the FRET or BRET signals can be done by evaluating variations in the intensity of the fluorescence emitted by the donor or acceptor, by ratiometry of these two values or still more elegantly using statistical measures of the partners’ excited state lifespans.

3.1.4. Other applications of GFP

Other applications of GFP take advantage of the natural changes in fluorescence intensity observed when certain cell conditions vary, such as pH, redox potential or concentration of calcium ions or ATP.

An interesting and bold strategy consists of recreating GFP activity based on two inactive fragments of GFP with the potential to be reconstituted into a functional whole. This approach is known as bimolecular fluorescence complementation (BiFC). The two fragments are associated with two proteins of interest, whose functional association is to be measured. This idea is similar to that used in the enzyme fragments complementation technology (EFC), widely employed in the pharmaceutics industry (see section 8.3), where GFP is replaced by luciferase or other enzymes.

Figure 3.4. Use of firefly luciferase (Photynus pyralis) in the reporter gene approach

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Another major application is, of course, the use of GFP as reporter gene. This is a rival strategy to the one using luciferase (or alkaline phosphatase) described below. Comparing the two, the GFP approach is often thought of to be of lower sensitivity but balanced by its greater ease of implementation.

3.1.5. The reporter gene approach

The reporter gene approach is applicable from the point at which the cell signal of interest is coupled to a genetic response. Indeed, this could open an infinite field of possibilities to the biologist. Above all, this concept relies on an understanding of the signaling pathways that connect the initial perception of a cell with respect to an external signal, the cascade of intracellular biochemical reactions (second messengers) induced and the activation of transcription factors (Figure 3.4). The latter proceed to bind themselves to DNA promoter sequences which induce genetic transcription. The reporter gene is preferentially chosen from among firefly luciferase of Photinus pyralis and sea pansy luciferase of Renilla reniformis (see an application in section 4.7). The aequorin photoprotein of Aequorea victoria is also used.

Response element

Signaling Pathway implicated Assays Section

ARE ROS/Nrf2 (anti-oxidant pathway)

ARE-Nrf2 (OECD 442D), LuSens

5.9; 6.6

CRE Gsα/ cyclic AMP/PKA/CREB Distal measure of cyclic AMP pathway

8.5

DRE Dioxin/AhR/ARNT CALUX 9.3

ERE Estrogen/ER STTA & TA ER BG1luc (OECD 455)

5.8

GalPV16 β-arrestin 8.2

NFAT Calcium Distal measure of calcium pathway

4.6; 8.5

SRE Raf/MAP kinase Distal measure on RTK 8.5

Table 3.1. Main approaches of reporter gene used in classic live cell assays


The reporter gene system based on luciferase uses luciferin as photon emitter substrate [FAN 07]. The aim is to couple the promoter sequence activated by the cell signaling pathway of interest to the luciferase coding gene and to integrate it in the genome of the chosen cell model. If the signaling pathway is activated, the luciferase enzyme will be produced. If firefly luciferin is present, it combines with ATP to form luciferyl-AMP, which becomes luciferase substrate. The presence of dioxygen generates the production of the new intermediary oxyluciferin, available in a state of high-energy electronic excitation. It is the relaxation to the ground state that ultimately produces the photon, which is reporter of the initial cellular activity.

The luciferase-based reporter gene approach is used (or has been used) in a great many high throughput cell assays, in particular those aiming to identify drug candidates modulating two large families of cell targets: seven-transmembrane domain receptors (see Chapter 8) and nuclear receptors.

An interesting aspect of the reporter gene approach is that it intervenes at the end of the reaction chain. It benefits both from the intracellular signal amplification and from the transcription of several copies of the gene in consideration, which, in theory, results in excellent sensitivity. In reality, this amplification parameter is sometimes accompanied by the generation of false-positives that reduce the test’s reliability. This problem can be avoided by the use of a second internal control based on the expression of Renilla luciferase, coupled to an independent signal, followed by ratiometric normalization.

An additional approach with the aim of improving signal quality consists of augmenting the luciferase expression dynamic by reducing its life span. This way, the persistence of the observed signal is limited, which allows for a closer regulation of the genetic expression. This can be implemented by adding sequences implicated in protein degradation to the luciferase gene, thereby limiting its functional life expectancy.

Another significant improvement came from mutagenic experiments that resulted in versions of luciferase in which both the level of expression and the sensitivity had been optimized significantly.

The reporter approach based on luciferase has become relatively ubiquitous, finding high-throughput applications in numerous cell lines, including in standard mammal cell models.

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The other reporter gene systems, such as excreted alkaline phosphatase (SeAP), whose activity is measurable in culture media, or β-galactosidase used in bacteriology, typically work in a similar manner.

3.2. Impedance variation in cell population

In certain cases, it is possible to use the cell’s own intrinsic physical properties without the need to add colorimetric, bioluminescent or fluorescent sensors. Such methods are known as label-free. Three essential properties have been exploited with this end: the electrical resistance and optical properties of evanescent waves and autofluorescence. These methods have the undeniable advantage of being uninvasive. They provide a workaround to the problems associated with more traditional technologies such as the intrinsic toxicity of fluorescent markers. In this way, the label-free methods leave the cell sample free of any interfering chemical. The sample can then be easily reused or followed over time on long-term kinetics. This method opens access to more information.

The first ingenious idea was to use the impedance measurement of a culture cell sample based on the principle that plasma membranes work as an electric insulator [WAN 12].

Figure 3.5. Impedance variation measurement: (1) plan of overall system; (2) the presence of cell movements or morphological changes

modify the electric current; (3) signal variations due to intracellular processes (plan after [GIA 84])


The use of fluorescent or other substances as labels for the sample is not required. In this case, the overall cell behavior in connection with any possible modifications of its cytoskeleton or adherence molecules will be exploited. Microelectrodes have to be placed under the cells’ attachment surface. Without cells, the electric current circulates freely. The presence of cells leads to an increase in resistance. Resistance is maximal if the culture is confluent. Disturbances in cell morphology, for example, induced following stress, toxicity or even a modification in cytoskeletal properties (as is the case during activation of certain signaling pathways), lead to variations in system impedance. However, this approach is limited to adherent cell cultures. Successive evolutions, advanced in large part by the increasing requirements of the pharmaceutics industry, have led to high-throughput tools, attaining previously unimagined levels of sensitivity.

The first use of impedance as a cell biology tool was published [GIA 84] in 1984 by Charles Keese and Ivar Glaever (1973 Nobel Prize for Physics), researchers at General Electric, the laboratory created in 1900 by Thomas Edison. In 1991, the two authors created the Applied BioPhysics company, which made use of the technology in the form of an instrumentation known by the acronym ECIS (Electrical Cell-substrate Impedance Sensing). This is an excellent, though all too rare, example of technology transfer between the realms of physics and biology. The initial target of the ECIS technology was the measurement of responses such as adhesion, migration or cell processes implicated in tissue healing.

The ECIS system relies on two golden electrodes, one of small size (10−3 cm2), upon which cells are cultivated, and one of larger size serving as counterelectrode [HON 11]. Cell membranes play the role of insulator, and the cells are to be considered as dielectric particles. The culture medium acts as electrolyte. The system also includes a signal amplifier, comprising an oscillator and measures the impedance variations via the application of a weak and noninvasive alternating current (1 µA in the 1 to 40 kHz frequency range) through a 1 MΩ resistance series placed between the two electrodes (Figure 3.5(1)). When the cells adhere to their electrode, the impedance varies according to the restriction in current. The signal is sensitive to any modification in morphology or movement of the cell layer (Figure 3.5(2)). Wells of 96 well plates include integrated electrodes.

The increasing interest of the pharmaceutical industry soon led other instrument suppliers into the race: Roche Applied Science/ACEA Biosciences who in 2008 launched xCELLigence, an integrated system that, like ECIS, manages the culture plate from the reader to the incubator, and

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MDS Analytical Technologies who in 2005 launched the CellKey system, more orientated to ultra-high throughput (384 well plates).

In addition, CellKey made for a significant and rather unexpected development. It is able to distinguish signals issued from the activation of two intracellular pathways, cyclic AMP and the IP3 attached to the seven-transmembrane domain receptors, a major class of therapeutic targets (see section 8.4). These are very subtly different signals, whose connection to impedance variations has not been well explained (Figure 3.5(3)). Gleaver and Keese already calculated that the impedance measurement could theoretically detect vertical cell changes at the nanometer level, which, in any case, was very far from the resolution of optical systems limited by the laws of physics to λ/2, or around 250 nm for typical applications. The spectacular advance of CellKey is not down to chance, but the addition of a spectral measurement mode. Consequently, impedance is not measured at a single frequency but along the 1 kHz to 110 MHz range, to which is added the confidential transformation output signal data, which seem to favorably influence the quality of the information provided.

3.3. Optical signals modified by state of cells

Another innovative option comes through optical manipulations on the lower surface of the plates on which the cells grow. Initially, the optical sensor approach used surface plasmon resonance (SPR) technology, more adapted to the measurement of macromolecular interactions than to cells, both due to the small sensitive surface area and the difficulty in analyzing cell flow in fluidic channels of 50 µm cross-section [COO 03].

Current cell tools use photonic crystals. The founding principle is based on the fact that cell samples, irrespective of the nature of environment or of any intracellular organelles present, have molecules, which, when exposed to electromagnetic waves in the optical spectrum, offer a greater dielectric permittivity than water. The photonic crystal (CP) used as sensor naturally modifies the propagation of electromagnetic waves. It is composed of a periodic rearrangement of dielectric material [ZHU 15]. The crystal geometry is conceived to concentrate light into tiny volumes so as to increase the intensity of the magnetic fields. When lit by a white light for example, the photonic crystal actually behaves like a reflection filter for discrete wavelengths. It reflects 100% of a unique, resonant wavelength, the value of which varies according to the biomass density or the local quality of the biological material [LIN 06, SHA 11].


This approach requires a dedicated tool, which emits, for example, in the infrared spectrum from an LED placed beneath the culture plate. Reflected light is captured by a spectrometer that records the resonant reflection spectrum, accompanied by analysis software that models the data to identify the emitted wavelength (Figure 3.6). A 96-well plate can be read in perhaps around 5 s, which allows for kinetics to be performed given the noninvasive aspect of the approach, and opens possibilities for high-throughput applications.

Currently, this apparatus is directly integrated to 96, 384 or 1,536 well plates. A spatial measurement of reflected wavelength is also possible by coupling with a suitable spectrometer, which allows for cell viability measurement applications for example [CHA 07b].

Since adherent culture cells are several micrometers in depth, considerably more than the 150 nm of the system’s exploitable analysis area, the attempt to extract pertinent morphological information was audacious. Nonetheless, it has proved to be fruitful. Several companies exploit this rival approach to impedance, particularly to meet the needs of the pharmaceutics industry. Corning’s EPIC system has been commercialized since 2005 for applications in the detection of interactions between biomolecules, like proteins. Its application in terms of live cell assays was developed shortly afterward. SRU Biosystems’s Biomolecular Interaction Detection (BIND) system, also launched in 2005, works along the same lines with a several minor differences. The technology, developed simultaneously by Zeptosens and later purchased by Bayer, is clearly centered on proteomic applications and does not seem to target the live cell assay market.

As for the BIND apparatus, it has been used through various sectors of the live cell assay market, such as in new drug discovery with cell models expressing therapeutic targets (seven domain receptors, RTKs, ion channels, see Chapter 8) in cell adhesion and also in the sector of cytotoxicity. In this last instance, the signal is characterized by a typical shift toward short wavelengths following reductions of the cell body and its loss of adhesion. Moreover, the wavelength profiles recorded in toxicology differ according to whether the toxin mode of action concerns genotoxicity, protein synthesis, steroid signals associated with endocrine disruptors, or the effect of detergents.

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Figure 3.6. Principle of cell status analysis by optical signals

In light of the considerations above, there is no doubt that the label-free cell assays based on impedance or optics provide a significant alternative to classic approaches based on fluorescence, bioluminescence or colorimetry. Their potential already exceeds that which their developers had imagined for them at their beginning. However, it should be noted that these technologies are opportunist, by which it is understood that the connection between cell event and signal detected is not controlled. They do not address any issue in particular a priori. Nonetheless, they can prove to be extremely powerful when a distinguishing signal has been identified, as demonstrated by the cyclic AMP and IP3 pathways.

The negative aspect of approaches based on impedance or optics concerns the prohibitive cost of these tools and the consumable items associated with them. New technologies such as these are far removed from the convenience of fluorescence readers associated with standard culture plates. This is why their use is limited to applications in new drug development, where the commercial stakes are high enough to potentially write off such investments.


3.4. Cellular autofluorescence

This property is characterized by the ability of cells to emit fluorescence when excited by light sources of sufficient wavelength. The list of intracellular fluorophores essentially comprises nicotinamide adenine dinucleotide phosphate in a reduced form (NAD(P)H), oxidized flavin (FAD), together with three aromatic amino acids (tryptophan, tyrosine, phenylalanine) and a few other molecules such as melanin, keratin or chlorophyll in plants. Each of these molecules has a distinctive spectrum of excitation/emission wavelengths, which can, however, partially overlap between each other and with molecules arriving in particular from the extracellular matrix like collagen. NAD(P)H and FAD are the two main metabolites used for the measurement of cell homeostasis. Both of these essential coenzymes are highly involved in redox control. Furthermore, they produce significant fluorescence. In the case of NAD(P)H, two measurement methods are exploited at the industrial level [HEL 06]: its light absorbing capacity (340 nm) or photon emitting capacity (445 nm when excited at 340 nm). Its fluorescence is considered to be more sensitive than its absorption. In both cases, the oxidized form NAD(P)+ does not share the fluorescent property. Accordingly, the fluorescent signature allows for the measurement of the observed cell sample’s redox status, or more precisely, for the quantification of metabolite conversion in stoichiometric reactions (or for which the molar relation is known) where:

lactate + NAD+ pyruvate + NADH

a reaction catalyzed by lactate dehydrogenase.

The ATP can also be measured in association with a NADH loss, according to the sum of the following reactions:

ATP + 3-phosphoglycerate ADP + 1,3-diphosphoglycerate

NADH + 1,3-diphosphoglycerate glyceraldehyde-3-P + NAD+ + P

catalyzed respectively by phosphoglycerate phosphokinase (PGK) and glyceraldehyde phosphate dehydrogenase (GAPD).

This sort of approach can easily be used with a good sensitivity and high throughput with fluorescence readers using photomultipliers as a signal amplification source.

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3.4.1. The case of chlorophyll

In the domain of plant biology, which we will only briefly mention here, the autofluorescence emitted by chlorophyll has been widely exploited, particularly in ecophysiology, to evaluate the photosynthetic performance of plant samples [MAX 00]. The chlorophyll molecule in solution is known to be highly fluorescent. However, the outcome for light absorbed by a chlorophyll molecule is divided between the photosynthesis in which energy is transformed to ATP, dissipation in the form of heat and emission of photons by fluorescence. This last mode only concerns 2% of the absorbed light, but remains easy to detect for modern tools, particularly modular fluorimeters that can be used in fields in direct sunlight.

The specific nature of this measurement exploits a remarkable property of chlorophyll’s fluorescence emission. The yield of this emission actually increases significantly through the first second following transfer of the photosynthetic sample from darkness to light (Kautsky effect). This is due to the gradual closure of the photosynthesis pathway, and is explained by a reduction in electronic acceptors like plastoquinone and the QA factor. In one of the steps following light absorption, QA accepts an electron and is incapable of accepting another before transmitting the first to the next electron carrier, QB. Electronic congestion follows, which blocks the photosynthetic system, thus favoring fluorescence. This phenomenon is not long in duration, diminishing through the following minutes according to a process known as quenching (fluorescence inhibition). The latter has two origins, one called photochemical, in connection to the stomatal opening and the activation of enzymes involved in the carbon cycle, and another in connection to the efficiency of the heat conversion pathway that improves over time.

The key to connecting emitted fluorescence to photosynthetic activity consists of blocking the photochemical pathway by a technique using a high-intensity flash of light, sufficiently short in duration to exert a transitory effect and not block photosynthesis over the long term. The intensity of fluorescence emitted is at a maximum during the flash. By comparing this value with the long-term yield and yield in the absence of light, an estimation of photosynthetic performance can be reached.

This knowledge, acquired through the 1970s and 1980s, has led to the development and marketing of a generation of instruments based on the principle of pulsation-amplitude-modulation (PAM), dedicated to measurements in the field, which measure photosynthetic energy based on


these yields. Moreover, current systems allow us to combine the chlorophyll autofluorescence with that of P700 (a pigment of the chlorophyll reaction center that absorbs light at 700 nm), P515 (another pigment that absorbs at 515 nm) and NAD(P)H along the lines set out above.

Approach Cell function targeted

Reading method Pertinence

ECIS, CellKey, xCELLigence

Possible cytoskeletal or adherence modifications

Electric (impedance measurement)

High throughput analysis

EPIC, BIND Biomass density Optical (electromagnetic wave reflection)

High throughput analysis

NAD(P)H NAD(P)H or ATP quantity

Fluorescence Adapted to 96 well plates

PAM Photosynthesis yield Fluorescence Low throughput but adapted to measurements in situ

Table 3.2. Label-free cell approaches

3.5. The different cell models and culture modes available

We saw in the previous chapter how the cell, as a tool in our understanding of the innermost workings of life, has been progressively separated from the individual in which it originated to be isolated in a dish of culture. This major conceptual development has been supported by works on the dissociation of extracellular matrices by trypsin, led by Rous in 1916, a hundred years ago this year. These achievements allowed for control over cell cultures, albeit at the price of a fundamental ambiguity. Isolation in a dish of culture certainly clothes the cell in a living unity, but simultaneously strips its soul away in snatching it from the whole to which it was attached.

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Moreover, the cells, and in particular the mammal cells, of interest for application use, do not readily lend themselves to life in culture. While a plethora of models are now available, the models themselves remain very limited. We have previously seen that the vast majority of lines are of cancerous origin or have been immortalized with the help of oncogenic viruses for example. However, other types of model are available, presenting superior or different information levels: primary cells, stem cells and cells obtained by differentiation of stem cells. Cultures of adherent cells, which have been treated thus far, typically present a classic monolayer organization called subconfluent, meaning they occupy most of their culture well without jostling each other or entering into competition. Two other modes are also available: cultures known as tridimensional (or 3D as opposed to 2D monolayers) and organotypic cultures.

Models derived from stem cells and organotypic cultures, whose applications are still emerging and whose potential remains unexplored, will be analyzed prospectively in sections 10.1 and 10.2, respectively. Here we will concentrate on the classic 2D and 3D models, both of which are widely exploited in the industrial and academic worlds.

3.5.1. Immortalized lines

Due to their oncogenic character, the immortalized lines have gradually drawn away from their original phenotype. They have, among other things, escaped from senescence and demonstrate elevated telomerase activity, which plays a role in their immortality. Nonetheless, they have retained certain attributes of their original organ or tissue, like enzyme expression or specific membrane markers, though can in no case be considered as representative of said organ or tissue. Such extrapolations are unfortunately not uncommon in the literature.

The immortalized lines have the particularity of possessing active oncogenes that confer on them stability for survival and proliferation in an environment far removed from their habitual living conditions. In terms of phenotype, these cells are typically cancerous, proliferative (their doubling takes on average around 24 to 30 h), incapable of redifferentiation and capable of supporting numerous cycles of freezing and cultivation. They grow in well-established nutritive media into which is typically added around 5 to 10% mammalian serum. For the most part, they are available commercially via organisms like ATCC at reasonable prices. Their cryopreservation at different passages also allows for them to be stocked and


reused in an almost infinite manner. It is worth recalling that HeLa cells have been used the world over since 1951.

Today, there are an impressive number of immortalized cells lines available. The American ATCC alone possesses around 4,000 lines of human origin together with thousands of lines covering more than 150 living species [ALL 05].

3.5.2. Primary cells

Primary cells are cells taken directly from individuals and subsequently placed in culture. These can be either circulating or adherent cells. In the latter case, they must first be dissociated by classic methods such as trypsin or measured mechanical action. The human being is composed of around 410 types of cell, 145 of which cover the diversity of neurons [VIC 06]. In principle, each of these cell types can be placed in culture. In application, culture cells include cardiomyocytes, hepatocytes, neurons, Langerhans cells and, of course, numerous circulating cells of both human and animal origin. These cells possess a significant advantage in that they are natural and retain, in phenotypic terms, numerous attributes native to their environment of extraction. It follows that these are the most pertinent models of the body’s physiological reality, unlike lines reflecting a pathological reality due to their own cancerous origin.

Unfortunately, numerous restrictions limit their use in the context of standardized assays. Unlike the immortalized lines, they are not cloned. When of tissue origin (biopsy), they can be contaminated by other cell types invariably present in tissues. Furthermore, the highly differentiated aspect that characterizes them and renders their great interest disappears completely after several hours. Moreover, their capacity for division is altered, their life span in culture remaining very limited, no more than several days at best. Finally, their use requires the donor’s informed consent and significant use precautions so as to manage the risk of viral transmission.

3.5.3. Three-dimensional cell culture

The majority of cell assays are performed on 2D culture support with the classic limitations as described in section 1.7. One step to circumventing some of these drawbacks of 2D culture is to proceed to a 3D structure, which intuitively seems closer to the physiological reality. Here, a distinction should

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be drawn between three-dimensional culture and organotypic culture [ELL 11]. The first refers to cell models cultivated on a 3D scaffold of more or less limited complexity, the second, to the reconstruction of complex tissue architecture, organized according to its many cellular components.

The 3D culture is characterized by a culture support that favors the exchanges between cells. These supports can be in the form of hydrogels or synthetic polymers, porous or sponge in structure. The prime materials are highly variable polymeric form arrangements based on polyethylene glycol, polyacrylamide, cellulose, polyurethane or vinyl polymers [AST 12].

Coating by components that favor adherence is essential. The reconstitution of basal membrane adherence is ideally achieved by tissue matrices formed at the junction of different cell types, such as epithelia, endothelia or stroma. However, such matrices are not routinely available, though standardized matrix models have been commercialized. The most well-known of these is Matrigel®, a matrix extract of secretions produced by a line of murine cells derived from EHS-type sarcoma (Engelbreth-Holm-Swarm) [BEN 11]. Matrigel® is a mixture of proteins including laminin 111, type-IV collagen, perlecan (proteoglycan), entactin and the main growth factors such as EGF, FGF, TGF-β, PDGF and IGF. It also contains numerous proteases and other factors. It can be used in both 2D and 3D cultures.

The wide variations in cell behaviors have led to the use of different 3D scaffolds. This results in specific morphologies, the implementation of varying signaling pathways and production factors, and more generally, in a reduced proliferation rate and characteristic differentiation profiles. These profiles can be used both in research and in the pharmaceutical industry for the identification of certain compounds, particularly in oncology where the approximation of the 3D structure to that of the tumor can be an interesting avenue for research. However, in this respect, interest must be tempered by the fact that these structures do not effectively mimic the intense vascularization that is characteristic of tumors.

4

Loss of Cell Homeostasis: Applications in Toxicity Measurement

4.1. What relevant information to use in the living cell?

The cell is at the heart of the living world. It is through the cell that life on Earth began. This fundamental role itself justifies the concerted research efforts of the biologists in their quest to unlock the inner workings that give cells a future (proliferation, differentiation, association), stability (homeostasis) and a means of ceasing to exist (apoptosis). This chapter will address cell homeostasis in so far as most toxicity live cell tests measure the disturbances that ultimately betray a cell’s inability to re-establish normal activity. Various conceptual problems arise as a consequence.

Walter Cannon coined the term “homeostasis” in 1926, describing it as a situation “in which organisms maintain their vital physiological variables at constant values despite environmental fluctuations. [CAN 32]”. It is quite logical to accept that the homeostasis concept applies to the different successive levels of a living organism, from the cell to the body or the population.

The measurement of cell homeostasis is problematic. Indeed, there are two opposing views. The reductionist view is the most common. This posits that global activity can be broken down into its parts and that knowledge of each of these parts should suffice to deduce knowledge of the whole. Many biologists hold the view that the molecule (or, going still further, the electron) is the smallest functional entity of the cell, and that everything can be explained according to molecular reactions [BRE 10]. However, it is not feasible to measure all of the reactions present in a cell under the effect of a


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stimulus. Mathematical models have been developed that integrate the vast bodies of experimental data shared online, and performed with occasional success. One example of such a success would be the model predicting apoptosis effects built according to the data of 7,980 intracellular events linked to the measurement of 1,440 response methods [JAN 05].

However, it is more generally accepted to measure one function, judged essential, and from there extrapolate the rest. The MTT assay is typically reductionist in interpretation. So long as respiration functions normally, the cell is healthy and in homeostasis. Nonetheless, it is clear that other aspects of cell function can go awry without disturbing respiration. Mutations in the DNA are an example of this occurrence. Certainly such shortcomings do not withstand scrutiny, even if ATP is a function, quite correctly, considered to be fundamental.

The holistic view is another school of thought. According to this view, cell function can be known only when apprehended globally, in its entirety. The cell is considered as a complex system, home to emergent properties. To study the individual parts separately would be inadequate. Partisans of this school of thought maintain that cell functions are, in part, stochastic and that the sum of all biochemical reactions is not equal but inferior to the whole. This approach lends itself to certain types of modelization, though not to experimentation [NUR 11].

We can conclude at this point that it is not currently possible to measure cell homeostasis by either reductionist or holistic approaches. On the other hand, it is possible to measure the major disturbances of homeostasis, known collectively by the term “toxicity”.

The cell is not a homogeneous environment. It is composed of clearly differentiated organelles fulfilling specific functions that define the cellular subcompartments. In connection with these cellular subcompartments, several fundamental parameters have been identified that affect the cell’s future when altered. These are principally oxidation reactions, calcium cytosolic rate, plasma membrane integrity, mitochondrial respiration, transcriptional and translational activity (protein production), going into apoptosis or even the capacity of the cell to eliminate undesirable substances, either by release towards the extracellular medium (via ABC transporters) or by intracellular digestion (in lysosomes). Each of these subcellular functions has prompted the development of multiple live cell assays used in the measurement of toxicity.

Loss of Cell Homeostasis: Applications in Toxicity Measurement 73

To these alterations common to most cells may be added some disturbances that are more specific in the activity of some specialized cells, such as, to provide two examples, the electric excitability of cardiac or nerve cells or the antibody production by immune cells. However, such alterations fall outside the remit of this book, which is limited to live cell assays targeting standard information.

Genotoxicity, however, is a cell alteration that, to a large extent, escapes the notion of homeostasis, at least in strictly cellular terms. The capacity of mutations to accumulate in the genome does not in general place the cell in any immediate danger. As we have seen in section 2.7, it is rather the accumulation of alterations over several cell generations that is ultimately expressed by dysfunctions at the level of the wider body. In fact, in terms of the maintenance of cell homeostasis, the majority of genotoxic aggressions are eliminated by a range of highly sophisticated reparation kits. The recent acquisition of this knowledge gives a good example of signals that could someday yield information concerning the nonlethal levels of toxicity to which cells are constantly being subjected.

At this point, we will briefly review the principal subcellular functions involved in toxicity. Where possible, we will also introduce the knowledge that leads to the existing cell assays and whether the acquisition of new knowledge could allow us to envisage the development of new and more performant assays in the near future.

4.2. Lysosomal activity

Lysosomes are a cell’s main digestive compartments. They were first described in the 1950s by the Belgian researcher Christian de Duve, a discovery that won him the Nobel Prize in Physiology or Medicine in 1974. For several decades, the lysosomes were considered as mere cellular waste management centers. Today, we know that their functions are far greater than this, being now considered as one of the key actors in cell homeostasis. They are particularly involved in the breakdown of extracellular and intracellular matter, also intervening in the regulation of apoptosis, reparation of plasma membrane integrity and homeostasis of membrane cholesterol. Their principal characteristic, which is, in fact, widely employed in cell assays, is to present an acid pH favorable to the activity of hydrolase enzymes, which specialize in the breakdown of biomolecules by acidic hydrolysis. They are essential in the maintenance of cell homeostasis for this reason [APP 13].

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Lysosomes are quite spherical organelles of a fair size (around 1 µm in diameter). They are present in practically all known animal cells, with the notable exception of erythrocytes. The lipid membrane that delimits them is simple though inside the organelle other membranes can be observed being degraded. They are easily identifiable thanks to a unique characteristic: the presence of 25 membrane proteins that are largely glycosylated, a characteristic called glycocalyx, which is believed to confer some protection against degradation on these specific proteins. Another characteristic of lysosomes is elevated levels of calcium, in the range of 600 µM, approaching the levels found in the intracellular storage sites such as the endoplasmic reticulum. This calcium is important for the various stages of intracellular lysosomal trafficking (generation, recycling, membrane fusion). This organelle also contains a receptor for the intracellular second messenger, nicotinic acid adenine dinucleotide phosphate (NAADP), a molecule specialized in the release of calcium and partner in its cytosolic regulation.

The recent discovery [SAR 09] of the CLEAR network of genes, which contain a specific nucleotide sequence, has shed some light on the poorly understood biogenesis mechanism of lysosomes. The key element seems to be a transcription factor named TFEB, which associates specifically with elements containing the CLEAR sequence and induces the expression of the genes implicated in the lysosomal cycle.

In terms of its activity, the lysosome is primarily specialized in the degradation of macromolecules, in partnership with the ubiquitin–proteasome system. Unlike proteasome, which mainly targets proteins of a short life span, lysosome is no shrinking violet and degrades all sorts of molecules of all sizes. This degradation work is performed by around 60 hydrolase enzymes that collectively cover the whole range of functions. The products of this degradation are finally brought out of the lysosome by transporters, and then recycled by the cell machinery. The most well-known lysosomal hydrolases are the cathepsins.

An acidic medium is the most characteristic signal of a lysosome. On the one hand, the acidic pH is optimal for assuring hydrolase activity. On the other hand, maintaining a pH between 4.5 and 5 has the advantage of making macromolecules lose their three-dimensional structure, which makes them more vulnerable to attacks from hydrolases, thus helping in their degradation. Indeed, the choice of acidic markers like neutral red, selected in 1930 to


demonstrate cell health through the development of the first cell assays, was highly appropriate. Today we know that these markers target lysosomes.

It is important to note the central role of ATP availability in controlling lysosomal pH. The difference in pH between the inside of lysosome and the cytosol is maintained by a multimeric transmembrane protein complex called H+-ATPase. This complex uses ATP hydrolysis as an energy source so as to pump cytosol protons to the lysosome against their electrochemical gradient. It is quite clear that cell assays based on pH markers, in fact, measure the health of the cells by verifying that this H+-ATPase complex functions correctly, a signal that can also be attributed to the availability of another key element, certainly the most essential to cell homeostasis: that of energy resources in the form of ATP.

The extreme dynamism of lysosomes within the living cell is another advantage for live cell assays that primarily seek the expression of cell dynamics. In general terms, lysosomes integrate into the endocytosis cycle, which allow the cell to constantly rejuvenate its plasma membrane. It has been estimated that with each passing hour, cells regenerate around 50% of their plasma membranes. This both complex and dynamic process requires the intervention of endosomes, vesicles formed directly from the plasma membrane by intracellular invagination, together with other organelles identified collectively as the trans-Golgi network. These elements exchange matter, ensuring the continuous recycling of the membrane, with the lysosome acting like a waste management and recycling plant. Without this fast and continuous system, the lysosome would stop working and no longer receive hydrolases and other essential membrane proteins supplied by the trans-Golgi network. Targeting the endocytosis system could prove to be a successful strategy for gathering information about the health of a cell in real time.

In conclusion, the lysosomal approach employed by live cell assays consists of verifying that the cell is healthy by measuring its capacity to correctly manage its waste with the utmost speed and regularity. This is the case of the wide spread and long-established NRU assay (see section 2.6), which draws on the intralysosomal pH difference in order to reveal its functional integrity. It is a colorimetric test. New fluorescent lysosomal markers named CAT have been developed to help harmonize the approaches into a multiplex analysis, and are available commercially for high-throughput applications.

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NRU Maintenance of intralysosomal pH differential

Colorimetry Adapted to 96 well plates.

CAT Marker of acidic organelle presence

Fluorescence Adapted to high throughput.

Table 4.1. Cell approaches for lysosomal activity measurement

4.3. Redox balance and oxidative stress

All cells functioning in an aerobic mode are continuously confronted by the production of partially reduced molecular oxygen species (O2) of high reactive value. These often highly aggressive reactive oxygen species (ROS) are normally neutralized immediately by cellular antioxidant systems. The main ROS are hydrogen peroxide (H2O2), singlet oxygen (1O2) and the free radical species like superoxide anion (O2

−.) and the hydroxyl radical (OH.). To this nonexhaustive list may be added the reactive species of azote, such as azote monoxide (NO.). ROS formation comes from the different degrees of reduction of molecular oxygen or azote by successive electron transfer. The primary source of ROS is the home of cell respiration, the mitochondrion, which uses molecular oxygen as an energy source as part of aerobic respiration.

Oxygen can also react with electrons liberated by the process of respiration to produce ROS, especially the superoxide anion. This is formed by the monoelectronic reduction of oxygen and is not-accumulated as a result of the action of enzymes like superoxide dismutase (SOD), which catalyzes its transformation to H2O2. When there are large quantities of superoxide present, SOD can favor the presence of Fe2+ and provoke the transformation of H2O2 into hydroxyl radical OH. as the result of the Fenton reaction. These different ROSs often have short life spans, which can significantly limit their action radius. The half-life of hydroxyl radical, for example, is in the order of a nanosecond. So it acts on the immediate vicinity of its production site. On the other hand, H2O2 with a half-life in the order of 10 microseconds can act at a certain range, even outside of the cell. These facets are significant in the


choice of species to evaluate in the context of live cell assay applications, with certain species being simply too labile for measurement.

Other ROS sources are available in the cell, in connection with different enzymatic activities like cytochromes p450 that metabolize xenobiotics, xanthine oxidase involved in the catabolism of purine bases, or cyclooxygenase involved in prostaglandin synthesis. All of these sources lead to the formation of ROS such as superoxide.

Faced with this arsenal of highly toxic molecules, evolution has established systems of defense that are equally sophisticated, allowing for ROS production and elimination to be held in homeostatic balance, and even for them to be used as signaling molecules. The defensive armada is comprised of favorable energetic reactions such as superoxide dismutation to H2O2 and also specialized enzymes like SOD or the very effective catalase that brings H2O2 back to the state H2O + ½ O2.

One of the essential actors in the balance of cell redox is glutathione (GSH), a tripeptide containing cysteine. GSH is the most important nonenzymatic antioxidant. Practically, all cells produce it in very significant quantities. GSH has several strings on its bow: it can act alone to neutralize OH., associate with glutathione peroxidase to degrade H2O2 or activate other antioxidants like vitamins C and E. Indeed, the latter exists in eight different forms, of which one, α-tocopherol specializes in scavenging radicals of lipid origin like the LOO. peroxyl radical.

Notwithstanding, the list of actors and strategies employed by the cell in the detoxification of ROS is extensive. However, for present purposes, it should suffice to retain that the loss of redox homeostasis results in deleterious effects for the cell, particularly via the degradation of the lipid membrane or DNA and protein modification, the consequences of which can prove to be catastrophic. Consequently, it is perfectly legitimate to accept the measurement of activities established by the cell to fight against oxidative stress as a signal of major disturbances in cell health.

The presence of intracellular ROS can be initially evaluated by a H2-DCF (2′,7′-dichloro-dihydro-fluorescein) marking, a non-fluorescent molecule that becomes the fluorescent compound dichloro-fluorescein (DCF) by intracellular oxidative conversion [FOR 10] (Figure 4.1). In order to avoid any leak of the signal to the extracellular medium, H2-DCF must be produced

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in the form known as CM-H2DCFDA, which possesses a chloromethyl (CM) and diacetate (DA) group, which allows for the passage of the plasma membrane and is cleavable by the intracellular esterases.

A new generation of three fluorogenic sensors have been released, commercialized under the name CellROX, featuring fluorescence activated by intracellular oxidation (Figure 4.1). These photo-stable sensors are capable of penetrating the inside of the cell and thereby entering into contact with ROS. The structure of these sensors remains confidential, though it is known that the green one is a modified nucleic acid sensor that, once oxidized, binds to the nuclear and mitochondrial DNA. However, there is little information available concerning the red (called deep red) and orange sensors other than their being cytosolic. In commercial terms, they were developed for image analysis applications, flow cytometry and high-throughput analysis on fluorescence readers. Their use is still recent, though opinions forums are divided.

The superoxide radical anion can be detected on living cells by means of a fluorescent hydroethydine sensor. The reaction of the two compounds produces 2-hydroxy-ethydium (2-OH-E+), which fluoresces at 400 nm. This reaction is accompanied in the cell by the production of ethidium, another fluorescent compound present in much larger quantities and not specific to superoxide action. This parasite presence complicates the interpretation of results obtained by fluorescence measurement alone [ZIE 10]. The commercialized MitoSOX red system (Figure 4.1) is based on this concept but employs a hydroethidine derivative including radical triphenylphosphonium (TTP+) captured by the mitochondrion.

The reduced glutathione (GSH) rate measurement represents a largely understood alternative approach for the assessment of cell redox activity (Figure 4.1). GSH depletion can be considered an unequivocal signal of the presence of an oxidative stress. The connection between thiol functions and fluorescence from bimane derivatives was established in 1979. Research for a fluorescent sensor that specifically associates with the GSH has led to monochlorobimane (MCB), a compound that crosses the plasma membrane [STE 02]. In unbonded form, MCB remains nonfluorescent due to quenching by its chlorine group. In the presence of glutathione S-transferase (GST), it conjugates with GSH, forming adducts. MCB does not seem to interact uniquely with GSH, which represents a limit in this approach.


Figure 4.1. Main methods for measuring cell redox activity

The spread of lipid peroxidation damage can be followed thanks to C11-BODIPY, an analog of fatty acid. This sensor that fluoresces in the red (590–595 nm), shifts to green in reduced form (510–520 nm) by oxidation of its polyunsaturated butadienyl part. A ratiometric measurement of the two emission wavelengths will register the membrane damage due to the peroxidation process depending on ROSs [PAP 99].

An alternative assessment for lipid peroxidation consists of measuring the presence of compounds directly issued from peroxidized lipid decomposition, such as malondialdehyde (MDA), a reactive carbonaceous compound. The commercialized test, called thiobarbituric acid reactive substances (TBARS), exploits the heated reaction (50°C, 3 h) between MDA and thiobarbituric acid (TBA), producing as final compound an MDA-TBA adduct of 2:1 stoichiometry, colored or fluorescent. In its cellular version, the assay is performed on lysates in solution.

Note that the autofluorescence emitted by oxidized NAD(P)H under the effect of oxidative stress can also be exploited (see section 3.4).

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MCB GSH depletion Fluorescence For image analysis

NAD(P)H NAD(P)H autofluorescence

Fluorescence Weak signal but adapted for high throughput

CM-H2DCFDA

Production of intracellular ROSs

Fluorescence For image analysis

MitoSOX red Superoxide production by mitochondria

Fluorescence For image analysis

CellROX Production of intracellular ROSs

Fluorescence Compatible with 96 well plates and fluorescence readers

C11 BODIPY Lipid peroxidation Fluorescence For image analysis, ill adapted to high throughput

TBARS Lipid peroxidation Colorimetry, fluorescence

Adapted to 96 well plates

Table 4.2. Classic cell approaches for oxidative stress measurement

4.4. Integrity of the plasma membrane

The plasma membrane forms the fundamental barrier that maintains the essential constituents of the cell’s milieu intérieur within a relatively isolated space. Any rupture of this would be perceived by the cell as on a par with the breach opened by Telamon during Hercules’ attack on Troy (during the first Trojan War), a breach of its innermost integrity.

Various toxins, such as those accompanying a bacterial infection, work to create pores in the plasma membrane. For a long time, it was believed that the reparation of these pores was connected to a spontaneous reorganization of phospholipid membranes to their more stable thermodynamic state. This is not the case, still less so if the lesion is of significant size (approximately >0.2 µm). Today, we know that the tension at work in the plasma membrane associated with the presence of cortical rigidity in the cytoskeleton is


opposed to this mechanism. The underlying process requires the provision of a new membrane by the fusion of intracellular lipid vesicles like endosomes or lysosomes recruited in the cytosol. These work as patches, which allow for the re-establishment of a certain balance inside the membrane.

In functional terms, the first major consequence of the rupture of the plasma membrane is manifested by a massive inflow of calcium, in turn provoking biochemical and structural disorders, which, if the problem is not resolved soon, provoke cell death. It is then logical that the cell uses the calcium signal as an alert to deploy the panoply of reparation mechanisms with which it disposes. Of course, these mechanisms can only be put into action if the situation is not too far gone, which corresponds to a reasonably high level of cytosolic calcium, no more than the 10 µM threshold. Indeed, most proteins involved in the plasma membrane’s reparation mechanisms, synaptotagmins, SNARES, ferlins and annexins have a fundamental connection to intracellular calcium. The synaptotagmins, for example, are activated by the presence of calcium and favor the fusion of intracellular lipid vesicles with the plasma membrane. They work together with the SNARES. These two families are present both in plants and in animals, suggesting a very early evolutionary introduction as reparation tools. Ferlins also possess numerous calcium-binding sites, and participate in the recruitment of vesicles around the plasma membrane.

In terms of developing live cell assays, the loss of membrane integrity is a common signal and a universal precursor of, or consecutive to, a homeostatic disturbance. It can be followed simply through observing the diffusion of compounds known for not crossing the plasma membrane to the cell interior. DNA colorimetric sensors such as Trypan blue, or fluorescent ones like propidium iodide, ethidium homodimer or Sytox are long known to penetrate the cell only after massive lesions to the membrane, usually associated with a critical cell condition.

The extracellular presence of enzymatic activities, normally confined to the cell interior, is the most common signal used in commercial approaches. Lactate dehydrogenase (LDH) activity is the most classic example of this. LDH catalyzes the reduction of pyruvate to lactate by oxidizing the NADH to NAD+ according to the reaction:

Pyruvate + NADH + H+ lactate + NAD+

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Figure 4.2. Main approaches for cell membrane permeability measurement

It is the consumption in NADH that is measured at 340 nm.

Long available high-throughput assays have taken this basic principle and adapted it to other enzymatic activities. One widely employed approach consists in measuring the extracellular ADP conversion to ATP under the effect of adenylate kinase (AK) followed by production of light from ATP and luciferin under the control of luciferase (Figure 4.2, also see sections 3.1 and 4.7). Another example is provided by monitoring the extracellular activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This enzyme is considered ubiquitous, stable and constitutive of cells of normal metabolism. It catalyzes the conversion of glyceraldehyde-3-phosphate (GAP), also intervening in glycolysis, in glycerate 1,3- bisphosphate (GBP) according to the reaction:

GAP + NAD+ + Pi GBP + NADH + H+


Here too, it is the reduction of the intermediate product NAD+ to NADH that is measured (Figure 4.2).

The extracellular activity of proteases can also be monitored. In the leading commercialized kit, the nature of the protease is not provided, with the authors of the editio princeps, cited in reference, maintaining that it is unknown [CHO 08]. What is known, however, is that the substrate used contains a tripeptide, alanyl-alanyl-phenylalanyl, which prevents luciferin (become AAF-aminoluciferine) being activated by luciferase. Following extracellular protease activity, the tripeptide is cleaved and luciferin liberated, which results in the emission of fluorescence (Figure 4.2). Apart from toxicity studies, this approach is also used in high throughput to search for kinase activity inhibitors for therapeutic purposes (see section 8.6).

The membrane behavior of annexins has also drawn the attention of researchers. These cytosolic proteins discovered in 1978 have the property of binding both calcium and membrane phospholipids. Highly sensitive to calcium, they are recruited to the plasma membrane from cytosol on as soon as the intracellular concentration of calcium rises. Other protein interactions together with proteolytic cleavages also modulate their activity. It is interesting to note that the 12 members of the annexin family present in vertebrates are active at various calcium concentration thresholds, making them sensitive and precise calcium markers, especially since the direct measurement of calcium in cell compartments open to the exterior remains particularly delicate [DRA 11].

Plasma membrane phosphatidylserines in the homeostatic cell are exclusively maintained on the internal leaflet by enzyme activities such as flippases (Figure 4.2). The loss of phosphatidylserine membrane asymmetry and their presentation on the outside leaflet is a well-established signal of plasma membrane disorganization. However annexin V is a protein known for its affinity for phosphatidylserine. Following a disturbance in membrane integrity, annexin V coupled, for example, to a compound such as FITC, will bind to phosphatidylserine and present a labeling limited to the cell edges. This marriage of affinity between annexin and phosphatidylserine is also used to evaluate cell apoptosis (see section 4.9).

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DNA markers

Influx of compounds following lesions to plasma membrane

Fluorescence Adapted to 96 well plates and fluorescence readers

Proteases Extracellular release of protease activity

Bioluminescence Adapted to high throughput (1,536 well plates)

GAPDH Extracellular release of glyceraldehyde -3-phosphate dehydrogenase

Optic density Adapted to high throughput

Kinases Extracellular release of kinase activity (including adenylate kinase)

Bioluminescence Adapted to high throughput

LDH Extracellular release of lactate dehydrogenase activity

Optic density Adapted to high throughput

Annexin V Presentation of phosphatidylserine following loss of plasma membrane integrity

Fluorescence by flow cytometry

Incompatible with high throughput

Table 4.3. Classic cell approaches for membrane permeability measurement

4.5. Cellular efflux

The rapid expulsion of undesirable substances from the cell is another major aspect in maintaining homeostasis. This function is performed by proteins of the plasma membrane known as ABC (ATP-binding cassette) transporters. These proteins form one of the largest known protein families. In fact, the human genome codes for 49 different ABC transporters. Only a few of them are known down to their inner workings. They consist of two transmembrane domains forming a channel along which the undesirable substance (known as substrate) is eliminated, and of two cytosolic domains for nucleotide binding that each has ATPase enzymatic activity. This ATP hydrolysis mechanism allows for the provision, when needed, of the requisite energy for the continuous working of the pump, thus releasing the undesirable compound by maintaining a negative concentration gradient between the inner and outer sides of the membrane.


ABC transporters are present at all levels of the living world. While in bacteria they perform different functions in substrate entry and egress, the versions of superior eukaryotes appear to specialize solely on the elimination of undesirable intracellular substrates. One of the working models accepted by specialists describes ABC transporters as hydrophobic vacuum cleaners, lurking around the internal surface of the plasma membrane [SHA 08].

ABC transporters are correctly considered to be a double-edged sword in the cellular armory. While they certainly protect the cellular integrity by eliminating undesirable compounds, they often fail to differentiate between xenobiotics and drugs, and in some cases lead to rejections, which disturb therapeutic treatments. It is this property, first described 40 years ago and named multi-drug resistance (MDR), which, in fact, led to their discovery. MDR still represents one of the essential challenges in the treatment of numerous cancers. Chemotherapy treatments are based on the use of aggressive compounds and are recognized as such by cancer cells, which adapt by overexpressing ABC transporters on their surface that then proceed to eliminate the prescribed drugs. In patients, ABC transporters present natural genetic polymorphism, which explains the variations in MDR observed from one patient to another.

Three ABC transporters belonging to three subfamilies of ABC protein are known to encapsulate the main differences in the efflux function. This is the product of the gene mdr called P-glycoprotein or P-gp discovered in 1976, together with the transporters MRP-1 and BCRP. P-gp has been extensively studied as it seems to be one of the transporters presenting the widest range of substrates. Its distribution is relatively ubiquitous, both through the body and at cell level. Moreover, it is particularly overexpressed in most cancerous tumors [BIN 13]. While P-gp and BCRP can export substances unmodified, uncomplexed to other intracellular transporters, the MRP-1 transporter manifests a significantly different activity. This protein is specialized in the transportation of intracellular molecules like glutathione, allowing for the elimination of different molecules complexed with it.

Note that the efflux function particularly consumes energy. A reduction in ATP availability is consequently soon associated with a massive accumulation of toxic substances.

The situation becomes more complicated when the indicator used to measure a given cell function is itself an ABC transporter substrate. This is the case of the calcein-AM used in the measurement of metabolic activity

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(see section 4.7). The interpretation of results has to take this into account. Efflux activity can indeed disturb the measurement of other cell functions.

On the other hand, as stated above, ABC transporters form a numerous family, and demonstrating the specific interactions of a marker with one or several members of the family is virtually impossible, particularly for living cells. Historically, efforts to develop specific ABC transporter modulators have mainly aimed to measure MDR activity in connection with clinical difficulties. Numerous inhibitors have been developed for different members of the ABC transporter family and are available on the market in the sector researching new therapeutic approaches in connection to multiple drug resistance. Their levels of specificity are still subject to debate and far from clear.

Figure 4.3. The three main methods for measuring cell efflux. LUCS: (1) the TO is predominantly kept out from the cell; (2) a weak quantity nonetheless reaches DNA; (3) TO photosensitization produces ROSs leading to (4) loss of homeostasis and efflux function and (5) a massive entry of TO. Calcein- AM: (1) transformation into fluorescent calcein; (2) efflux; (3) efflux inhibition; (4) cell accumulation. Hoechst: (1) entry and efflux, (2) efflux inhibition; (3) accumulation in nucleus


In terms of live cell assay development, a loss in function of the ABC transporter system represents a major sign of homeostasis loss. Indeed, there are several fluorescent cell markers that are themselves ABC transporter substrates and thereby represent excellent potential indicators of cell efflux activity in real time.

An elegant option is to make use of calcein acetoxymethyl ester (calcein-AM). Unlike native calcein, which possesses six negative and two positive charges, its esterified form is lipophilic and readily penetrates the cell. Once inside the cytosol, the ester of the molecule is cleaved away by the intracellular esterases, the new formed calcein becomes fluorescent and finds itself trapped in the cell. However, with calcein being a substrate of ABC transporters, a loss in fluorescence will reveal functional efflux activity [FEN 08]. Moreover, intracellular fluorescence retention after the addition of a selective inhibitor (with the nuances described above) will attest to the activity of P-gp or MRP-1 ABC transporters. Notwithstanding that the leaking of fluorescence into the extracellular medium is not easily interpreted in a live cell assay and that the method of retention by inhibitor addition complicates operational protocols. As a result, the approach is limited for the time being to flow cytometry and resists the standardization required for 96 well plates. Calcein fluorescence is also highly dependent on the presence of calcium ions (see section 4.7).

Rhodamine 123 is sometimes used for efflux measurements. It is primarily a fluorescent marker used at the macroscopic level by industries in tracing water flow. What is more, it is known for accumulating in the functional mitochondrion (see section 4.7). Rhodamine 123 is also reputed to be a substrate of the P-glycoprotein, but its application in efflux measurement is complicated by the fact that the compound is metabolized to rhodamine 110 by the intracellular esterases, which modifies both its spectral properties and its quality as an ABC transporter subtype selective substrate. Indeed, a recent study [FOR 12] demonstrated that the intracellular transfer of rhodamine 123 is complex, requiring the intervention of a passive and an active transport together with an intervention of OATP transporters (see section 8.7).

The Hoescht assay on CHO cells uses the fluorescent nucleus marker Hoechst 33342, known since 2002 to be the substrate of the ABC transporter subtype called BCRP [SCH 02]. So long as the efflux system functions, the Hoeschst marker is rejected from the cell before reaching its nucleic target. The addition of an efflux inhibitor blocks its egress. As a consequence, it accumulates in the cell and binds massively to the DNA, presenting strong fluorescence (Figure 4.3). This type of approach is particularly used in flow cytometry.

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A new approach called light-up cell system (LUCS) completes the picture. As starting point, it resumes the same logic of Hoechst marking. LUCS uses thiazole orange (TO), an ABC transporter substrate and nucleic acid marker whose quantic yield increases significantly after binding with its target, two features that it shares with Hoechst 33342. However, TO possesses a third distinguishing feature: when bonded with nucleic acids, it metamorphoses into a powerful photosensitizer. Its relaxation is accompanied with ROS production together with the cleavage of nucleic acids [THO 00]. As TO efflux activity consumes a great deal of ATP here, any loss in cell homeostasis necessarily leads to a disturbance in ABC function. The idea behind LUCS is to measure the loss in cell homeostasis by relying on the major disturbance triggered by TO relaxation after photosensitization (Figure 4.3). In the altered cell, TO is diffused freely and photosensitization is without effect. However, in the normal cell, TO is essentially maintained outside of the cell due to a major efflux by the ABC function. Only a very small quantity penetrates the cytosol and the nucleus. This acts like the horse used in the second Trojan War. Its photosensitization visibly provokes a loss in ABC transporter activity, via alterations of oxidant origin, followed by a massive entry of marker along with a large increase in fluorescence signal [FUR]. Though conceptually complex, the LUCS assay is particularly straightforward to implement. It requires no more than the introduction of TO into the culture medium along with a photosensitization flash. Indeed, this is the first efflux disturbance assay to be standardized on 96 well plates for the high throughput market [FER 13a]. It is presently at the prevalidation stage with the ECVAM (see Chapter 5) for regulatory applications [ECV XX] under the name Valitox [PRO]. Unlike technologies based on calcein-AM or Hoechst 33342, LUCS does not require the intervention of transporter inhibitors to validate the loss in ABC functions.

Approach Cell function targeted Reading method

Pertinence

Calcein-AM Disturbance of ABC transporter activity

Fluorescence Adapted to high throughput.

Hoechst 33342

BCRP activity on CHO cells

Fluorescence Adapted to high throughput. Preclinical use

LUCS/Valitox Disturbance of ABC transporter activity

Fluorescence Adapted to high throughput. Undergoing ECVAM prevalidation

Table 4.4. Classic approaches for cellular efflux measurement


4.6. Homeostasis of ion exchanges

4.6.1. The calcium ion

Maintaining calcium homeostasis is achieved at body level by means of stringent hormonal control of its extracellular concentration in different organs. At cell level, the calcium ion intervenes in the control of numerous functions, such as enzymatic activities, cytoskeletal dynamics and allosteric protein regulation. Its cytosolic concentration is maintained at low levels, around 10–100 nM, known as physiologic, thanks to the activity of ATPase pumps present both at plasma membrane level and at the surface of intracellular reservoirs as endoplasmic or sarcoplasmic reticulums. Exchanges with the outside of the cell are also controlled by Na+/Ca2+ membrane ion channels.

The other fundamental aspect of intracellular calcium is its major role in the transduction pathways associated with hormonal messengers or neurotransmitters. In such cases, the calcium signal is characterized conversely by high concentration waves of calcium (around 0.5–1 µM) that spread through the intracellular space, developing in a short space and time (around a second). This aspect of calcium regulation has found numerous applications, particularly in new drug development, which will be addressed in section 8.4.

While a distinction must be drawn between calcium homeostasis and its role as a second messenger involved in the transduction mechanism, the measurement methods, on the other hand, are common to both. The most widespread rely on the use of the Fura-2, Indo-1 or Fluo-3 fluorescent colorants, developed by Roger Tsien’s team between 1985 and 1989 [TSI 99]. All of them announce the calcium-binding sensitivity by a very significant increase in fluorescence (often by more than 100 times). They were first used in flow cytometry before being applied to high-throughput instrumentation. Fluo-3 and Fluo-4 work without spectral modification. Indo-1 excitation is at 350 nm followed by a drop in its peak wavelength from 475 to 400 nm according to the Ca2+ concentration. This property allows for ratiometric measurement, which qualifies the result with great precision. The spectral behavior of Fura-2 in the presence of Ca2+ is somewhat different. When the emission channel is fixed at 510 nm, it shows a progressive drop of around 30 nm from its maximum absorption peak. Note that these different sensors applied to real-time fluorescence imaging are the foundation of the major discoveries on the role of intracellular Ca2+, particularly concerning its activity as second messenger.

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Different genetic constructions have since been developed in order to stabilize the monitoring of the intracellular signal [PAL 06]. This is the case of the cameleon system developed by Atsushi Miyawaki (Wako, Japan) in 1997. This is an intramolecular FRET signal (described in section 3.1 and Figure 3.3), emitted between two GFP proteins. The chimeric protein was made from four genes placed end to end, coding successively for a GFP (CFP), for calcium-sensitive calmodulin, for a calmodulin-binding peptide present on a myosin light chain (M13) specific kinase, and for another GFP (YFP), FRET partner of the first. The Ca2+ bond to calmodulin provokes the chimeric protein to fold completely and narrows the distance between the GFP to within the Förster radius range.

One advantage of the cameleon approach is that its modest cellular expression disturbs calcium homeostasis less than the massive addition of classic fluorescent sensors.

It is also possible to address the FRET signal to target different cell subcompartments like mitochondria or endoplasmic reticulum by selecting certain regulation proteins of calcium pathways specific to these organelles. This level of subcellular precision remains unavailable to other fluorescent sensors.

Aequorin, a naturally bioluminescent protein that uses resonance to supply energy to GFP in Aequorea victoria, has been associated with the measurement of intracellular Ca2+ for almost 50 years since the avant-garde works by Ridgway and Ashley published in 1967 [RID 67]. The molecular complex of aequorin is described in section 3.1. In this case, the BRET transfer energy is not used, but the bioluminescence property of coelenterazin, a prosthetic group of aequorin. Coelenterazin’s oxidation in the presence of calcium ions and dioxygen leads to a conformational change, resulting in the emission of light at 470 nm. Since the 1990s, cloning of the apoaequorin gene has allowed for the protein to be expressed in numerous cell models. Incubation with coelenterazin allows for the reconstitution of the complex, which then becomes a biosensor for the presence of intracellular calcium.

Gene reporter systems based on luciferase have also been developed using, for example, the activation of the NFAT-RE transcription factor associate with the presence of intracellular Ca2+ (see section 8.5). The advantage of these systems is in producing more satisfactory light signals. Indeed, the quantic yield of aequorin is in the order of 0.15 whereas that of luciferase reaches 0.88. However, the reporter system expresses a rapid and


transient event by a response that is delayed (sometimes by several hours) and stable over time (see section 3.1).

The calcium-dependent bioluminescence resonance energy transfer (BRET) signal, natural in Aequorea victoria, has been successfully replicated through the creation of a fusion protein between the two BRET partners, aequorin and GFP [GOR 04] (see section 3.2).

4.6.2. Maintenance of membrane potential

Calcium regulation aside, the maintenance of the resting membrane potential, on both sides of the plasma membrane, is another essential parameter of the cell’s equilibrium. Here, the main actors are sodium, potassium, calcium and chlorine ions. The active movement of these ions against their concentration gradient is ensured by enzymatic pumps called Na+/K+-ATPases, present in the plasma membrane. For example, the energy of each ATP molecule allows for the entry of two K+ ions and the egress of three Na+ ions. In a typical mammal cell, the resting potential is maintained at around −70 mV.

There are numerous varieties of ion channels with the role of reinitializing resting potential or of participating in the generation and propagation of action potentials, characteristic of excitable cells. Unlike ATPases, these channels allow for ions to proceed passively in accordance with their electrochemical gradient. The channels open and close due to various signals, such as transmembrane voltage, ligand binding or mechanical stress. In application terms, these activities can be evaluated by means of the patch clamp technique, which consists in measuring the electric current on both sides of the plasma membrane using glass capillary microelectrodes. This technique is still widely employed and has been optimized to attain a certain throughput on platforms like PatchXPress or IonWorks Quattro.

Furthermore, membrane potential can be advantageously measured using a high-throughput fluorescence technique called VIPR, developed around 2002 as part of ion channel activity measurement required in industrial programs. This live cell assay uses two FRET partners, one coumarin-linked phospholipid (energy donor) and a negatively charged oxonol marker such as DiSBAC2(3) (energy acceptor). The first is loaded on the external leaflet of the plasma membrane. The second is the true current sensor. Its distribution on external and internal leaflets of the plasma membrane varies according to electric potential. This results in variations in the distance between the two

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membrane markers, which is expressed by a FRET signal. Sensitivity at the mV level can be reached (1–3% of the FRET ratio variation). Applications developed by the company Aurora Bioscience, now Vertex (Vancouver, Canada), are presented in Chapter 8.

An alternative strategy to FRET can consist of using an oxonol fluorescence quencher that does not cross the plasma membrane. Consequently, only the oxonol present on the external leaflet of the membrane would be quenched. This strategy is sold in kits called Brilliant Black.


Fluo-3, fluo-4, Indo-1, Fura-2

Fluorescent sensors sensitive to Ca2+

Fluorescence Adapted to high and ultra-high throughput.

Cameleon GFP chimera protein sensitive to Ca2+

Fluorescence (FRET)

Adapted to high throughput.

Aequorin Luminescent protein activated by calcium

Bioluminescence Adapted to high throughput.

NFAT-RE Activity of transcription factors activated by Ca2+

Bioluminescence Adapted to high throughput.

Aequorin/GFP Luminescence signal activated by calcium

Bioluminescence/ Fluorescence (BRET)

Adapted to high throughput.

VIPR Membrane electric potential

Fluorescence (FRET)

Adapted to high and ultra-high throughput.

Brilliant Black Membrane electric potential

Fluorescence quenching

Adapted to high and ultra-high throughput.

Table 4.5. Classic approaches for measurement of ion exchange homeostasis within the cell

4.7. Metabolism and cell respiratory activity

The most straightforward way to assess a cell’s health is by observing its metabolism and level of respiratory activity, which allows for the creation and conservation of energy in the form of ATP. In principle, many steps can


be exploited between glycolysis and ATP production, from the NADH-collecting electrons, to their ultimate capture by the dioxygen molecule. One of the first and most famous live cell assays, the MTT assay, exploits this activity (see section 2.6), The Alamar blue assay works on a similar mode, in this case measuring the reduction of resasurin to fluorescent resorufin following cellular reductase activity. Both of these approaches simultaneously use mitochondrial and cytosolic enzymatic activity. For the purist, this means that they do not measure respiratory activity in the strictest sense. Perhaps the simplest way of measuring this function is by the direct measurement of cellular oxygen incorporation by means of oxygen sensitive fluorescent indicators. Attempts in the 2000s to standardize this measurement on 96 well plates were successful, in particular with the use of water-soluble probes whose fluorescence has been quenched by oxygen [HYN 06]. This method requires the culture plates to be sealed.

The internal membrane of the mitochondrion contains complexes organized in electron transport chains that ensure ATP synthesis by means of an oxidative phosphorylation process. The energy produced by the electrons leads proton transport from one side of the internal membrane to the other, creating an electrochemical gradient. Mitochondrial membrane potential (ΔΨ) value generated by the electric potential is consequently a measure of mitochondrial function. Different cell approaches based on fluorescent and membrane-permeable cationic agents allow for the measurement of this parameter. Among these are MitoTracker Orange, DiOC6 (also used to mark the endoplasmic reticulum), tetramethylrhodamine methyl ester (TMRM), JC-1 and rhodamine 123. These markers are trapped in the active mitochondrion, being redistributed electrophoretically in the mitochondrial matrix in response to the electric potential. Furthermore, they present a red fluorescence shift through their excitation emission spectra, which can be exploited for measurement purposes [SAK 12].

Another option is to exploit the universal presence of esterases as a signal of normal metabolic activity. In this case, the indicator used is calcein-AM, described in section 4.5 in the context of cell efflux measurement. Once cleaved by the functional cellular esterases, the new-formed calcein is trapped in the cell and binds to intracellular calcium, inducing a strong fluorescence indicative of metabolic activity.

Protease activity can be exploited using the same model. This is the case of the CellTiter Fluor, a high throughput commercially developed assay that uses glycylphenylalanine-aminofluorocoumarin (GF-AFC), a peptidic

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substrate capable of crossing the plasma membrane, which, once cleaved by cell proteases, produces the fluorescent metabolite AFC.

To date, ATP determination is by far the most perfected metabolism assessment assay. ATP, whose generation and consumption must invariably be kept in balance, can be considered a key element in cellular homeostasis. ATP synthesis pathways require the coordinated interaction between multitudes of interconnected enzymes along multimolecular complexes, control of which is greatly disturbed in case of cell function alteration. Consequently, any infringement of synthesis pathways represents a critical signal of toxicity. The first studies using bioluminescence emitted by oxyluciferin in the presence of ATP were published in 1984. The typical reaction used to generate the light signal is as follows:

ATP + D-luciferin + O2 oxyluciferin + AMP + PPi + light

The reaction is catalyzed by luciferase and Mg2+. Typically, the luciferase used comes from the Photinus pyralis firefly. The chemistry above is only possible in solution, outside of the cell context. The cells must be lysed prior to measurement, the only constraint of a system that has otherwise proved to be extremely reliable and easy to implement on miniaturized and ultraminiaturized culture supports. This makes it a preferred tool for high- and ultrahigh-throughput applications routinely used by the pharmaceutics industry in particular. Various optimizations were required before finalizing the kits now available commercially (ATPlite). For example, the intracellular ATPases had to be deactivated since they act independently of the syntheses control pathways, disturbing the measurement. This was resolved by balancing the pH between the lysed solution and the solution containing the luciferase substrate. In 2007, an optimized version was developed by Promega, called ultra-glow rLuciferase (Cell Titer GLO), based on the Photuris pennsylvanica firefly with the aim of circumventing the rather short bioluminescence life span. This parameter was successfully improved to 5 h by coupling the new enzymatic version to 5′-fluoroluciferin, a luciferin-derived substrate.

As a point of comparison, the ATP detection improvements gained using luciferase for high throughput have, perhaps, allowed for a sensitivity 100-fold over the MTT assay. Suppliers assert that current systems should be capable of detecting the activity of a dozen cells in a culture well.



MTT Measurement of mitochondrial enzymatic and cytosolic activity dependent of NAD(P)H

Colorimetry As method for measuring viability, integrated in different OECD approved assays

Alamar blue Measurement of reductase activity

Fluorescence Adapted to high throughput as MTT assay

Oxygen quenching

Cell oxygen concentration

Fluorescence Adapted to 96 well plates

MitoTracker Orange, DiOC6, etc.

Indicator of mitochondrial membrane potential ΔΨ

Fluorescence Image analysis, can be adapted to 96 well plates

Calcein-AM Measurement of esterase activity

Fluorescence Adapted to 96 well plates and readers

CellTiter fluor Measurement of protease activity

Fluorescence Developed for high throughput

ATP lite, CellTiter GLO

ATP presence measurement

Bioluminescence Developed for high and ultrahigh throughput

Table 4.6. Classic approaches for measurement of cell metabolism

4.8. Genotoxicity

Aside from the massive genomic damage described in section 2.7, DNA is constantly subjected to numerous types of aggressions that touch its innermost workings. It is estimated that the DNA in each of our cells accumulates millions of lesions each day. In the main, these are base incorporation errors, adduct formation, the presence of damaged bases, together with single or double strand breaks that occur along the whole DNA backbone.

For each of these lesions, the cell possesses a series of specialized repair kits in its biochemical arsenal. There are six main kits. Single-strand lesions are eliminated by the BER kit (base excision repair). This intervenes primarily so as to oppose ROS effects derived from the environment or the respiratory metabolism. Inappropriate bases are eliminated by the combined action of a DNA glycosylase, an endonuclease and a ligase. This mechanism

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is fundamental to prevent mutations from establishing themselves in the DNA permanently.

Certain adducts together with errors in nucleotide matching are repaired by the mismatch repair (MMR) system, which with significant support from complex proteins, ensures the excision and re-synthesis of the mismatched nucleotide.

The nucleotide excision repair (NER) system engages against adducts and lesions that provoke distortions in the DNA’s helicoidal structure. This system also employs complexes whose proteins cooperate, recognize and excise incriminated regions. One form of NER even provides a system that continuously inspects the whole DNA.

The translesion synthesis (TLS) system is more innovative. It introduces a certain tolerance of errors with the rationale that it is better to risk mutation than to cause major chromosomic aberrations.

The final two reparation systems, nonhomologous end-joining (NHEJ) and homologous recombination (HR) react to double-strand breaks. Indeed, when the previous systems are powerless, single strand breaks accumulate, ultimately leading to much more problematic double-strand breaks. These breaks are known to provoke oncogenic transformations, particularly when their reparation was not performed correctly. NHEJ provides the simplest solution for resolving this problem since it requires no DNA matrix. It is, however, quite unsafe, sometimes itself generating mutations, deletions and chromosomic rearrangements. HR is the more sophisticated system. It uses exonucleases, recombinases and polymerases, relying on the information provided by the corresponding DNA matrix. Without a doubt, it is the more effective tool, though while the NHEJ system can intervene at any moment, the HR system’s action is contained to the phase M and phase S of the cell cycle, which limits its usefulness.

When taken together, these natural repair kits form an extremely competent network that ensures the maintenance of DNA identity, and as a consequence, corroborating cell homeostasis. Furthermore, these systems organize themselves both in time (according to the cell cycle stage) and in space. This system of multilevel intervention relies on protein kinases, which, through a series of phosphorylation cascades ensure enzymatic activation both at the repair site and at chromatin level to surround the lesion and thereby facilitate reparation.


Access to reliable measurement of this newly acquired knowledge would clearly increase the information level of cell assays, placing the impetus on sublethal aggressions. Other genotoxicity measurement approaches in connection with apoptosis are addressed in the following section.

4.9. Apoptosis

In general, the culture cells here in question are well equipped to repair or compensate aggressions derived from the environment. Their attributes as cancerous cells also ensure them a good capacity for survival and resistance to apoptosis. Nonetheless, when cells have exhausted all of their resources, they inevitably do lapse into apoptosis.

Within the body, apoptosis is primarily a sort of collective cellular suicide with the purpose of satisfying the cohesion of the whole. Unable to ensure its own homeostasis, the cell commits to a process of programmed death to ensure global homeostasis. This is indeed a fine lesson in altruism, which always deserves approval. This regulation mechanism is extremely widespread in the living world. Day by day, the human body maintains a constant number of cells thanks to a mechanism balancing mitosis and apoptosis involving around 10 billion cells [REN 01]!

The term “apoptosis” was introduced in 1972, though it was mentioned in earlier works, and since then, the underlying molecular mechanisms have been particularly well studied. Apoptosis can be surmised as a coordinated cell process that uses energy and initially requires activation of the proteases called caspases. These enzymes are among the key actors of apoptosis. Hundreds of their substrates are known and can be classed into two groups. The first group is made up of proteins that act on cells’ structural and morphological component such as nucleases or cytoskeletal proteins. The second group is involved in the death blow or termination of apoptosis. This operates on all cell aspects necessary for survival such as metabolism, DNA transcription regulation, growth signals, etc. Caspases are present in the cytosol in the form of precursors called pro-caspases. Several caspases are substrates of other caspases and the progression of apoptosis can be resumed as an irreversible proteolytic cascade resulting in cell death. Caspases can be classed according to their order of apparition in this chain of events. Accordingly, there are initiators (caspases 8 and 9 in mammals) and performers or executors (caspases 3 in 7 in mammals).

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It is important to note that, unlike in necrosis, which implies brutal, sudden and passive death, apoptosis takes place without any loss in membrane integrity. This difference may be illustrated by perceiving apoptosis as a series of centrifugal events spreading from the nucleus to the plasma membrane, and necrosis as a series of centripetal events, starting from the loss of membrane integrity.

The decision making leading the cell to its suicide can, however, be external or internal in origin. Once the process has begun, first there is a generalized shrinking of the cell, densification of the cytoplasm and chromatin condensation. The cell is then the subject of budding and produces fragments, known as apoptotic bodies, each carrying its own part of cytosol, organelles or nucleus fragments. They are ultimately phagocyted by macrophages or other neighboring cells.

What molecular mechanisms are at play in apoptosis? Three pathways have been identified according to the type of stimulus, each irreversibly directing the cell towards suicide.

The first pathway is known as extrinsic. This depends on extracellular factors secreted by other cells in the body, which bind to membrane receptors of the family known as death receptors. Some refer to these ligands as social signals. It is accepted that in this mode of apoptosis, the body gives the order.

The second pathway is called intrinsic or mitochondrial. It is characterized by independence with respect to the extracellular signals acting on the membrane or cytosolic receptors. This is the pathway that can be qualified as self-induced. It is accepted that in this case the cell acts for itself. The initiating center is the mitochondrion.

The third pathway is called granzyme B. It is triggered by the cytostatic T lymphocytes, which use the expression of a membrane pore, the perforin, to inject the targeted cell with granules containing granzyme proteases.

It is remarkable that all three pathways converge on the activation of one protease, the caspase 3, which proceeds to execute a program that results in DNA fragmentation, cytoskeleton dismemberment, structural protein degradation, apoptotic body formation, chromatin condensation and finally in the expression on the plasma membrane surface of signals (ligands) that are specifically recognized by the cells destined to phagocyte them.


So as to digress no further from the subject of live cell assays, only the mitochondrial pathway will be addressed here in further detail. Indeed, the models used for live cell tests are culture cells, living in forced exile, far removed from their original body. As a consequence, the extrinsic pathways and granzyme B in connection to regulations on a higher level, such as tissue or organ, are a priori inoperative, even if it can be reasonably envisaged that the paracrine death signals could still function. Accordingly, in vitro apoptosis can initially be limited to being triggered by the mitochondrial pathway, after exposure to a toxic molecule for instance.

The mitochondrial pathway is not initiated by the classic ligand/ receptor/signaling pathway that is found in the majority of cellular interactions. Instead, it responds to aggressions that escape from this action mode, such as the effect of toxins, radiations, oxidative stress, hypoxia, hypothermic problems or even viral infection. It can also be triggered in a more novel fashion by negative signals, like the absence or reduction in supply of growth factors, hormones or cytokines. These various positive or negative stimuli initially cause a pore to open that leads to transitory mitochondrial permeability. This causes rupture of the transmembrane potential and the release of a whole family of pro-apoptotic proteins from the intermembrane space to the cytosol.

The first group called apoptosome is comprised of a seven-protein multimer known as Apaf-1 that recruits the cytochrome c and activates caspase 9. Mitochondrion and cytochrome c are primarily involved in the cellular respiration function. The discovery of their role in this major apoptotic pathway, totally independent of the respiratory pathway, surprised a great many. Once more, this demonstrates the functional plurality of cellular organelles and their components.

A second set of proteins is then freed from the intermembrane mitochondrial space. These are mostly endonuclease G, AIF and CAD. These three actors intervene later on. Having penetrated the nucleus, they begin DNA fragmentation, and thereby participate in chromatin condensation. Control of these events of mitochondrial origin is achieved by a large family of regulation proteins, also mitochondrial, of which the most well-known is Bcl-2. The Bcl-2 family includes at least 25 members, whose roles, on which a great many research teams work, are complex, pro- or anti-apoptotic. Their intervention is recognized as being at the heart of the apoptotic decision process due to their close relationship with another star of cellular destiny, p53, which presents loss of function mutations in the majority of cancers.

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We conclude with the molecular details of the final phase of apoptosis execution. This happens via activation of caspase 3, which then activates CAD endonuclease together with the cytoskeletal reorganization that results in the formation of apoptotic bodies. This phase is accompanied by phospholipid asymmetry, particularly of phosphatidylserine on the external surface of the plasma membrane, a signal participating in the recognition of the apoptotic bodies by phagocytic cells.

Live cell assays describing the key steps of apoptosis are particularly informative to those interested in the fate of culture cells. DNA fragmentation is one of the most characteristic signals of the cell in apoptosis. The first step in degradation produces DNA fragments of high molecular weight of around 300 kb, which can easily be detected by conventional electrophoresis techniques, in pulsed or inversed field. These methods make use of DNA extracts precipitated using polyethylene glycol, which are then deposited on agarose or polyacrylamide gels and subjected to an electric field.

Single-cell gel electrophoresis (SCGE), more widely known by the name Comet, constitutes an extremely powerful assay to visualize the quantity of degraded DNA present in a single cell. Strictly speaking, it is not a live cell assay, since the measurement is not performed on plates of cell culture but after lysate migration on electrophoresis gel (Figure 4.4(1)). As a consequence, the Comet assay is limited in terms of industrial use by its complex procedure that requires significant expertise. It is difficult to standardize and low throughput. The approach remains informative nonetheless. The electrophoretic migration of cell DNA decomposes in the form of a comet head and a faint tail, more or less present (Figure 4.4(2)). Normal or necrotic cells show significantly nuclear and compact DNA, which remains concentrated in the head of the comet. Inversely, cells in apoptosis show a reduced head of compact DNA and a significant tail of degraded DNA. Measurement can be highly sensitive and quantitative if the appropriate analysis software is used. In neutral pH conditions, the assay can detect double breaks as well as single breaks and can even inform on repair mechanism in place.

TUNEL is the most widespread method for apoptosis assessment. It measures the DNA fragmentation level according to discontinuities (adjacent nucleotides having lost their phosphodiester bond, also known as nicks or nick-ends) present on the DNA double strand. The assay uses nucleotides (dUTP) marked with a fluorophore coupled to the activity of the terminal deoxynucleotidyl transferase (TdT) that catalyzes the addition of dUTP on the free nucleotides’ 3′-hydroxyl endings. The method covers single and double strand lesions. For detection of just single-strand lesions, an alternative would be to use the incorporation of nucleotides by DNA


polymerase or its Klenow fragments. Since the DNA fragments arise very early in apoptosis, prior to even the first morphological signs, their detection could provide information about the early stages of the process.

Figure 4.4. Comet assay: (1) principle of assay, (2) CHO cells subject (2B) or not (2A) to DNA photodegradation. Treatment with thiazole orange 8 µM, 15 min, illumination by LED 480 nm during 30 sec, NaOH alkaline lysis, fluorescent marking with YOYO 100 nM. The comet tail present in 2B reveals an electrophoretic delay due to DNA degradation (photo Sylvain Derick, LAAS/CNRS)

Morphological alteration of the cell is another characteristic criterion of apoptosis. Here, flow cytometry provides a useful choice, particularly since it allows for cells to be sorted and counted on a multiparametric basis. It is possible for example to mark the sample with DNA markers that do not pass through the plasma membrane, like propidium iodide or acridine orange, whereby the absence of marking would be a signal of plasma membrane integrity observed at least during the initial stages of apoptosis. Cytometry has the other advantage of placing apoptosis and the cell cycle stage together. In terms of disadvantages, note that the instrumentation is still onerous despite significant progress and the experimental protocol is time-consuming.


Unlike in microplate analysis, cells must be extracted in order to be guided toward the flow, a disturbing step as in most cases culture cells are adherent to their support.

The preceding approaches share the same inconvenience. The measured signals are indeed apoptosis selective, but not exclusive and confusion with necrosis, another process of cell death, is always possible. The activation of caspases, particularly of caspase 3, is a far more specific signal of apoptosis. Its protease activity can be readily detected by intramolecular FRET on the model described in section 3.1.

As mentioned in section 4.4, the execution phase of apoptosis is initiated by the presentation of a phosphatidylserine on the outside leaflet of the plasma membrane following a loss of the enzymatic activities that maintain asymmetry. Indeed, as described above, phosphatidylserine and annexin V have a strong affinity for one another. The presence of annexin V (coupled with fluorescent FITC, for example) in the membrane environment can then be interpreted as an apoptotic signal. However, we encounter the same co-localization as described in section 4.4 in the context of membrane integrity loss. Accordingly, in order to qualify apoptosis, it is necessary to complete the annexin V marking with a nucleic acid fluorescent marker that does not normally pass through the plasma membrane, such as 7-AAD. In this way, a positive annexin V and negative 7-AAD faithfully announce the apoptotic status of the analyzed cell. This approach is readily quantifiable by flow cytometry.


Comet (SCGE)

Single or double DNA strand degradation

Electrophoresis + fluorescence

Unadapted to conventional cell assay measuring tools

TUNEL Double DNA strand degradation

Fluorescence Requires low throughout image analysis

Caspase 3 Presence of activated caspase 3

Fluorescence (FRET)

Compatible with high throughput

Annexin V/ 7-AAD

Phosphatidylserine translocation on the external surface of the plasma membrane

Fluorescence by flow cytometry

Incompatible with high throughput

Table 4.7. Classic cell approaches for apoptosis measurement

5

The Replacement of Animal Testing: A Driving Force in Live

Cell Assay Development

Alternative methods to animal tests form their own category of assays. They are frequently based on cell approaches, typically with the aim of measuring toxicity, though they seldom involve the methods described in the previous chapter. These assays have a history of their own. Their singularity comes from the fact that they are essentially elaborated in opposition to animal tests, particularly those used as routine methods in the regulatory sector to evaluate genotoxicity, phototoxicity, reprotoxicity and both ocular and cutaneous irritation/corrosion. Unlike industrial applications that almost instantaneously translate acquired scientific knowledge as cell assays, regulatory applications suffer, to put it lightly, from a chronic delay in the transfer of the same knowledge. Nonetheless, in Chapter 6, we will see how the application of European Directives such as REACH or Cosmetics creates a non-negligible future market for alternative methods.

Moreover, should cell assays, or more generally, in vitro methods replace animal tests in the future, the real impact would be to replace the use of animals in the laboratory in the broadest sense. Indeed, 91.25% of animals used for experimental purposes fall outside of the regulatory context. The latest available European statistics [RAP 13] (7th commission report, 2013) indicate that the total number of animals used for experimental purposes in Europe in 2011 is evaluated at 11.5 million. Mice are the most widely used species (60.96%), followed by rats (13.96%), cold-blooded animals (12.47%), birds (5.99%) and rabbits (3.12%). Primates represent 0.05% of this number. Concerning the distribution between experiment types,



fundamental biology covers 46.1% of use, R&D in human and veterinary medicine 18.8%, production and quality control in these same sectors 10.97%. Toxicological tests and other safety assessment are only in the fourth place with 8.75% (i.e. 1 million animals), a proportion that has remained stable over several years, slightly declining since 2002.

5.1. On the pertinence of in vitro assays

As stated before, the cell constitutes the simplest living entity. As such, it should follow that representation of the cell is in itself a good appraisal of the nature of life [NUR 08]. This point of view, though substantiated by various theoreticians in biology, is not enough by itself to consider the study of the cell model as sufficiently pertinent.

Indeed, debate surrounding the place of the cell in the living world is as old as cell theory itself. It was through this debate that the cell became a science of its own, cytology, which broke away from the science of tissues, histology. Established in the 19th Century, cell theory resolved the problem of body composition [CAN 52]. All living organisms are composed of cells and the cell is held as the vital element containing the characteristics of life (Theodor Schwann, 1838). Cell theory also resolves the problem of genesis of organisms. All cells are derived from an anterior cell “Omnis cellula e cellula” (Rudolf Virchow, 1858).

Progressively, cell theory took hold of the scientific community. Indeed, it was widely accepted from the end of the 19th Century, though not without any resistance [CAN 52] such as at the Toulouse University of Medicine where Professor Edme Frédéric Tourneux refused to teach it until his death in 1922. Notwithstanding, Claude Bernard (1813–1878) affirmed as early as 1874 that “in the inner analysis of a physiological phenomenon the same point is invariably attained, we always reach the same elementary, irreducible, organized element: the cell [BER 74].” He would even describe it as the “vital atom.” He went still further, considering the living being as a city, accepting that within the individual, cells behave in the same way as in isolation, assuming their milieu would remain similar. In philosophy too, the world of cell is perceived as a society. Bergson wrote in Creative Evolution (1907) that “very probably it is not the cells that have made the individual by means of association; it is rather the individual that has made the cells by means of dissociation” [CAN 52].

The Replacement of Animal Testing 105

Clearly, the ground had already been laid to establish cell culture and, consequently, live cell assays as analysis elements and proofs of the interrelations between living organism and their environment. However, animal tests were to reign supreme, remaining the benchmark approach throughout the 20th Century, even once cell culture had been mastered in the 1950s. It would seem that the general consensus was that health risk assessment is too serious a matter to be entrusted to cells.

Ultimately, the breakthrough in live cell assays was due to society’s growing awareness of animal suffering. Indeed, this constituted the first driving force in the elaboration of in vitro methods, to this day known as alternatives. The basis of the live cell assay is then essentially elaborated as a means of opposition to animal tests.

5.2. On the pertinence of animal tests

Vivisection has been practiced since antiquity by doctors and researchers who, like Galen, used it principally for descriptive purposes. It was François Magendie (1783–1855) and moreover his pupil, Claude Bernard, who introduced the practice of animal experimentation into biological research. In epistemological terms, animal experimentation is a form of hypothetico-deductive reasoning. Bernard repudiated the structure–function relationship, for him the body was defined by the milieu intérieur, the crossroads of physiological regulations. While he was conscious of the distance separating humans from animals, Claude Bernard was convinced: “in sum, I conclude that the results of animal experimentation are not only applicable to theoretical medicine in physiological, pathological and therapeutic terms, but it is my belief that without the comparative study of animals, practical medicine can never assume the traits of a science [CLA].” On the moral question, Claude Bernard sidestepped the issue of man’s right to life and death over animals or that of pain arising from treatments or vivisection. He delivered himself “to the laws of morality and of the state” that placed no restrictions on animal experimentation. However, many were indeed shocked by his public experimentation on dogs, vivisected without anesthesia. None less so than his own daughters and wife who were so horrified and traumatized by his experiments that they paid for a dog cemetery after his death [CHA 09].

It was at this time that an antivivisection movement arose, attempting to oppose the practice on strictly moral grounds. At the end of the 19th Century, legislation began to afford animals some protection, though animal


experimentation remained legal. The procedure of anesthesia that came into practice calmed the situation for some time, though the battle between these rival camps continued throughout the 20th Century, still persisting today. For Hannah Landecker [LAN 07], the concept of life applied to research and its applications changed between the 19th and 20th Centuries, moving from the milieu intérieur (in vivo) to the milieu extérieur (in vitro), all the while retaining animals, considered as an intermediate system, at the heart of the arena.

5.3. The problem with extrapolation

In terms of measuring effects, it would seem a priori that the advantages of the animal model are clear. It integrates the action of the compound tested at body level. However, this impression is based on a belief concerning a significant functional proximity between species that are far removed from one another in phylogenetic terms [HAR 13a]. The intellectual lure of this model lies in the fact that while there are remarkable similarities between the functions of the different organs, fundamental differences are to be found between the inner functions and interrelations between these functions, if only at genetic and protein sequence level. But it is precisely at this level of subtle functions that drugs work. This is also the level where their adverse effects apply and where toxic agents exert their effect. It follows that one species does not have predictive value for another species. Today, this belief would raise a smile with the knowledge of the variable effects of drugs or xenobiotics observed even within individuals of the same species, due to genetic polymorphism or environmental factors, for instance. The prediction rate of animal models for human effects, particularly that of murine models, is observed to peak at around 60% [TRA 12]. This relatively low level above all reveals the interspecies barrier. In terms of the extrapolation to toxic effects in humans, there should be no favorable a priori toward animal tests being used alone with the pretext that animals are entire, intact organisms.

5.3.1. The interspecies barrier

Over the course of evolution, all species have developed their own adaptation mechanisms according to their own environment. Each reacts in its own manner to xenobiotic aggression, to pathologic agents and to the pathologies themselves. Typically, the metabolic pathways used by the organism for detoxification are, for the most part, native to the species. The most prominent example is that of the P450 cytochromes involved in the


metabolism and elimination of toxic compounds. Each species expresses different subtypes of P450, which consequently influence their tissue distribution, metabolization kinetics and orientation toward one metabolite or another [SMI 91]. This point is crucial in the knowledge that, in terms of drugs, it is often the metabolites themselves that exert toxic or adverse effects. Paracetamol, for instance, is well absorbed in humans but toxic in cats, which do not possess the glucuronyl-transferase required for transformation to an inactive glucuronide conjugate. Another example is that of pH balance in the digestive tract. This differs significantly in herbivores and carnivores, resulting in different absorption capacities for compounds. A further example would be plasma composition, which affects the capacity of drugs or toxic agents to bind to proteins, thereby altering their bioavailability. The results of measurements performed on animal models, even on species close to humans in phylogenetic terms, like primates, cannot be, or can seldom be, extrapolated to human beings. The interspecies barrier poses a significant problem to the reliability of animal tests.

5.3.2. The striking example of TGN1412

Concerning drugs, the most recent example is that of TGN1412. This drug is a monoclonal immunomodulator antibody tested on healthy patients through phase one of clinical trials. The product had shown no toxicity to this point through preclinical trials on animals, including on primates. Six healthy patients were treated with TGN1412 in London on March 13, 2006. All developed a severe, rapid inflammatory response associated with a massive release of proinflammatory cytokines followed by cardiogenic shock. After 12–16 h of treatment, all the patients were in a critical condition with lung infiltration and damage, disseminated intravascular coagulation and kidney problems. The patients required immediate transfer to intensive care, where two of them remained for 8 and 16 days. The inquest made it clear that the cytokine storm syndrome triggered by TGN1412 had been caused by a difference in toxic effect between two species, in this instance, between the long-tailed macaque (Macaca fascicularis) and human beings [PAL 10].

The idea of the therapeutic use of TGN1412 was based on the observation of rodents in which this type of antibody provokes a functional activation together with an increase in the number of naturally occurring regulatory T cells (Treg). TGN1412 indeed appeared to be an excellent candidate for applications in the sector of inflammatory and autoimmune pathologies. In mechanistic terms, TGN1412 is, in fact, a humanized monoclonal antibody specific to the protein CD28 (Cluster of Differentiation 28), on which it


exerts a superagonist effect. The factor CD28 operates as a costimulator of the T lymphocytes in interaction with the TCR receptor. The T lymphocyte stimulation typically requires their combined action. But TGN1412 is a superagonist of CD28. This means that it circumvents this requirement for combined action, activating T lymphocytes directly.

It would be worthwhile to briefly consider the details of the preclinical trials. These trials took place in three independent experimental phases: two in vivo studies performed on rodents and the macaque to which up to 50 mg/kg of TG1412 had been applied with no observed adverse effect (NOAEL), and one study in vitro employing human peripheral blood mononuclear cell (PBMC) culture that also gave negative results.

How can these results be explained? In the in vivo tests, the scientific investigation initially consisted of duplicating the same experiments in an independent manner. These experiments attained the same results. The various investigations performed since have demonstrated that in the murine model, it is the specific activation of Treg cells that protects against the cytokine storm [GOG 09]. This is not the case in humans. As for the primates, the CD28 gene is indeed present in the macaque genome. Its sequence had been verified prior to beginning clinical trials: 100% identical to that in humans. The interaction between TGN1412 and CD28 also showed an identical affinity between the two species. An explanation was produced in 2010. The type of T lymphocytes that react massively to TGN 1412 in humans quite simply do not express CD28 in macaques [EAS 10].

Why was it then that the in vitro assay for cytokine measurement did not predict the rapid release of TNF, interferon γ and other cytokines under the effect of TGN1412? There are numerous formats of cytokine release cell assays in use through the pharmaceutics industry and its subcontractors. All of these assays use whole blood or PBMCs derived from healthy donors. Antibodies like TGN1412 are presented in soluble phase or solid phase. For soluble-phase approaches, certain laboratories use cocultures with human umbilical vein endothelial cells (HUVEC). A recent analysis [FIN 14] indicated that unfortunately these varied approaches led to responses to TGN1412 showing wide variations in results. Nonetheless, it emerged that the PBMCs are preferable to whole blood and moreover that presentation in solid phase provides results more indicative of human data. A further study showed that a high-density PBMC preculture, unlike the low density used in the initial TGN1412 study, allows for in vitro detection of the cytokine storm. The authors concluded that in accordance with the mechanism described above, high cell density favors the maintenance of the second TCR cofactor


in tonic phase, via the multiplication of molecular exchanges between cells, thereby favoring the release of cytokines [RÖM 11].

This example is indicative of the present context. Interspecies differences invalidate animal models as tools evaluating human risk. However, cell models do not seem any more (or less) apt in performing this task. Globally, studies show that the prediction rate of cell assays spans the 50–85% bracket, according to the nature of the approach, values that are noticeably increased by the implementation of simultaneously performed assays in batteries.

5.4. Toxicological assessment of substances

Animals have been extensively used since the 1920s for research purposes. Along with a series of repeated health scandals, they have since participated in the emergence of a new science: toxicological risk assessment. Initially, this science was focused on two indicators: eye irritation standardized in 1944 in the form of the Draize test and acute toxicity derived from studies combining effect and doses on animals. This last measurement was standardized around the notorious lethal dose 50 (LD50), introduced in 1927 by the British pharmacologist John William Trevan (1887–1956), which would ultimately be accepted as an international benchmark.

At this time and through the following decades, cell methods were not yet sufficiently well understood to provide applications in health risk assessment. On the other hand, animal tests had been standardized, laboratory animals were cheap and their social and legal standing of small bearing. All of this contributed to the propagation of their use. Indeed, their use would be generalized until the 1970s in both regulatory and research applications. Generally, the number of animals sacrificed reduced considerably in the 1970s before seeing an increase at the beginning of the 1990s with the use of laboratory mice in the context of transgenic studies, particularly following the creation of knockout models in which the gene under study had been removed from the genome.

The project to ban animal trials and replace them with substitute methods can be traced back to 1959 and the publication of Bill Russel and Rex Burch’s book The Principles of Humane Experimental Techniques [RUS 59]. In this work, the authors develop their philosophical view based on the inhuman treatment forced on animals. They argue their point in terms of pain, distress, suffering and anguish. The book established the principle of the 3Rs: replacement, reduction and refinement. Replacement refers to the will to


substitute, wherever and whenever possible, animal testing (a term limited at the time to vertebrates) for inanimate systems. Reduction proposes to redefine the number of animals sacrificed or, inversely, to increase the information level while keeping a constant number of animals. Refinement encourages any changes in experimental protocols that could allow for an improvement in animal well-being through the entirety of the experimentation. The ethical principle of the 3Rs for animal well-being was initially intended for fundamental research though very soon would resonate internationally. This founding text broadly encouraged the development of new methods, particularly in the sector of health risk assessment, essentially in the form of live cell assays.

Trusts were soon made available for the development of alternative methods [STE 14]. The oldest of these, Humane Research Trust, was born in 1961. Non-governmental organizations began to be established with FRAME (1969) in the United Kingdom and CAAT (1981) in the United States. In legal terms, alternative approaches were progressively integrated to national legislations in Holland (1977), Switzerland (1981) and the United States (1985) followed by Europe (1986). Specialized scientific reviews followed along these same lines with the creation of Alternative to Laboratory Animals (ATLA) in 1973, ALTEX in 1984 and Toxicology in vitro in 1986. The first symposium on the subject, entitled “The Future of Animals, Cells, Models, and Systems in Research, Development, Education, and Testing” was held in the United States by the National Academy of Sciences (NAS) in 1975. The first worldwide symposium on alternatives was held in Baltimore (United States) in 1993. Since that date, it has been renewed every 2 or 3 years. Despite these advances, the concretization of live cell assays in the regulatory sector still leaves much to be desired. For the most part, practitioners must be satisfied by the procedural application of the 3Rs.

One example of the application of the 3Rs is provided by the evolution in LD50 evaluation protocols. Until 1989, this evaluation required the sacrifice of around 150 animals (10 females, 10 males, 7 different doses). After 1989, the number of animals was reduced by half for the same result. In the 1990s, researchers finally realized that it was not necessary to treat the animals simultaneously with all the doses, and that it was more effective to act sequentially. Indeed, a negative test at maximum dose, or a positive test at minimum dose make further testing quite unnecessary. At the same time, it was demonstrated that three rats suffice where 10 had previously been used. As a consequence, today on average only 8–12 animals are sacrificed for an LD50 on the altar of toxicological risk evaluation. What is more, now anesthesia is applied as soon as the animal suffers.


In order to limit health risks that may arise due to extrapolation problems, two strategies have led to the animal being retained as a near exclusive assessment model despite the principle of the 3Rs. The first is the addition of a parallel measurement of DL50 on a second species. The second is to satisfy the principle of precaution by determining the last no-observed-adverse-effect-level (NOAEL) in the animals, and then applying a dilution factor of x100 to determine the maximal tolerable dose in humans.

5.5. Irritation and eye corrosion: the long (ongoing) quest for an alternative to the Draize test

Much of the demand for alternative tests comes from consumers, who have become increasingly reticent to purchase products tested on animals. Consequently, the principal manufacturers of cosmetics and consumer products have had to invest heavily in alternative tests in response to this pressure. The process consists of developing different cell approaches, conducting mechanistic studies that support the credibility of these approaches, performing prevalidation studies presented to ECVAM and financing international workshops. This incontestable effort has been followed by numerous other governmental or associative actors. The first historic advance concerns the Draize test.

The following brief summary (to go into detail the subject would require a book for itself) shows that even with the overt support of the market’s major multinational actors, more than two decades had to pass before forcing the lines of the regulatory institutions concerning Draize. An ECVAM estimation counted nearly 70 alternative methods (in the broad terms this includes in silico methods, live cell assays, ex-vivo methods) proposed for the Draize issue [HUH 08]. The complexity of these approaches and the maze of validation procedures, together with the general inertia of the evaluation system, are symptomatic of a situation that, with the notable exception of genotoxicity measurement (see section 2.7) can be found in all regulatory applications targeted by in vitro methods.

The difficulty of assessing eye irritation dates back to the 1930s. In 1933, a lash and eyelash mascara was marketed under the name Lash-Lure. A scandal broke out soon after a series of severe adverse reactions, which led to several patients losing their sight. In fact, Lash-Lure contained paraphenylenediamine, an ingredient that would later be known to provoke bilateral keratitis resulting in corneal opacity. Eye irritation tests had indeed existed since the 19th Century, but none were obligatory. It is worth remembering that these same eye


irritation tests were developed based on military applications that used them to identify irritant and blinding substances. One such compound, dichloroethyl sulfide, better known as yperite or mustard gas, was first used in July 1917 by the German army near Ypres in Belgium, with both sides releasing many tons of it through 1917–1918.

In 1935, the American army recruited the toxicologist John Draize (1900–1992), in order to find solutions to the mustard gas problem. Several years later, Draize standardized an assessment test based on the injection of the assessed substance into the eye of white New Zealand rabbits. These rabbits have peculiarity of big eyes, a low price and ready availability. The test was based on six animals initially, which was later reduced to three. The animals are followed over 21 days for signs of opacity, irritation, edema, corneal redness. The test is validated by an OECD guideline. In its most recent version from 2012, the application of analgesics and anesthetics is recommended in case of suffering. If the animal is severely impaired prior to the end of the study, it is recommended to sacrifice it by euthanasia.

5.5.1. The CM test

The research for alternatives to the Draize test [HAR 10b] also enjoyed the support of biotechnology companies like Molecular Devices. Created in 1983 as a university offshoot, the company commercialized its first plate reader for high-throughput applications in 1987. In 1992, Harden McConnell, research fellow at Stanford University presented a new tool in Science, the cytosensor microphysiometer (CM) that allows for the measurement of living cell metabolism on an automated mode but with a very low throughput [MCC 92].

It was Alan Goldburg from the CAAT who piloted eye toxicity measurements on the CM device. When success came soon after, Procter & Gamble (P&G) invested the funds to establish the first industrial laboratory. The CM test is based on the detection of small changes in cell metabolism. Such changes are announced by an increase of extracellular acidic subproducts derived from the energetic metabolism. Any modification of the intracellular ATP status is signaled by a modulation in proton release measured using the LAPS technology devised by Molecular Devices and based on a potentiometric sensor piloted by LED excitation. P&G ultimately associated with Microbiological Associates to refine the protocol, develop a major database and submit the procedure for ECVAM validation. In total, there were six validation studies performed between 1991 and 1997 on


various alternatives. None found the right arguments to replace the Draize test in full.

In 2002, on the initiative of its new director Thomas Hartung, the ECVAM widened the validation of alternative tests to different approaches based on the concept of factual medicine, which is founded on evidence: retrospective meta-analysis and weight of evidence (WoE) approaches. The example of eye irritation, which was studied with great frequency through the 1990s yielding great quantities of data, is particularly well suited to retrospective meta-analysis. Following numerous discussions between the ECVAM and its partners, the validation body chose to assess the CM methodology in association with three other cell tests by retrospective analysis. This would take two and a half years, leading to a report by the ECVAM management, validated by ESAC, the consulting scientific subcommittee that indicated that the approach is reliable though only for the study of water-soluble substances. Moreover, the test’s application was limited to subcategories of compounds according to the Globally Harmonized System (GHS) of classification and labeling [SYS 13] (Table 5.1). Still no full replacement was found for the Draize test.

Another hurdle in the complex process required for even partial validation of in vitro tests is that the methods should be both commercially available and into the public domain. However, these tests were under patent to Molecular Devices, which ultimately accepted to release the manufacturing designs of the CM device into the public domain. It should be noted that due to its throughput limitations, this dispositive had been obsolete for some time on the cell assay market and was no longer commercialized! Nonetheless, it was not until July 2010 that the CM approach was taken under consideration by the OECD [OEC 10]. To this day, it has not been validated.

5.5.2. Ex vivo approaches

During this time, several new eye irritation tests were developed that are essentially at the midway point between animal and cell models. Two of these tests have been taken under consideration for OECD validation. The first is the isolated chicken eye test (ICE). In 2007, this was the subject of OECD guideline 438. The eye is taken from an animal destined for consumption purposes. This is then maintained enucleated in an isotonic controlled environment, treated with the test substance and washed. The corneal opacity is then measured. This test has received OECD approval for the evaluation of substances classed in category 1 of the GHS (Table 5.1) and


may be employed prior to the Draize test in order to reduce the number of animals used. The second, called bovine corneal opacity permeability (BCOP), uses bovine cornea along the same lines. It was also validated in 2007 by OECD guideline 437.

GHS Category Criteria

1 Serious damage/irreversible

effects to the eye

Substance provokes: (1) in at least one animal: effects on the cornea, iris, or conjunctivitis, not

reversible over 21 days (2) in at least two animals: (1) clouding of the cornea or (2) iritis after 1, 2 or

3 days

2 Eye irritation/reversible effects

2A Substance provokes on at least two of the three animals (1) clouding of the cornea (minor) and/or (2) iris irritation and/or (3) conjunctivitis redness and/or conjunctivitis edema after 1, 2 or 3 days

and reversible over 21 days

2B Within 2A, substance considered slightly irritating when aforementioned effects are reversible over 7

days

Table 5.1. GHS categories concerning eye damage and irritation

There is also another approach that makes use of a non-ocular model: the Hen’s Egg Test on the Chorio-allantoic Membrane (HET-CAM). The principle of this test is to employ the respiratory membrane present in the fertilized chicken egg, the vascularization and inflammatory response of which is close to those of rabbit conjunctive tissue. The test is yet to be validated though is already widely used in qualitative or optimization studies. The more exotic mucosal irritation test uses the land slug Arion lusitanicus, which is not protected by legislation. This test was developed in Belgium in 1999 and is based on the observation that these slugs produce mucus and lose weight when in contact with an irritant.

5.5.3. 3D culture models

The eye surface is an environment essentially comprising the cornea, the conjunctival epithelium and a subjacent stroma. This complex system is difficult to mimic or reconstitute outside of the natural context. Nonetheless,


different approaches have been successfully developed. All are based on the use of cell systems: monolayer cell models, 3D reconstituted epitheliums (an example is provided in Figure 5.1) and multicellular cornea equivalents.

One model of 3D cell culture was developed and commercialized by MatTek Corporation between 2009 and 2012. The cell line used, OCL-200, is derived from human keratinocytes taken from the prepuce of new-born babies. The culture cells are organized in the form of a stratified epithelium called EpiOcular. Cytotoxicity is measured using the MTT assay. The EpiOcular assay is used instead of the Draize test, albeit prior to the regulatory phase, by actors in the cosmetics or maintenance products industries essentially for development purposes.

The SkinEthic system from the eponymous laboratory (Nice, France) was also developed through 2006–2007. It uses a reconstituted corneal epithelium, in this case established from immortalized human cells derived from corneal epithelium mucus. The culture cells are self-organized according to a very similar structure to that of the corneal mucus in the human eye.

Both the EpiOcular and SkinEthic 3D models were offered for ECVAM validation for use according to the GHS classification. The EpiOcular model successfully met the criteria for certain applications and was the subject of a 2014 OECD prepublication that should soon lead to a guideline in due form. Two new models from Japan and South Korea are currently in development. Unlike the aforementioned models, these use normal human corneal epithelial cells isolated from remains of surgically extracted human cornea, then transplanted onto layers of nourishing cells. These models express both the biomarkers and the typical morphology of intact human corneal epithelium. Both of these models approaching the true human physiology are also undergoing prevalidation.

The detractors of 3D cornea models point out that they are fragile and do not predict the most profound effects on the stroma and endothelium or the possible inversion characteristic of irritation due to the absence of any immune or hormonal regulation.

5.5.4. Recent attempts and validations

Recently, two more live cell assays have been adopted by the OECD: the fluorescein diffusion assay published in 2012 and the short-time exposure


(STE) assay published on July 28, 2015. Their principle will be presented briefly at the end of the chapter in section 5.9. Both of these assays are limited in use to certain categories of the GHS.

Between 2007 and 2010, the ECVAM and its American counterpart, the ICCVAM, researched combinations of alternative methods, albeit without any further success. Indeed, for the time being, there is no comprehensive alternative strategy to replace the regulatory applications of the Draize test. Opponents of cell strategies in this sector often seize on classic objections to voice their criticism: even integrated in organized multilayers, culture cells lack the complexity of a living body, for example aqueous humor or lacrimal flow. Furthermore, the natural protections provided by mechanical movements (blinking eyelids) that evacuate the toxic substance are not taken into account, leading to false positives. According to official reports, cell methods successfully predict the absence of effect or severe effects, but do not (or just barely) distinguish intermediate classes of irritants (category 2, Table 5.1) according to the GHS. Moreover, in commercial terms, the reduction in cost is not abundantly clear, particularly when a combination of assays is required. As a consequence, no alternative to the Draize test has yet been validated.

5.6. Measurement alternatives for skin absorption, corrosion and irritation (2004–2010)

The replacement of animal tests used in assessing dermal absorption, corrosion and irritation more or less followed the path described above. Here, the alternative method came from models of animal or human skin derived, for the latter from surgery, or cultivated in the laboratory. Several models have proven to be as valid as the rabbit skin treated in vivo, though none have managed to replace animal tests in all regulatory applications [ESK 12]. Indeed, in this respect, the point of reference is guideline 404 that upholds the test on rabbits, but out of concerns for their “well-being” (sic), recommends the use of a tiered testing strategy integrating validated in vitro and ex vivo approaches.

Approaches known as ex vivo were developed from tissue taken from animals. The RET test for example (OECD guideline n°430) is based on the measurement of transcutaneous electric resistance using disks of skin taken from sacrificed rats. Corrosive substances produce irreversible damage to the integrity of the stratum corneum, announced by a reduction in electric resistance. The test has been validated since 2004 for distinguishing corrosive and non-corrosive substances.


5.6.1. Skin absorption: in vitro method (OECD guideline no. 428)

Strictly speaking, this is not a live cell assay as the cells extracted from excised skin are not placed in culture but used directly. In this context, absorption is understood as referring to the diffusion of a substance into and through to the deep layers of the skin, logically as far as the blood systems present in the derma. As early as 2004, the OECD noted that skin absorption measurement methods in vitro had been used for many years, with experts adjudging the volume of evaluated data sufficient to establish a guideline.

Method 428 measures the diffusion of a substance through the skin to a fluid reservoir. It is recommended for a first quantitative evaluation of cutaneous penetration, but, according to the OECD, is not necessarily suited to all situations and substance classes. It can also prove necessary to complete the study using in vivo data. The experimental system comprises a skin membrane of 2.54 cm2 placed on a medium surrounded by two chambers, one as a donor chamber for the application of the chemical substance, the other as a receptor chamber to receive the fluid for analysis. The skin extract used can be of human or animal origin, and can be viable or non-viable. Skin strata of 200- to 400-µm thickness derived from dermatome can also be used. It is recommended to maintain a temperature of 32°C and to apply a range of substance concentrations that are realistic in terms of potential human exposure.

5.6.2. Reconstituted skin models for corrosion and irritation

In terms of corrosion and irritation measurements, the closest model to physiology consists of using biological skin equivalents. The development of highly organized, differentiated, viable and standardized 3D cell systems has constituted a challenge for several decades, which only in the last decade has resulted in positive spillovers in regulatory and commercial terms. This success can also be interpreted as a major technological breakthrough in the history of live cell assays. These reconstituted skin models demonstrate that standardizable experimental conditions can be found for ensuring tissue structure formation from multilayers of differentiated cell cultures (see section 10.2). OECD guideline no. 431, initially adopted in 2004 and updated in 2014, follows the same schema to a certain extent. It combines four methods based on the four main commercially available models of human epidermis.


5.6.3. In vitro skin corrosion: human skin model test (OECD guideline no. 431)

The EpiDERM (MatTek corp.) model is composed of normal human epidermal keratinocytes (NHEK). These are placed in culture so as to obtain a 3D structure that presents significant tissue differentiation approaching that of human skin. The ultrastructure reveals the presence of keratohyalin granules, tonofilaments and desmosomes together with the various intercellular and lipid strips characteristic of the stratum corneum. EpiDERM also reproduces the multilayer organization of the skin, presenting the specific markers of the epidermis such as involucrin and profilaggrin. Moreover, the system remains active at the cellular level both in metabolic and mitotic terms. The model also reproduces the main properties of the functional barrier, characterized by the qualitative and quantitative presence of lipids and ceramides, revealed by chromatographic analysis. The model can be used in the estimation of both irritation and corrosion.

The SkinEthic reconstructed human epidermis (SkinEthic RHE) model is also based on the culture of normal human keratinocytes. The medium employed is an inert polycarbonate filter. As previously stated, reconstructed human epidermis announces the differentiation markers, such as transglutaminase 1, keratin 10 and locricine. Also present are other typical markers of the basal membrane such as type IV collagen, integrins and different laminins. Ultrastructural analysis shows epidermal arrangements together with the formation of a permeable barrier. The model can be used in the estimation of both irritation and corrosion. It is also exploited in the form of the protocol known as 42 bis, though this has not yet been the subject of an OECD guideline.

epiCS or EST-1000 (CellSystems) is also based on the culture of primary human keratinocytes. First, the cells are cultivated in a liquid medium in 0.6 cm2 inserts. Then, they are brought to the air–liquid boundary to begin differentiation. This interfacing step on the edge of the liquid compartment allows for the provision of nutriments and keratinocyte stratification. Immunodetection studies helped in verifying the presence of the main differentiation markers. Here we find, as before, the different strata of epidermis as basal membrane, keratinocytes and a stratum corneum, which is functional as a barrier. The keratinocytes proliferate, expressing, for example, cytokeratin 14 (suprabasal layers). Filaggrin, transglutaminase and involucrin are also expressed. The model can be used in the estimation of both irritation and corrosion.


EpiSkin (EpiSkin, L’Oréal) is the latest model to be recognized by the regulatory bodies. It is certainly also the best characterized model. Again, this is a three-dimensional model of reconstituted epidermis whose keratinocytes are derived from healthy donors during breast plastic surgeries. The cells are maintained for 3 days in a culture medium, then 10 days at the air–liquid boundary. After 13 days, the epidermis is multistratified, comprising the main attributes of human skin. DNA analysis by biochip revealed the expression of all of the differentiation markers mentioned above for preceding models. The overexpression of certain markers, such as keratin 1/10 and 5/14, loricrin, filaggrin, corneodesmosine or caspase 14 have been observed. Furthermore, the different classes of constitutive lipids of the human epidermis are also found in EpiSkin. Immunohistological studies have effectively confirmed the results above. The reproducibility of each sample is measured by scores given according to six precise criteria: epidermal stratification, size of intracellular spaces, adhesion of the basal lamina to the support, the quantity of granular cells, the thickness of the hair layer and the nucleation of the basal lamina. The test substance is applied topically for 3 min, 1 h or 4 h. The guiding principle is that the substances diffusing in the stratum corneum exert their toxicity on the cells in the layers below. This toxicity is assessed by viability measurement using the MTT assay. The correlation with in vivo data is excellent (93%).

Figure 5.1. Model of reconstituted epidermis. The example of Episkin commercialized by L’Oréal (drawn from L’Oréal website)


As in the case of the Draize test, the development of alternative tests that are standardizable and useful in regulatory terms, has been a laborious process, with prevalidation stages dating back to the 1990s. EpiSkin and EpiDerm are the two benchmark methods initially recognized by the OECD. Both are recommended for distinguishing between corrosive and non-corrosive substances. Only the EpiSkin model was initially validated to subcategorize chemicals. According to the OECD, postvalidation studies performed between 2012 and 2014 have improved performances (reliability, pertinence, limitations to recommended uses) of the three other models. Since then, the four models can even be used to subcategorize substances according to the GHS, with only EpiSkin not over-ranking substances from 1B to 1C (Table 5.2). A wide range of chemical classifications and substance physical states is available for assessment: non-aqueous or aqueous liquid, water-soluble or insoluble substances, semi-solids, crushed solids, waxes. Gases and aerosols are excluded.

GHS Category Criteria

1 Skin corrosion

Destruction of skin tissues (necrosis) from the epidermis to the dermis in at least one animal following exposure of <4 h

1A Corrosive reactions in at least one animal following exposure of <3 min during observation period of <1 h

1B Corrosive reactions in at least one animal following exposure between 3 min and 1 h during observation period of <14 days

1C Corrosive reactions in at least one animal following exposure between 1 h and 4 h during observation period of <14 days

2 Skin irritation 1. Score between 2, 3 and 4 for rashes and eschars or edema in at least two out of three animals at 1, 2 or 3 days after

removal of patch 2. Inflammation persisting after 14 days in at least two

animals 3. Lower scores when the responses vary significantly

between one animal and another despite indicating positive result

3 Score between 1.5 and 2.3 for rashes and eschars or edema in at least 2 out of 3 animals at 1, 2 or 3 days after removal of

patch

Table 5.2. GHS categories for skin irritation and corrosion


Again, the guidelines do not replace animal tests through all their applications, but instead integrate the cell approach in to multitier test procedures included in guideline no. 404.

5.6.4. In vitro membrane barrier test method for skin corrosion (OECD guideline 435)

In this context, corrosion is understood as any of the irreversible skin lesions visible from the epidermis through to the derma. The cell parameter associated with this event is necrosis. Method 435 was adopted in 2006. It is not a live cell assay in the sense that the test substance is applied to a sealed Corrositex membrane, composed of a biological macromolecular protein gel placed on a permeable support membrane. The protein-based product is composed of keratin, collagen or another mixture. The OECD notes that the damage observed on the membrane are seemingly due to one or more corrosion mechanisms similar to those that would act on living skin. The test is based on colorimetric measurement of the time taken by a substance to cross the biomembrane. It follows that the essential information of this test resides in the kinetic evaluation of the corrosive effect. The method was validated in conjunction with guideline 431 described above. The OECD specifies that guideline 435 allows for a more precise classification than 430 or 431 for the three categories of corrosiveness in the GHS. As a consequence, the method represents a partial replacement of animal tests, particularly in terms of the corrosive and non-corrosive effects of acidic, basic substances or their derivatives.

5.6.5. In vitro skin irritation: reconstructed human epidermis test method (OECD guideline no. 439)

Unlike corrosion, irritation only concerns reversible lesions. In animals, skin irritation tests are traditionally performed on shaved rabbits. The apparition of edema or rashes is assessed after 1 h, 1, 2 and 3 days. Since rabbit skin is particularly more sensitive to irritants than human skin, the pertinence of the animal test may be called into question.

Guideline 439 adopted in 2010 and reviewed in 2013 aims to identify the substances classified in category 2 of the GHS (Table 5.2). Guideline 439 sets out to “determine the skin irritancy of chemicals either as a stand-alone replacement test for in vivo skin irritation testing or as a partial replacement test within a tiered testing strategy”. The skin models used are the same as


those developed in guideline 431. Skin irritation is manifested by rashes and edemas. Initially, the substance penetrates the stratum corneum. Lesions of keratinocyte subjacent layers can appear. The damaged cells reject the inflammation mediators or, conversely, trigger an inflammatory cascade. An MTT reading of the viability of the cells involved in the damaged tissue provides the test’s final measurement point. The current OECD guideline is limited by the fact that it does not allow for classification in category 3 of the GHS. However, in terms of the substance types available for assessment, guideline 439 is very expansive: solids, liquids, semi-solids and waxes. Liquids can be non-aqueous or aqueous, solids water-soluble or insoluble. Gases and aerosols are not concerned.

Once again, the 439 test seems to replace the animal test but is ultimately applicable only on one subcategory of substances according to the GHS classification.

5.7. The live cell test for phototoxicity measurement (2004)

OECD guideline no. 432 concerning phototoxicity assessment was adopted on April 13, 2004. It is, in fact, the first and only validated method to replace an animal test over the whole of its applications. In this respect, it is the most successful regulatory in vitro test to date.

Phototoxicity can be defined as the toxic effect of a substance triggered or accentuated by exposure to light (UV/visible). According to the laws of photochemistry, a threshold of a certain quantum of photons must be attained before triggering a photoreaction. The value of the substance’s molar extinction/absorption coefficient determines if this presents a photoreactive potential or not. Guideline no. 101 establishes this physical parameter. Method 432 is based on the comparison of cell toxicity with and without exposure to a non-toxic dose of light emitted by a solar simulator. The idea is to select the areas of visible and UVA light. UVB rays, which are by nature cytotoxic, are not concerned with this analysis. Accordingly, the solar stimulator can be devised from xenon arcs or mercury/metal halide arcs. The emission must be filtered in both cases so as to filter wavelengths <320 nm and thereby attenuate UVB emission.

The phototoxicity assay uses murine fibroblast cells balb/c 3T3, clone 31 maintained in culture over 24 h. Eight distinct concentrations of substance are


used. After 1 h of incubation with the test substance, half of the culture wells are subjected to irradiation, typically 5 J/cm2 (1.7 mW/cm2 during 50 min), while the other half is kept in darkness. Exposure to the substance is maintained over 24 h, with toxicity revealed by the NRU assay (see section 2.6). Since the assay requires a positive control, a phototoxic chemical, chlorpromazine, must be integrated into each assay.

5.8. Assays for endocrine disruptor tracking (2009–2011)

In 1998, the OECD launched an activity of high priority in line with screening for endocrine disruptors. These are substances that exert effects on reproductive organs or function. The issue had been raised by numerous epidemiologic studies that questioned the influence of various disturbing agents present in the environment. These studies were for the most part correlative, with the causal connections between presence and effect not always well established and the role attributed to them in epidemiologic observations remaining a controversial subject both in scientific and societal terms.

Over a decade went by before the emergence of the first validated approaches. Published in 2012, the OECD’s official document No. 150, presents the approved strategy on the matter. First of all, it is accepted that certain mechanisms of action observed in rodents are not pertinent for humans. To this date, 13 cell assays providing information on interference with the endocrine system had been initially selected for further study. Of these, five essays obtained national or international recognition, two of which were accepted by the OECD. It is interesting to note the criteria used to exclude certain assays. The assay on thyroid hormone dysfunction, for instance, was eliminated as the thyroid gland’s function is considered highly complex, and the proposals insufficiently so, with the number of assays in battery required to cover the gland’s complexity beyond reason. These points are debatable. The document encouraged the use of these assays in order to accumulate information that could later prove useful for the procedure known as the weight of evidence (WoE). Indeed, this is the crux of validation strategies by regulatory bodies. At present, we will only address the two cell assays that were approved at international level.


5.8.1. Detection of estrogenic agonist-activity of chemicals (OECD guideline 455)

Guideline 455 adopted in 2009 covers two equivalent cell assays with the purpose of detecting estrogenic receptors ERα and ERβ agonists. Antagonist activity is not exploited. It has been recognized that the positive interaction of substances with ER receptors can lead to physiopathological modifications, particularly in terms of fetal development and reproductive function. The first assay, called STTA used the hERα-HeLa-9903 cell line, a subclone of the human HeLa line in which the human receptor gene ERα has been stably transfected. This assay was validated by the Chemicals Evaluation and Research Institute of Japan. The second assay, named TA ERBG1Luc, made use of the BG1-Luc-4E2 cell line, immortalized from an ovarian adenocarcinoma of human origin, and which expresses the two ER receptors endogenously. This assay was validated by the American validation bodies NICEATM and ICCVAM.

Both are categorized as reporter gene assays. The ER receptors are cytoplasmic proteins, which, when complexed with their ligand, become nuclear and bind to DNA response elements on which they act as transcription factors. ER gene activity when complexed to its ligand leads to the expression of the reporter gene, firefly luciferase for instance (see section 3.1), inducing the apparition of a specific luminescent intracellular signal. Other cell reporters like fluorescent proteins or β-galactosidase can also be considered. The fact that the metabolism capacity of the cells employed is limited can lead to false-negatives and could, in certain cases, not describe the effects of substances for which only metabolites exert an estrogenic activity. Unfortunately, this significant limitation is common to many live cell assays.

5.8.2. H295R steroidogenesis assay (OECD guideline 456)

Published in 2011, this OECD guideline uses the H295R cell line of human adrenocortical carcinoma. The OECD states that this is a “level 2 in vitro assay, providing mechanistic data to be used for screening and prioritization purposes.” The aim is to identify the substances influencing the production of two steroid hormones, 17β-estradiol and testosterone, considered as the end products of the steroidogenesis pathway. The specificities in the activity of certain enzymes (particularly cytochromes


P450) involved in steroidogenesis can vary between species, and the choice has been made for H295 human cells, the enzymatic equipment of which is faithful to the main steroid synthesis pathways in humans. Otherwise this line behaves like fetal adrenal cells, and in this role possesses the unique property of being able to produce all steroid hormones of the adult adrenal cortex and gonads. The assay allows for the measurement of positive and negative variations in testosterone or estradiol level and, above all, the impact of these variations on cell viability. This point is considered essential as it provides information on both of the main action mechanisms expected in the test substance: cytotoxicity and/or interaction with the steroidogenesis pathways.

However, there are some limits that arise due to the adrenal origin of the line, among others, its capacity to produce other types of hormones like glucocorticoids and mineralocorticoids that can influence the level of testosterone and estradiol measured. Also, the model does not allow for testing effects in connection to the hypothalamic–pituitary–gonadal axis, although this is a classic limitation and typical of all live cell assays that by themselves cannot integrate the overall organ functions. Exposure to the test substance is set to 48 h. The signals measured are cell viability and testosterone and estradiol concentration. The determination of cell viability is open. It is established by any assay that measures the percentage of viable cells, such as MTT for instance.

5.9. The four last live cell assays to be validated (2012–2015)

In the last 3 years, the OECD has validated new live cell assays with an average frequency of one guideline per year. This is very little. The four last assays to date do not concern new domains of application but complete the initial battery of available tests on eye corrosion, skin irritation and genotoxicity.

5.9.1. Eye corrosion: fluorescein leakage test method (OECD guideline 460)

The 2012 adoption of assay 460 by the OECD is limited to eye corrosion and strong eye irritants according to the GHS category 1 (Table 5.1). The method is not, then, validated as a complete means of substitution for in vivo


tests on rabbits, but is recommended for classification and labeling and as an element in tiered approaches. Validation was undertaken by the three major bodies, the ECVAM (EU), the ICCVAM (USA) and the JacVAM (Japan). The assay uses tubular epithelial cells called MDCK CB997. When they are maintained in confluent monolayer culture on an insert, they present tight junctions and desmosomes between themselves that block the way for solutes. In this way, they mimic the cell barrier that prevents exogenic substances from penetrating the corneal epithelium. It is thought that the loss of this sealing function is an early effect of eye irritation. The time of exposure to the test substance is very limited, around 1 min. The medium is then replaced by a fluorescein solution and incubated for 30 min. The corrosive effect, characterized by the loss of the tight junctions and desmosomes, is quantified by the quantity of colorant found on the other side of the insert.

5.9.2. Mammalian cell micronucleus test (OECD guideline 487)

First recognized by the OECD in 2010, it was subsequently the subject of revision in 2014. This is a genotoxicity measurement that targets aneugenic and clastogenic effects. Essentially, micronuclei can be fragments of chromosomes lacking a centromere, or even whole chromosomes that have lost the capacity to migrate from the cell equator to the poles during the anaphase. This test measures the major chromosomic lesions that occur during or following exposure to the test substance. The issue of more precise classification into clastogenic and/or aneugenic effect can be resolved by hybridization with fluorescent sensors, specific to centromeres (the FISH method presented in section 9.1) or by kinetochore marking with specific antibodies. Since the micronuclei are necessarily being transmitted from the carrier cell to the daughter cell, any genotoxic information obtained is complementary to the other tests adopted previously (see section 2.7), the chromosome aberration test in particular (OECD 473). The addition of a cytochalasin treatment stage simplifies reading of the results, as in this case daughter cells are binucleated and easy to count. Concerning compatible cell models, there is a wide range, covering numerous lines (CHO, V19, L5178Y, TK6, HT-29, CaCo2, HepaRG, HepG2), including primary cells of either human or animal origin. It is of note that the test can be performed on human lymphocytes derived from peripheral blood, and so could potentially find applications in public health, in epidemiology in particular.


5.9.3. ARE-Nrf2 luciferase test method for in vitro skin sensitization (OECD guideline no 442D)

This method, validated on February 5, 2015, is used to distinguish skin sensitizers from non-sensitizers. A skin sensitizer is a substance that provokes an allergic response after contact with the skin. It is interesting to note that for the first time the strategy deployed in the TOX21 procedure (see section 6.4) is chosen to position the test in its area of application. Indeed, guideline 442D takes care to describe the different consensual paths (a notion from adverse outcome pathway, see the TOX21 program, section 6.4) in association with skin sensitization, namely (1) an initiating event, covalent bond between electrophile chemical substances and skin protein nucleophile centers, (2) the inflammatory response and gene expressions at keratinocyte level linked to antioxidant response, (3) the activation of dendritic cells, one signature of which is the expression of chemokines and cytokines and (4) the proliferation of T. lymphocytes. The ARE-Nrf2 method covers step 2 described above using a reporter gene approach targeting the ARE response element activated by the ROS/Nrf2 pathway.

For the first time, this is an assay based on a mechanistic approach. It seems clear that from the regulatory point of view only the development of a battery of tests covering all of the various stages of the toxicity pathways described could result in the replacement of animal tests by a generalized in vitro approach. The OECD is very careful when it comes to authorizing applications. This method contributes some help in the identification of skin sensitizers, and only in the context of an integrated approach. As ever, according to the OECD, the guideline cannot be exploited alone, not for classifying substances into subcategories of the GHS, nor for predicting sensitization power in the context of safety assessment. Only a positive result could be considered to suffice for substance classification. In short, this guideline is barely exploitable as is.

5.9.4. Short-time exposure in vitro test method for identifying (1) chemicals inducing serious eye damage and (2) chemicals not requiring classification for eye irritation or serious eye damage (OECD guideline 491)

Everything is already almost explained in the title of this guideline adopted July 28, 2015! The method described, called short-time exposure (STE), is still not an alternative to the Draize test. At best it is “acceptable


[sic] as part of a multi-level assay strategy”, and only for substances entering category 1 of the GHS (Table 5.1) and the uncategorized substances. Simply put, the assay resumes the MTT protocol (see section 2.6) applied to the SIRC line, a model of fibroblastic cells of rabbit cornea called Statens Seruminstitut, cultivated in 96 well plates. The method consists of simply exposing the cells to the substance over 5 min.

We may quite legitimately wonder why it took until July 2015 to see the international regulations adopt a method using a cell line registered to the ATCC in 1957 and associated with a viability test that has been routinely applied since the 1960s.

6

Regulatory Applications and Validation

6.1. Brief history of the validation process in Europe

It should be noted from the outset that animal tests have never had to pass through validation. They were imposed as the natural means of assessment. This status has made animal tests the de facto gold standard and it is proving difficult to dislodge them from their place of prominence. Alternative tests, on the other hand, have to be proven. The validation process for alternative tests came of age at the end of the 1980s. The first symposium on the topic, organized and sponsored by the CAAT and European Research Group for Alternatives in Toxicity Testing (ERGATT), was held in Amden, Switzerland in 1990. This resulted in a first definition of validation. In 1994, the second symposium introduced the notion of prevalidation and statistical prediction models together with a governance structure. In 1995/1996, the ECVAM process was recognized by the European Union, the United States and the OECD. However, soon a chorus of dissatisfaction denounced the inertia and rigidity of the regulatory body. It was Thomas Hartung who revitalized it. His clinical pharmacology background brought new concepts, like meta-analysis, retrospective validation (based on the simple notion that tests under assessment have already proven their worth in other contexts, so why start again from zero), the modular approach, which allows us to use the data independently, leading for example to the separation of reproducibility (multilaboratory) from pertinence studies (where one laboratory suffices).

One of the main problems facing the validation strategy is that of reference, as mentioned above. This question presented itself through the



second half of the 2000s and remains unanswered. It has still not been demonstrated that the use of animal data is pertinent as a base reference. The AcuteTox program (2005–2010), financed by the European Union, has partly addressed this problem by providing researchers with a monograph of 97 substances for which the acute toxic effects (lethal and sublethal) are known in humans [SJÖ 08]. This project required the retrospective analysis of 2,800 human cases of acute intoxication (accidental ingestion, suicide, overdoses). For each monograph, the human lethal dose 50 (LD50) is available, deduced from blood doses obtain pre- and post-mortem. The data were assembled from information provided by Swedish and German poison control centers, various specialized databases, together with the 50 first monographs published by the MEIC [CLE 99], the previous European program (1989–1996). By correctly comparing cell or animal LD50 with human LD50, this database can help establish more pertinent prediction models.

6.2. The validation process of a live cell assay

The validation process for regulatory applications is established by the assessment of the various criteria [JUD 13, LEI 10] recognized by the main international validation bodies.

The test’s definition is one first key point in the validation process. The protocol must be described in the form of a standard operating procedure (SOP) that states each step in detail. Next, the retained positive and negative controls must be defined along with the nature of the end point measurement. A prediction model and a data interpretation procedure must be established. It is also required to document the mechanistic basis at work in the sequence of molecular events as far as possible. And there is still more. The expected limitations have to be analyzed, the performance of standard compounds has to be assessed and, finally, the application sector has to be defined.

Regulatory Applications and Validation 131

The reliability criterion includes the notion of reproducibility, which is understood as the capacity to obtain a statistically similar result from a SOP when the experimental act is applied on multiple occasions. This notion must cover cell batches or consumables and different experimenters in the laboratory. Then the notion of transferability applies, which evaluates reproducibility in another laboratory. Lastly, the variation of results obtained by different experimenters in different laboratories (2–4) is assessed.

The pertinence criterion describes the relationship that exists between the information produced by the test and the expected effect on human beings. Pertinence assessment is ultimately a measure of how well a test can predict the targeted biological function and define if this information level is sufficient and appropriate for the regulatory application under consideration. According to the paper by Kandarova and Letasiova, pertinence assessment considers different aspects of sensitivity (that can be defined by the percentage of correctly identified active substances), specificity (defined by the percentage of inactive substances correctly identified) together with an appreciation for predictability and precision (defined as the percentage of correct classifications compared to standard data) [KAN 11].

These validation criteria were formulated by the main actors involved: European Centre for the Validation of Alternative Methods (ECVAM), Interagency Coordinating Committee on the Validation of Alternative Methods, United States (ICCVAM), Japanese Center for the Validation of Alternative Methods (JaCVAM) and the OECD for international harmonization. Other independent expert partners like Center for Alternatives to Animal Testing (CAAT) at John Hopkins University, Centre for Documentation and Evaluation of Alternative Methods to Animal Experiments, Germany (ZEBET) and Fund for the Replacement of Animals in Medical Experiments (FRAME), UK, also participate in the many debates on the subject.

Traditionally, there are two types of validation processes: prospective and retrospective. Retrospective study uses the body of existing data on the subject to obtain the information required to qualify the relevant validation criteria. An example of this is described in section 5.5. Prospective study is more frequent. This requires the generation of new experimental data according to a predefined question framework addressing the criteria of reliability and pertinence.

There are traditionally six key steps characterizing the validation process over time: research, development, prevalidation, validation, peer review and


approval (Figure 6.1). If the time required to pass each stage is added up, the result for total duration required to acquire validation can be anything from 9 years (total of minimal time) to 23 years (total of maximal time).

6.3. Live cell assays adopted by the OECD

The great majority of regulatory tests are still performed on animal models. At present, there are only 14 live cell assays that have been adopted by the OECD. This is a very small number, and moreover, their application is typically limited to certain subcategories of substances. Note that the first four tests to be validated in 1986, all addressing genotoxicity, were all retired in 2014 due to obsolescence. Of the 14 current tests, four address genotoxicity measurement, six corrosion and irritation, two the issue of endocrine disruptors, the last two target phototoxicity and skin penetration (Table 6.1). Several ex vivo tests may be added to this list, such as the BCOP (OECD 437) and the ICE (OECD 438) tests described in section 5.5.

Figure 6.1. Steps for a prospective validation study (after [JUD 13])

How can the validation rhythm be accelerated to allow the areas of application to expand and ultimately replace animal tests entirely? According


to Thomas Hartung, it is utopian to believe that in a near future risk evaluation could depend solely on alternative tests [HAR 13]. Nonetheless, a driving force could stem from the market itself, in particular with the European regulatory constraint REACH, which alone represents a projected expense of $1.3 billion per year over 10 years for a global market evaluated at $3 billion per year.

Application OECD ref

Name Year of adoption

Described in sections:

Genotoxicity 471 Ames 1983 (1997)

2.7

473 In vitro mammalian chromosome aberration

1983 (1997)

2.7

476 In vitro Mammalian Cell Gene Mutation

1983 (1997)

2.7

487 Mammalian Cell Micronucleus

2010 (2014)

5.9

Phototoxicity 432 Phototoxicity on 3T3 NRU 2004 5.7

Skin absorption

428 Skin absorption 2004 5.6

Skin corrosion 431 Episkin, Epiderm, EST-100, Skinethics models

2004 (2013)

5.6

435 Corrositex (acellular model) 2006 5.6

Skin irritation 439 Episkin, Epiderm, Skinethic RHE models

2010 (2013)

5.6

442D ARE-Nrf2 2015 5.9

Endocrine disruptors

455 Transcriptional Activation Assay for Detection of Estrogenic Agonist-Activity

2009 5.8

456 Steroidogenesis H295R 2011 5.8

Eye corrosion 460 Fluorescein leakage 2012 5.9

491 STE – short exposure 2015 5.9

Table 6.1. Live cell assays adopted by the OECD for regulatory applications


6.4. The future of regulatory cell tests: the TOX21 and SEURAT programs

All things considered, it is clear that there is a certain resistance to the changes taking place in the validation of new regulatory methods for health risk assessment. Thomas Hartung wrote concerning this that “it is probably the only area in the life sciences where crucial approaches have not changed for 40–80 years. These approaches are apparently difficult to revise because they represent a belief system rather than a scientific approach with self-critical renewal system” [HAR 11].

The current system of assessment is quite simply unfit for purpose. Ethical problems still abound, the quality of predictions is poor, the throughput is too low, knowledge of cytotoxicity mechanistics is lacking and specific problems like immunogenesis, endocrine disruptors, neurological toxicity or others are not taken into account, or barely so [HAR 11]. The massive demand for new regulations, such as REACH or the 7th amendment of the Cosmetics Directive banning animal tests could well sound the death knell for the current assessment system.

Faced with this reality, several initiatives have been approved, the two of greatest ambition being TOX21, on the American side of the Atlantic, and SEURAT, driven by the constraint of the blanket ban on animal tests in the European cosmetics market.

6.4.1. TOX21, a new paradigm in the assessment of health and environmental risks

From the 1980s until the turn of the century, the traditional approach for assessing health risks was based on an established sequence: identifying a danger, assessing exposure to the danger, measuring toxicity (on the eye for instance) and characterizing the risk. The methods employed were for the most part animal tests, which were developed before the implementation of regulatory constraints. As previously stated, they were used de facto, thereby avoiding any validation procedure of the relevant intergovernmental bodies.

The design of health risk assessment was in need of a total reconfiguration in its heuristic approach. It was not by chance that between


2007 and 2010 numerous fresh initiatives began to emerge with this aim together with the formulation of a new paradigm via the TOX21 project. The document that would mark a turning point was published in the United States. It was produced by the Environmental Protection Agency (EPA) and the National Academy of Sciences (NAS), titled Toxicity Testing in the 21st Century: A Vision and a Strategy [NAT 07]. The text unequivocally proposes to look to the future by setting out a new vision based on in vitro tests, whose pertinence is imposed by their direct links to the workings of human biology. According to this vision, future tests will be based on the knowledge of upstream biological pathways, disturbances to normal cell responses and the events that connect them. The TOX21 project adopts the concept of toxicity pathways. It aims for the implementation of a network of integrated knowledge, targeting the biological pathways that intervene in the expression of toxicity. The hope is that such a tool will be capable of penetrating the complexity of the toxic response. To ensure this project’s success, the scientific process must start at the disturbance begun by exposure to a toxic substance (the concept of molecular initiating event…) to end up at the adverse effect on the cell, a concept that can initially be described as the loss of cell homeostasis as explored in Chapter 4. Indeed, this is no less than the beginning of a new paradigm, accompanied by new experimental approaches, with the declared ambition to use the results in the assessment of risk for health and environmental applications.

Furthermore, this program is also guided by the ambition to ultimately end all tests on animals. This would imply their total replacement by in vitro methods and indeed, as part of the impetus on the launch of TOX21, the main American risk assessment bodies (EPA, NIH, NTP) signed a memorandum in February 2008 about ending animal tests within 10 years. The consortium established by this memorandum agreed to use batteries of supplementary tests, establish effect doses and associating these means with computerized extrapolation models As there remains an immense gulf between the number of substances to be tested and actual means, both in experimental and financial terms, high throughput and miniaturization represent attractive options. From this point, cell models are certainly the model of choice, both in terms of understanding the underlying mechanisms of toxicity and as the medium for new tests proposed in risk assessment for health and environmental applications.


In scientific terms, the new TOX21 paradigm is structured around advances in molecular biology, the availability of significant databases, new modeling approaches and the emergence of computerized tools capable of integrating all of these variables. From this mine of information, the project will ultimately map the signaling pathways implied in toxicity expression [SEI 09].

Launched in 2011 in the United States as the initiative of CAAT, this project to map the cell pathways involved in chemical toxicity is indicative of the sea change in approaches to the discipline. It is financed by the NIH. The project, also known as “The Human Toxome Project,” is set to become a cornerstone of sorts in the response to the fundamental question of chemical risk assessment [BOU 15].

The idea is to no longer rely on dispersed and independent initiatives or observations for the development of new tests, but rather to describe the mechanisms and inner workings of chemicals in a systematic way. In 2012, the OECD harmonized the definition of terms concerning this process. The pathways of toxicity (PoT) correspond to the series of key events beginning at the initial chemical interaction with a (or a collection of) biomolecule(s) and ending at the final toxicity signal. The concept of mode of action goes further, integrating the toxicity pathway and its effects downstream of the cell signal, that is, at the level of the organ or individual. This concept does not require a profound understanding of the mechanisms of action, instead addressing only key events [WIL 14]. Consequently, knowledge of all the intermediary mechanistic steps is not required to advance in the validation process and find applications for a test. Of course, the more complete the understanding, the greater the pertinence in connection with assessing a health risk [HAR 11]. In parallel to this process, the Human Toxome Project has adopted the idea of standardizing approaches, centralizing and sharing results and establishing a public database to be used as reference for approaches in regulatory matters [BOU 15].

The process adopted by the OECD proposes to define a key event critical in the cell signaling pathway (defined as a sequence of biochemical events) that must be measurable and pertinent in the development of future in vitro tests. The notion of adverse outcome pathway (AOP) encompasses the preceding concept in that it describes the whole sequence of events beginning with exposure to the test substance and ending with the individual effect.


Two other concepts are emphasized by the TOX21 initiative: cross-sectional research and evidence-based toxicology [WIL 11]. The first prescribes the use of clinical and/or toxicological observations to establish a series of experiments that explain them, and then using these results to devise new in vitro methods that meet the requirement of predicting effects on the body. Evidence-based toxicology is founded on the rigorous analysis of published data, prescribing to use only those established to be scientifically valid. This is precisely the weak point of the current validation process. Concerning the identification of modes of action, new toxicology focuses on mechanistic validation [HAR 13] of the relationship between the two observed events. The Hill criteria [HIL 65], as set out in 1965, are reaffirmed: strength of association between the two events, relevance of the established proof, specificity of the physical and temporal relationship, dose–response relationship, plausibility evaluation, coherence of the proof and taking putative alternative explanations into account.

These last notions, as recalled with great force in successive workshops, are indeed fundamental. In a way, they describe how any good science is practiced. Nonetheless, it was not until the 2010s that these fundamental values would have a place in the debate. And if the causal approach so needs to be held in esteem in this new process, it is because previously it was largely absent.

It is also interesting to note that this new process in experimental toxicology, as established in 2008 and based on the description of cell mechanisms induced by chemical substances, ultimately mimics the process initiated in the pharmaceutics industry during the 1980s for new drug research. Indeed, the greater part of live cell assays used in campaigns for the identification of new compounds for therapeutic purposes make meticulous use of the mechanistic knowledge connecting therapeutic targets and cell response. However, we will see in Chapter 8 that this approach also has its limits, with various specialists recommending a return to more phenotypic observations.

An illustration of TOX21shift towards high throughput process was very recently given by a first large scale study using crossed information from10,000 substances assayed at 15 different concentrations through 30 different nuclear receptors and stress response cell assays. Combining structure and activity data allowed us to generate mode of action hypotheses and, more interestingly, to build models that perfom better than animal tests in predicting human toxicity [HUA 16].

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intracellular signaling pathways. The proposed approach is both experimental (cellular) and computerized (database analysis, modeling). According to SEURAT website, it plans (1) to identify pertinent biosensors by means of sophisticated and physiologic cell models, (2) to develop functional human systems from various cellularities (sic) using stem cell models, (3) to apply the biosensors identified in cell systems in screening programs, (4) to connect the in vitro responses to tissue dosimetry, (5) to use mechanistic knowledge to extract prediction models for in silico approaches and (6) to establish proof that the strategy correctly predicts repeated dose toxicity, at least in the case of the hepatic model.

The project is founded upon six building blocks, four of which primarily use cell models. NOTOX is dedicated to the development of in silico models derived from organotypic cultures (HepraRG, liver primary cells). The cells are studied by structural, transcriptomic, epigenomic, metabolomics, proteomic and fluxomic analysis performed, in this last case by carbon-13 isotope marking. The Scr&Tox block is focused on the upstream development of new cell assays based on pluripotent stem cells models. The role of the HemiBio block is to develop a bioreactor from sinusoidal endothelial (HSEC) and stellate (HSC) hepatic cells in culture combined with a microfluidic device. And the DETECTIVE block resumes the omics approach to identify human biosensors on cell cultures in connection with repeated dose toxicity. The last SEURAT report [GOC 15] to date expounds on the situation in each of these blocks, albeit without signaling that any decisive advances have been made.

6.5. The REACH regulatory context

Registration, Evaluation, Authorization and Restriction of Chemicals (REACH) is an European legislation promulgated with the aim of ensuring the protection of citizens and the environment against the potential risk posed by chemicals. Its implementation should ultimately allow for the free circulation of registered substances and thereby safeguard the competitiveness of economic actors. The two main innovations of REACH compared with previous legislations are its range (generalized assessment of all European produced or imported substances over 1 ton/year, bringing to more than 100,000 the number of substances for assessment) and the shift in responsibility, which had previously fallen on the public authorities. Under REACH, it is the industry that must finance assessment, manage risks and provide the information collected to both the public regulatory authorities and to users.


In terms of applicable assessment tests, their requirement varies according to the annual tonnage of product placed on the market. The tonnage is divided into four categories, 1 to 10 t, 10 to 100 t, 100 to 1,000 t and more than 1,000 t. Each of these categories, organized by annexes VII to X, respectively, has a corresponding list of toxicity tests that extend according to production volume. Whether they are in vivo or in vitro, the tests employed are for the most part derived from OECD guidelines. The promotion of alternative methods is clearly a priority, with measures adopted to limit use of in vivo, such as the obligation to inform other applicants of all results obtained on vertebrate species. The presence of in vitro test is overwhelming in the tiered test strategies, where in vitro tests are used to provide the most information possible prior to animal testing.

One significant innovation concerns the information derived from crossed-referenced data, in silico structure/function data (also called QSAR), human retrospective data and even information obtained independently from tests validated by the OECD that can henceforth be taken into account as the weight of evidence.

6.5.1. Assessment approach by weight of evidence (WoE)

The notion of weight of evidence (WoE) proposed in annex XI of REACH provides a unique opening in the regulatory assessment of chemical substances. The text states that the in vitro method must be both reliable and adapted to the assessment of the substance tested, whether it is used alone or in a battery with other approaches. Section 1.4 of the annex stipulates that all new in vitro methods are adapted as soon as their level of development has reached the international criteria necessary for acceptance in the prevalidation phase. Indeed, all the innovative methods can be accepted as soon as the applicant provides sufficient proofs validating the results in the context of REACH’s requirements. This crucial point means that REACH accepts virtually all the live cell assays described in this book in so far as the reliability studies (intra- and interlaboratory variation assessment, for instance) have been performed.

6.5.2. Up-date on the use of live cell assays under REACH

Two articles, both by Constanza Rovida and Thomas Hartung, published simultaneously on 26 August 2009, in the magazine Nature and the review ALTEX estimated the real cost of REACH in terms of substances to test,


animals to sacrifice and funds to be deployed [HAR 09, ROV 09]. The figures for the next 10 years (2010–2019) are mind-boggling: from 68,000 to 101,000 substances to test, up to 54 million vertebrate animals used and expenses evaluated at between $9.5 and $13.4 billion. This is 20 times more, in terms of animal sacrifices and six times more in terms of cost than had been anticipated at the creation of the REACH program. These articles caused significant commotion, being debated during the 7th International World Congress on Alternative Methods, which perchance took place several days later, from 30 August to 3 September 2009 in Rome. This was truly a momentous transformation as at the time the European Union used around 90,000 vertebrate animals at a cost of $60 million. The results of the study show that 90% of the cost in animal lives and 70% of the financial cost are projected to come from only one sector: that of reprotoxicity assessment. This great demand will arise from the need to assess the test substance over two generations and two species, a European particularity as American regulators consider one species sufficient. This equates to 3,200 rats per substance tested used for the purposes of assessing reprotoxicity. The authors conclude that without major investment in alternative high-throughput methods the very feasibility of REACH may be called into question. Moreover, they posit that any alternative methods validated for reprotoxicity have very little chance to see any use before 2018 and the end of the first phase of REACH.

The second report on the implementation of alternative tests in REACH was published [EUR 14] in June 2014 by the European Chemical Agency (ECHA), the body responsible for REACH file management. This report analyzes each of the 10 areas of evaluated toxicity, concerning both human health and the environment, with retrospective analysis (2011–2013) using the registered applications. Their number grew from 24,560 (28 February 2011) representing 4,599 substances, to 38,711 (1 October 2013) or 8,729 substances. Beyond the repeated assertions promoting alternative methods, what is the reality for alternative methods? What does their role actually consist of according to the REACH project? Overall, these approaches are still not very widely used.

6.5.3. Acute toxicity

There is no regulatory in vitro method positioned on this aspect. Only one live cell assay, the 3T3-NRU, is available but limited to the monitoring of non-classified chemical agents (that is, chemical agents whose oral toxicity in animals is >2 g/kg of bodyweight!). Moreover, the ECHA recommends only


using 3T3-NRU in an integrated strategy, that is, combined with other methods, or in a WoE process.

6.5.4. Skin corrosion and irritation

The rules vary according to tonnages. Applicants for low tonnages (1 to 10 t) only require live cell assays. They have not entered the deposition phase. For higher tonnages, confirmation by a test animal is required. Nonetheless, several in vitro methods can be used here in a tiered approach that can totally replace animal tests according to the WoE procedure. This strategy is only used for 12% of the total applicant number using in vitro methods.

6.5.5. Eye irritation and severe damage

The regulatory constraints are the same as above. Some ex vivo tests are used, such as BCOP and ICE. The live cell test called fluorescein leakage (OECD 460) is used, but, like the previous two, only covers extreme classification situations like severe damage and not intermediate classifications according the GHS. ECHA analysis shows that in 2010 only 3.6% of applicants have managed to present an application using exclusively in vitro data, and 7.3%, using in vitro data together with old in vivo data.

6.5.6. Skin sensitization

Animal tests like the LLNA (product is applied into the ear of a mouse followed by an intravenous injection of tritiated thymidine to measure cell proliferation in the auricular lymphatic nodules), the GMPT (intradermic injection) or the Buehler patch test (by occlusion) in guinea pigs are used. The authors of the report state that in vitro approaches are currently undergoing the validation process, although their performance in terms of reproducibility and predictivity is not yet established. In principle, one of them, the ARE-Nrf2 luciferase test (OECD 442D) validated in 2015 (see section 5.9) is now available.

6.5.7. Repeated doses (long-term effects)

No in vitro approach is employed or in development.


6.5.8. Genotoxicity

This is the verification of the mutagenic potential of the test substance. Mutagenesis is understood to be the DNA lesions that can lead to cancers or provoke damages that are transmissible between generations. Clastogenicity and aneugenicity are also assessed. For tonnages from 1 to 10t./year, only the Ames test (OECD 471) or equivalent is required. For tonnages between 10 and 100t./year, an additional cytogenetics in vitro test is required, such as the micronucleus test (OECD 487). If both tests are negative, a mammalian cell gene mutation test (OECD 476) is required. If only one of these three tests is positive, an animal mutagenicity test is obligatory. For tonnages between 100 and 1,000 t./year, the applicant must also file a proposal for a genotoxicity test study on somatic cells in vivo. Beyond 1,000 t./year, a second test on somatic cells in vivo is required. In addition, the report’s authors state that in terms of alternative tests the Comet assay (see section 4.9) was the subject of an OECD submission in April 2014. In the area of genotoxicity, WoE concerns 21.9% of applications lodged.

Genotoxicity is the area of toxicological assessment in which the most in vitro (live cell) assays have been adopted by the OECD. Nonetheless…, their validity is debatable due to their frequent oversensitivity that can result in a high incidence of false-positives, at least in comparison to the animal approaches. A false-positive does not by itself pose a problem for human health, but can lead to increased cost and increased reliance on the sacrifice of animals to confirm or overturn the result. This lack of reliability can be explained by the use of cell lines often presenting alterations in their DNA repair kits (see section 4.8) or in their capacity to metabolize the test substances [DOK 14].

6.5.9. Reproductive toxicity (reprotoxicity)

The two accepted measurement points in this area are toxicity on prenatal development and toxicity for reproduction, itself spanning at least two generations. These tests are traditionally performed on mice, rats or rabbits. No alternative test is envisaged at this time.

6.5.10. Carcinogenicity

Here, the idea is to identify carcinogens, their mode of action and their efficacy. The benchmark test is the monitoring study over 2 years performed


on rodents. Under REACH, only tonnages more than 1,000 t./year are of concern. No alternative test is envisaged at this time.

6.5.11. Bioaccumulation and toxicity in fish

According to the authors of the report, research is undergoing to develop an in vitro test for bioaccumulation based on a metabolism study. To date, the only tests recognized are those on fish. Concerning short-term toxicity, a study is required for tonnages > 10t./year. According to the classification of the substance, and in particular for substances of low water solubility, long-term tests are required beyond 100t./year. As for alternative methods, the fish embryo toxicity (FET) test (OECD 236, see section 9.3) was adopted in July 2013, although its applicability under REACH is not yet decided.

6.5.12. Long-term toxicity and reprotoxicity in birds

No alternative test is applicable to this last point, measured under REACH to tonnages >1,000 t./year.

6.6. Implementation of the 7th amendment to the Cosmetics Directive

European Regulation 1223/2009 governs the commercialization of cosmetic products. This stipulates that products available on the market must be safe to human health. The 7th amendment, implemented on 27 February 2003 following much back and forth, stipulated in its article 4a for a total ban within 10 years on the use of animal tests in the context of health risk assessment for cosmetic products. From 11 September 2004, the global ban began with ingredients or combinations of ingredients, with progressive implementation over 6 years according to the availability of validated and adopted tests. This saw the formation of major public–private partnerships, involving the European Commission and COLIPA (today called Cosmetics Europe, an association representing industrial interests), to actively research alternative methods.

On 11 March 2009, the ban on ingredients was complete, irrespective of the availability of alternative tests. Nonetheless, an exception persisted for repeated dose toxicity tests (long-term effects), reprotoxicity and toxicokinetics. The directive would definitively apply on 13 March 2013, for


these last three areas. On this date, all products whose toxic effects are not tested exclusively by alternative methods are banned for commercialization throughout the European Union. A report prepared in early 2010 by the Joint Research Center (JRC), in which many specialists and scientists participated, was presented to the European Parliament in order to postpone the deadline, arguing that it was impossible to validate alternative tests covering all the areas concerned by health risk assessment for that date. The European Commission invited the petitioners to analyze the status and outlook for alternative methods. Specialists confirmed that more than 5 to 10 years would be required for the development and validation of methods for skin sensitization and toxicokinetics, and a nondetermined time for repeated dose toxicity, carcinogenesis and reprotoxicity. A scientific document of 119 pages was subsequently published [ADL 11] on 1 May 2011. On 11 December 2012, an official European body, the Scientific Committee on Consumer Safety (SCCS) expressed the opinion that the alternative tests available in the EU are insufficient to ensure consumer safety. Different legislative options were then studied, but the European Commission decided nonetheless to maintain the deadline. After 2 years, what is the situation?

It is difficult to discern. However, the same approaches validated by the OECD, and already used in the REACH regulation, are still used with no additions. Still no developments concerning the measurement of repeated doses or reprotoxicity. Of course, the obligation of testing toxicity without relying on animals should lead to the emergence of new strategies, but the ambiguity persists as to their adoption by official bodies.

6.6.1. Acute toxicity

The conclusions are the same as for REACH, no tests validated. The 3T3-NRU test is not considered to be a reliable alternative.

6.6.2. Eye corrosion and irritation

The challenges with regards to this issue are presented in section 5.5. In addition to the classic in vivo and ex vivo tests applicable for REACH (BCOP, ICE, fluorescein diffusion), the company BASF (test provider) and the Cosmetics Europe association have performed internal studies, defining a strategy that combines the BCOP test and EpiOcular or SkinEthic tissue models used in a precise order according to the expected classification of the


test substance in the GHS categories. The authors have judged their results to be sufficiently predictive [RAM 14].

6.6.3. Skin irritation and corrosion

The various in vitro tests described in section 5.6 and routinely used through the industry are applicable here, at least for certain GHS categories.

6.6.4. Skin sensitization

Here, sensitization corresponds to an immune reaction of the skin (contact allergic reaction). While the cascade of biochemical reactions resulting in this reaction is complex, different live cell assays have been developed and adopted by the OECD or are in the process of ECVAM validation. The ARE-Nrf2 luciferase (OCDE 442D) test is now available. As a side note, another test, biochemical in this case, called direct peptide reactivity assay (DPRA), has also been recognized (OCDE 442C). Three other cell approaches that have not yet been presented are undergoing validation or prevalidation: h-CLAT (Human Cell Line Activation Test), LuSens and MUSST.

The h-CLAT test measures modifications in the expression of two surface markers, CD86 and CD54, present in the plasma membrane of the THP-1 line. The cells are treated with the compound for 24 h (exposure phase) and then marked with specific fluorescent antibodies CD86 and CD54. The fluorescence signal is read by flow cytometry. The approach has been accepted into formal OECD validation in 2014.

The MUSST test is akin to the h-CLAT test. It is based on the sensitization of the myeloid line U937 and combines CD86 expression measurement with assessment of cell viability by propidium iodide exclusion. Its progression at ECVAM is less significant.

The LuSens is a reporter gene test that uses a line of transgenic keratinocytes presenting the antioxidant ARE response element from the NADPH/quinone oxydoreductase 1 gene associated with the luciferase gene [RAM 14]. The test’s developers claim that the predictability of the three test battery LuSens, DPRA and h-CLAT can reach an equivalent level to that of the in vivo LLNA approach.


Indeed, the battery strategy (live cell tests + QSAR + biochemical tests) is also employed by technology providers such as BASF or Kao Corporation (Japan) in their in vitro service/product range for regulatory toxicity assessment in cosmetics (see their website). This type of offer attempts to fill the legal gap created by the lack of synchronization between regulatory constraint and the availability of implementation tools. It is not based on a regulatory agreement but on internal studies, or cross-reference studies, that show the soundness of the approach in terms of predictive assessment. These examples represent concrete examples of the WoE approach, although it is still too early to know exactly how the relevant regulatory bodies will accept them.

6.6.5. Genotoxicity

The issues with live cell assay reliability evoked in the REACH section can also be evoked in precisely the same terms for cosmetic assessments. Here, a combination of tests adapted to the problem, such as the micronucleus on reconstituted skin or a modification of the Comet test, are in the validation phase.

6.6.6. Skin absorption

The topical pathway is the classic exposure pathway for cosmetic products. Skin absorption measurement is regulated by the in vitro approach OECD 428 described in section 5.6.

6.7. Food safety and biocides directive

6.7.1. Food safety

In terms of food safety, the main substances to evaluate for toxicity are technological auxiliaries and flavor enhancers, collectively known as food additives [TRA 15]. These are subjected to an authorization procedure. A European Food Safety Agency (EFSA) document specifies the tests required. Concerning genotoxicity, any effect observed in vitro must be confirmed in vivo and becomes a criteria for exclusion of the substance. Assessment is performed according to a tiered approach. Stage 1 is based on four OECD guidelines, of which two are live cell assays, guidelines 471 (Ames test) and 487 (micronucleus test), and two are animal tests, guidelines 408 (oral


toxicity on rodents, doses repeated over 90 days) and 407 (oral toxicity on rodents, doses repeated over 28 days). Beyond this basic information, a positive result on either guideline 471 or 487 or on either guidelines 407 or 408 leads to stage 2, performed essentially in vivo and comprising the absorption, distribution, metabolism and excretion (ADME) study together with genotoxicity, chronic toxicity, carcinogenicity and reprotoxicity data.

In industrial terms, live cell assays are currently in development to measure not only process quality but also the presence of germs or toxins like aflatoxins or pesticides [KIN 15]. One of the only commercially available tests is DR CALUX that assesses the presence of dioxins and related species. Also for use in the environment, it will be described in section 9.3.

6.7.2. The biocides directive

The new European biocides regulation was implemented on 1 January 2013. The development of this new version includes the obligation to share animal data obtained on vertebrates. Overall, animal tests remain a large majority, with 6,000 animals divided between dogs, rabbits and rodents required for the assessment of each biocide [FER 12]. For alternative approaches, the tests adopted by the OECD apply.

Approach Cell function targeted Reading Method Pertinence

h-CLAT Expression of skin sensitization markers

Flow cytometry Undergoing validation

MUSST Expression of skin sensitization markers + cell viability

Flow cytometry Undergoing pre-validation

LuSens Anti-oxidant activity on keratinocytes

Bioluminescence (reporter gene)

Undergoing pre-validation

Table 6.2. Other assays used in the regulatory context but not adopted by the OECD

7

Cell Signaling: At the Heart of Functional Assays for

Industrial Purposes

The industrial sector is characterized by the use of sophisticated cell assays, ultraminiaturized at high to ultra-high throughput, performed on live cells. For the most part, these are functional tests, which means that they use one of the key steps in the cell response induced by modulation of the therapeutic target as the measurement signal.

In Chapter 8, we will describe the various models that apply through the industrial sector, essentially represented by the pharmaceutics industry. Initially however, we will return to the key discoveries that have punctuated the history of cell signaling exploration, in particular the two key concepts of therapeutic receptor/target [LIM 04] and second messengers and transduction, on which most live cell assays for industrial purposes are currently based. While we will limit the present treatment to discoveries in cell biology, it is worth recalling that other research sectors such as molecular biology and genomics have also contributed to current developments.

7.1. Membrane receptors, the primary target of drugs

The central question of how active substances interfere with the body was first addressed in the middle of the 19th Century through the works of Rudolf Virchow, one of the fathers of cell theory, who established that pathology can be interpreted as resulting from a dysfunction at cell level, a concept that he named cellular pathology in his eponymous work published in 1858.



But it was not before the end of the 19th Century that the connection between biological activity and molecular structure became clear. The cell then became the uncontested site of action for most compounds, with various experiments indicating that active molecules concentrate their efforts on discrete cell surfaces. In 1878, the British physiologist John Langley developed the receptive substance hypothesis, which could be involved in the effect of pilocarpine and atropine on saliva secretion. He postulated the existence of a receptive substance comprising the site of action for neuromodulators such as nicotine and atropine [BEN 00].

7.1.1. Development of the therapeutic target/receptor concept

It was Paul Ehrlich (1854–1915), the German immunologist, 1908 Nobel Prize winner and father of chemotherapy, who introduced the idea of a chemical anatomy of the cell. Ehrlich was the prime mover in the shift from a cellular position to a molecular position in matters of biology [DEB 83]. Ehrlich is an eminent representative of the German school of microbiology, known for differentiating itself from the Pasteurian school by its more biochemical vision of life. In 1897, he published his side-chain theory, elucidated with the visual metaphor of a lock and key, which he adapted from Emil Fisher’s studies of carbohydrate stereochemistry. For Ehrlich, toxins, for instance, have a strong affinity for their binding sites, or side-chains, present on the cell surface. In 1908, Ehrlich finally replaced this notion with the more general one of receptor [EHR 08]. In a borrowed style for contemporaries, he wrote: “Corpora non agunt nisi fixate”, bodies do not act unless bound [LIM 04].

The torch passed to the pharmacologists of the 1920s and 1930s, including the Scotsman Alfred Joseph Clark (1885–1941), who, even in the absence of direct demonstration, would ever maintain the existence of hormonal receptors and of their potential role as therapeutic targets. Throughout this period, the existence of drug receptors remained a pure speculation.

In the after war years, another pharmacologist, Raymond P. Ahlquist (1914–1983) attempted to publish an article in which he demonstrated that the relaxing and contracting effects of sympathomimetic amines can only be explained by the presence of two clearly distinct types of adrenergic receptors present in different tissues. He named them α and β. However, the source of his demonstration was mathematical and his manuscript, which went against the received theories of the time, was rejected in 1948 by the Journal of

Cell Signaling 151

Pharmacology and Experimental Therapeutics. Ahlquist then presented it to his friend Hamilton, a physiologist and editor of the American Journal of Physiology who ultimately decided to proceed with its publication. Nonetheless, the article went unheeded.

In 1954, Ahlquist was invited to write the chapter on adrenergic receptors in a reference book. He seized this opportunity, developing his theories on α and β receptors in detail through the chapter. The work found its way into the hands of a professor of pharmacology, Sir James Black, who, first intrigued but soon convinced, decided to teach it to his students. Before long, Black realized that if the Ahlquist’s β-receptor indeed existed, it should be implicated in the development of several coronary cardiac pathologies. He set to work researching substances possessing β-blocking properties, and discovered the compound ICI 45,520, or propranolol, which became the first β-blocker, launched in 1964 and administered with great success to patients afflicted with cardiac ischemia and arrhythmia and, later, hypertension. The propranolol revolution, besides its clear incidence in the clinical monitoring of cardiac pathologies, resulted in attention being drawn to the importance of these receptors, on which much more research was needed, initially to simply demonstrate their physical existence.

The discovery of propranolol won Black the Nobel Prize in 1988. As for Ahlquist, he never obtained international recognition. He may have assuaged his disappointment in sharing it with his contemporary Oswald Theodore Avery, who had demonstrated the hereditary character of DNA in 1944, that is, 9 years before the famous publication of Watson and Crick on the double helix. Although in this latter case, as a gesture of recognition to a misfortunate Nobel candidate, the International Astronomical Union named a lunar crater Avery, an uncommon fate.

7.1.2. Purification, sequencing and heterologous expression

It was not until the 1970s that two international teams, those of Jean-Pierre Changeux from the Collège de France in Paris and of Robert Lefkowitz from Duke University, North Carolina, imagined a first method of detection and activation for membrane receptors. This is based on the construction of radioactive ligands whose energy can be used as a marker to indirectly visualize the receptors along with they associate. Before exploiting the radioactive ligand binding affinity, receptors, to be studied, have to be isolated and separated from the thousands of cell constituents. Moreover, they need to be extracted from the plasma membrane lipid environment that


literally sticks to them. This step requires the use of detergents that have the troublesome property of denaturing proteins and making them lose all affinity for their ligand. In response to this, Lefkowitz had the idea of using digitonin, a plant-derived glycoside that had been used successfully in the purification of rhodopsin, the light receptor. Digitonin proved itself to be tremendously effective. The purification of functional adrenergic receptors was achieved in 1982 using amphibian erythrocytes with the support of affinity chromatographic matrixes ensuring the capture, and thereby, the purification of the functional receptors.

Subsequent analysis of these purified receptors proved that they were, in fact, glycoproteins. The cloning of the β2-adrenergic receptor gene was achieved in 1986, again by Lefkowitz’s team. The result amazed everyone. The sequence revealed a protein crossing the cell plasma membrane seven times. Curiously, this unique characteristic invited the comparison of the β2-adrenergic receptor with that of the light receptor discovered 3 years earlier. It appeared then that these specific receptors whose stimuli, as far removed as hormones and light, share significant sequence identities and structural homologies, which would seem to indicate that they are derived from a common ancestor. From this point, only a small step remainded before imagining that all sorts of messengers communicate between cells across structurally universal seven-transmembrane receptors, which indeed Lefkowitz’s team made in their May 1986 publication. The hypothesis was partially confirmed four months later by Ross and Ullrich who found the same serpentine structure in the β1-adrenergic receptor, then by Numa the following year who observed it on the muscarinic acetylcholine receptor. Indeed, if the cell is Loch Ness, then Nessie not only exists, but exists in many varieties whose seven protean forms emerge here and there on the plasma membrane, ensuring the intracellular transfer of information. 7.1.3. The therapeutic importance of seven transmembrane domain receptors

In 2011, the family of seven domain receptors, also known as G protein-coupled receptors (GPCRs) alone represented 82 established therapeutic targets (on which at least one drug binds) or 17% of the total 435 identified targets [RAS 11]. What is more, seven transmembrane domain receptors represent around 36% of the action sites for all existing drugs. Six (or 30%) of the 20 best-selling drugs, Plavix (clopidogrel, Sanofi/BMS), Seroquel (quetiapine, AstraZeneca), Zyprexa (olanzapine, Lilly), Singulair, (montelukast, Merck),

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Abilify (aripiprazole, BMS) and Diovan (valsartan, Novartis) target seven domain receptors.

Today, stable cell lines expressing one of the hundreds of receptors of this family, all considered as potential targets, are used on a daily basis in high-throughput screening campaigns. Such are the stakes in this type of cell-based assay for the pharmaceutical industry.

Before exploring the jungle of live cell assays associated with these therapeutic targets, we will focus for a moment on the discovery of the intracellular signals measured on the implementation of these complex approaches. Second messenger and signal transduction are the foremost of these concepts.

7.2. Second messenger, base unit of the functional live cell assay

The second messenger signals the initiation of the cellular response following activation of therapeutic targets of receptor family. As such, it represents the first functional intracellular event. Its proximity to the target allows for the establishment of a causal link between activation of the receptor and the cell response. Numerous live cell assays exploit this connection to demonstrate the functionality of a drug candidate. Up to the 1950s, the information transfer from the exterior to the intracellular compartment posed a major conceptual problem, which was not helped by the uncertainty surrounding receptors. The breakthrough was made following a series of elegant studies at a time when the functional unity of the cell could not be used experimentally.

7.2.1. The second messenger concept

It was Earl Wilbur Sutherland (1915–1974), a young doctor recently demobilized from the Medical Corps, who took the first steps toward the discovery of second messengers in 1947. He had earned his medical diploma just as Pearl Harbor was attacked. On mobilization, he enlisted as a field surgeon in the European theater. At the end of the war, he directed his attention to the study of the mode of action of hormones. Along with his colleague Theodore Rall, he was interested in the cellular interaction of two factors, adrenaline and glucagon. On 5 November 1955, for the first time, Rall observed the action of adrenaline on an intracellular enzyme, phosphorylase. Many years later, Rall told how Sutherland, despite his


typically cool demeanor, was in raptures at this result, which for the first time opened a pathway to the well-reasoned research of the cellular compartments implicated in the hormonal effect. The excitation continued to the weekend, which the two men had planned to spend together with both of their families, which duly transformed into an interminable scientific discussion.

By fractioning the different cell compartments, the two researchers soon demonstrated that the adrenergic effect exerted on the cytoplasm. A young Belgian researcher named Jacques Berthet, who had spent 4 years developing a technique of cell fractioning by differential centrifugation, freshly arrived at the laboratory and not overly convinced by Rall’s method, placed a new protocol in plain view on his desk. Rall had it performed and lost all of the adrenergic activity that he had worked so hard to obtain. But Berthet would not yield. He was convinced by his protocol. If the activity was lost, then it had to be elsewhere. So he offered to recuperate it, testing the membrane fraction remaining at the bottom of the test tube. The activity was found there, intact.

Clearly, in one way or another, adrenergic activity stuck to the environment of the receptors, provided that they indeed exist as indicated by the contemporary works of Ahlquist. In which case, how could information be transmitted between the membrane and cytoplasm containing the phosphorylase? Sutherland then envisioned producing the molecule or molecules responsible for the intracellular information transfer using the plasma membrane then incubating the product obtained with an independent cytoplasmic preparation containing phosphorylase. Hope resided in the fact that the compound responsible for the information transfer is thermostable, since the protocol included a denaturation stage in boiling water. The result was clear. The activity was found, and again intact.

The scenario was that of a membrane hormonal action followed by the liberation into the cytoplasm of a thermostable factor capable of itself activating the cytoplasmic phosphorylase. For the time, it was a conceptual watershed.

The year was 1956. The unknown factor was only present in a very small quantity. It would prove to be extremely difficult to identify. It seemed to be part of the nucleotide family but corresponded to none of them. The mystery persisted, so Sutherland decided to write to Leon Heppel of the NIH who had developed nucleotide identification methods. He soon received the enzyme that he had requested from Heppel, who happened not to have the tidiest desk around, with letters from colleagues tending to pile on it. That Saturday,

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Heppel had set aside time for a sorting session, when he noticed two letters both atop adjacent piles. Mechanically, he proceeded to read them in tandem. One was from David Lipkin, a friend and former colleague at Washington University, who described an experiment in which ATP, following treatment with a barium hydroxide solution, had been transformed to an unusual nucleotide, adenosine 3′,5′ monophosphate or cyclic AMP. The other letter was that of Sutherland. In a flash of inspiration, all seemed clear. What if the two men were working on the same molecule? He immediately put them in contact with each other [PAS 71].

Lipkin’s cyclic AMP was structurally identical to the natural nucleotide produced under the effect of adrenalin. Theodore Posternak then synthetized dibutyryl cyclic AMP, an analogue capable of traversing the plasma membrane, which was successfully used to mimic the hormonal effect. Other works followed on from these, demonstrating that cyclic AMP intervenes in the cell action mechanism of various hormones, including glucagon, corticotrophin (ACTH) and the antidiuretic hormone (ADH). Progressively, the nucleotide acquired the stature of universal second messenger. But what was the mechanism that produced it?

7.2.2. Adenylyl cyclase and phosphodiesterase regulate the concentration of cyclic AMP

It was again Sutherland and his team who identified the system that produces cyclic AMP. It is an enzyme, adenylyl cyclase, which is similarly stuck to the plasma membrane. However, it was this property that led Sutherland to an incorrect model.

Today, it is known that adenylyl cyclase forms a family of enzymes whose structure reveals a short cytosolic segment, six transmembrane segments (M1), a long cytosolic domain (C1), a second set of six transmembrane segments (M2), followed by another long terminal cytosolic domain (C2). While this type of structure with 12 transmembrane domains can be indicative of transporter activities, no function would be detected that corresponds to this activity. From a biochemical point of view, the adenylyl cyclase catalyzes the intramolecular cyclization of ATP at the level of the 5′-adenyl portion of the molecule, giving birth to both 3′,5′-cyclic AMP and two inorganic pyrophosphates. In fact, the energy required to activate the enzyme favors the production of ATP from cyclic AMP instead of the other way round as could well be believed. In reality, it is the presence of hydrolases in the immediate vicinity that tip the balance of the reaction toward the production of cyclic AMP by eliminating pyrophosphates that may “stay”, “hang around” or “dawdle” around the enzyme.


Cyclic AMP produced by the cell is rapidly destroyed as a result of the phosphodiesterase activity which transforms the cyclic nucleotide into 5′-AMP, an inactive compound. Of course, the action of cyclase and phosphodiesterase is strictly regulated by the cell, but these mechanisms were still highly confused through the 1970s, with the lability of cyclic AMP significantly slowing the speed of discovery. How could this system of regulation be shifted in favor of cyclase? The solution would come from afar, in terms of both time and space.

In 1973, the Makandi (Coleus forskohlii) was the subject of an organized collection in the region of Dehradun (North India) [BHA 77] by a German pharmaceutics company involved in ayurvedism and traditional pharmacopoeia. Ayurveda (the Sanskrit word signifying life-knowledge) is an Indian traditional system of medicine based, in large part, on a rational process combining diagnosis, prognostic and therapeutic indication. According to ancient Sanskrit texts, the roots of makandi, a distant cousin of mint, were prescribed by ayurvedic doctors for the treatment of various ailments, including heart illnesses, intestinal spasms, insomnia and painful urination. The plant was described in the mid-18th Century by Pehr Forsskal, a Danish botanist and physician, who would later be made famous by his account of his role in a particularly chaotic scientific expedition [HAN 02] to Arabia in 1761.

In 1977, a heterotricyclic diterpene called forskolin was purified from makandi roots. It was soon demonstrated that this active substance inhibits arterial pressure and exerts a strong positive inotropic cardiac effect. Two Americans, Kenneth Seamon and John Daly, soon showed that forskolin’s site of action is adenylyl cyclase. Before long, this universal stimulator had become a tool of choice in the understanding of cyclic AMP action.

7.3. The concept of cell transduction

Two fundamental questions remained unresolved. Above all, how did cyclic AMP exert its effect? Next, more prosaically, can the synthesis and degradation kinetics of intracellular cyclic AMP explain fast cell effects, such as those observed in the case of adrenaline on myocardial contraction?

Yes was the answer of Sutherland and his colleagues to the second question. To demonstrate this they showed that the increase of cyclic AMP induced by adrenaline can be revealed after only 3 s of incubation. However, the first question was another matter.

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7.3.1. The protein kinase A, the (near) universal target of cyclic AMP

Here Edwin Krebs (1918–2009) enters the fray. Like his compatriot Earl Sutherland, whom as a young student he briefly encountered on the University of Washington tennis courts, Edwin had been destined for a career in medicine. According to his autobiography, the young Ed showed no real interest for life sciences at a young age, only recalling his childhood memories of fish in the family aquarium, watching them, and feeding them when he was told to.

In 1944, Ed was recruited by the Navy and sent to the Pacific as a doctor. On demobilization, he accepted a post as biochemistry professor in Seattle, a town that had left its impression on him during a port call. In 1953, he was joined by Edmond Fischer, a young Swiss researcher (born in 1920). Seemingly destined to follow music, Fischer had enrolled in the Geneva Conservatory where he followed the piano lessons of Johnny Aubert, whose interpretation of Beethoven’s 5th concerto had so overwhelmed him. For a time he hesitated, considering a career as soloist, but ultimately opted for biochemistry. In 1955, the two Eds demonstrated the biological importance of the phosphorylation by protein kinase that favors the transfer of an ATP phosphate to proteins. They soon intended to demonstrate the role of cyclic AMP on kinase activity, but this would have to wait until 1968 when Donald Walsh and John Perkins, two researchers at the laboratory, purified the kinase activated by cyclic AMP [KRE 93, KRE 98].

The understanding of intracellular signalization had taken a substantial step. The protein kinase A (PKA) is widespread through the body, catalyzing the phosphorylation of numerous proteins. The stage was set for a new star. It soon became the most famous kinase, the first whose gene was sequenced (1981), then the first whose structure was revealed at an atomic level by crystallography (1991).

The structure and function of PKA were unveiled in 1971 in tandem with the action mechanism of cyclic AMP. The enzyme is constituted of functional catalytic (C) and regulatory (R) subunits. The cyclic AMP bond to the R subunits dissociates them from the C subunits, thereby freeing enzymatic activity.

The Nobel Prize in Physiology or Medicine was awarded to Earl Sutherland on 11 December 1971 for discovering the role of cyclic AMP in the transmission of the hormonal message. The same Nobel Prize would be


awarded in 1992 to Edmond Fischer and Edwin Krebs for their discoveries concerning protein phosphorylation as a biological regulation mechanism.

Finally, in 1998, it was demonstrated that the protein kinase A is not the only intracellular target of cyclic AMP and that the latter regulates another protein known as exchange protein activated by cAMP (EPAC).

7.3.2. Decrypting the transduction pathways

Naturally, it fell to Earl Sutherland to synthesize these various advances. In 1967, he proposed a model explaining how the hormonal message results in intracellular production of cyclic AMP. Sutherland thought that adenylyl cyclase was a transmembrane protein composed of two subunits, one regulatory (the hormone receptor) in contact with the exterior, and one catalytic (the cyclic AMP synthesis enzyme), in contact with the cytoplasm [SUT 66]. This model was particularly attractive in its simplicity, still more so as it provided a fine example of the allosteric mechanism that had just been demonstrated by Jacob, Monod and Changeux.

This did not account for the fact that nature does not always choose to take shortcuts. From 1969, Martin Rodbell (1925–1998) and his associate Lutz Birnbaumer (born in 1939) began to seriously doubt Sutherland’s minimalist hypothesis of the two subunits enzyme. Effectively, they had proof that the adenylyl cyclase of adipocytes could be stimulated simultaneously by at least six receptors and struggled to envisage how all of these elements could live together. In addition, they noticed that when these same cells were treated with the various hormones at maximum effective concentration, they had no additional effect on cyclic AMP production, a result also obtained around the same time by the Bär and Hechter team on different cell models with different hormones. Rodbell was increasingly convinced that the truth was elsewhere. Again and again, he mulled the question in new ways. Birnbaumer later told of how Rodbell arrived almost every day with a new model under his arm, and how the foreign travels of the globetrotting Rodbell were appreciated as they left his team the time to test the various models [BIR 99].

Around 1968 and 1969, Sutherland’s model was widely recognized, but Rodbell persisted. During a congress of the NIH organized in November 1969 in honor of Sutherland, Rodbell spoke with Oscar Hechter, a biochemist specializing in steroid hormones, whose theories had greatly influenced Sutherland in the development of his second messenger concept. Through

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long and engaging discussions in a bar in Washington D.C., Hechter initiated Rodbell to certain aspects of cybernetic theory and the analogies that he found there with the supposed mechanisms of the hormonal message. Rodbell found satisfactory expression for his model in the abstract language of cybernetics. The receptor could be identified with the discriminator and the cyclase with the amplifier. There remained the missing link that the father of cybernetic theory, Norbert Wiener, had called the transducer. Rodbell seized on this and spread it without hesitation. The addition of the term “transduction” to molecular vocabulary would coin a name and thereby typify the lacking entity in the intracellular transmission of the hormonal message. From then on, Rodbell would affirm continuously that the theory of information described the organization of biological systems [ROD 95]. He set himself the unique goal of identifying the cellular transducer.

He soon demonstrated that ATP, already in use for cyclic AMP production, is also necessary and sufficient to ensure the fast and, above all, reversible bonding of a hormone, in this instance glucagon, on its receptor. Consequently, the receptor environment should contain a regulatory entity capable of regulating its activation. Rodbell wanted to be certain of this as he understood that commercial ATP preparations were by no means pure, containing non-negligible quantities of other nucleotides such as GTP, GDP or ITP. More trial and error followed. In January 1970, Rodbell discovered that it was indeed GTP contaminating the ATP preparation that was behind the observed effect. These studies culminated in a series of five articles published in the Journal of Biological Chemistry on 25 March 1971.

In hindsight, it appears that it was also this ATP contamination with GTP that allowed Sutherland to discover cyclic AMP. Indeed, Sutherland had used the ATP as adenylyl cyclase substrate without suspecting that his preparation contained the little GTP required for the upstream activation of the transducer, of which he was totally unaware.

Speaking in Stockholm on 11 December 1971, Earl Sutherland, visibly trapped in his model, made no mention of Rodbell’s works.

In 1974, Rodbell’s team showed that without GTP, glucagon exerts no effect on adenylyl cyclase activity. In 1975, the same team developed a non hydrosable analogue of GTP, Gpp(NH)p. They had shown within a few months that the analogue stimulates adenylyl cyclase activity. It began to be known as a universal hormone substitute. Yoran Solomon, from Rodbell’s


team, brought it to a new dimension with the addition of a radioelement, phosphorus-32, to the analogue in order to locate its action site. As suspected, GTP was indeed found to be associated with the plasma membrane. In 1976, Orly and Schram demonstrated the physical separation of the receptor and adenylyl cyclase. In the following years, Cassel, Levkovitz and Selinger showed that the transducer not only bonds the nucleotide, but in fact functions as a GTPase, capable of catalyzing GTP hydrolysis to GDP. Clearly this is indeed the missing link, but 10 years have gone by since Rodbell emitted the transducer hypothesis although its precise nature remained elusive. Despairing of his cause, he called his unidentified transducer N for nucleotide regulatory protein.

From the 1980s, Rodbell spent increasingly long periods abroad. He became a philosopher of sorts in the cell world, using, through his various conferences, the language of cell communication to describe the society of men. He came to be seen as an indefatigable researcher, a poet, a specialist in French existential literature, in particular of Gide, and a humanist biologist, through his later years instigating a vast program of humanitarian action in Ethiopia.

7.3.4. G proteins, the missing link in cell transduction

The mystery surrounding Rodbell’s notorious unfindable transducer drew the interest of another American specialist of adenylyl cyclase, Alfred Gilman (1941–2015), who in 1975 was joined by Elliot Ross, a biochemist specialized in membranes. Ross and Gilman hoped to obtain two preparations, one of which contained the cyclase, the other the receptor, then to artificially reconstitute the whole in order to isolate the element, or elements, that supposedly acted as an intermediary. They were assisted by Bourne and Tompkins, who in 1973, using a cell line of S49 lymphoma, isolated a genetic variant insensible to cyclic AMP though in possession of the β-adrenergic receptor. They named their presumed variant adenylyl cyclase deficient (cyc−).

In 1977, Ross and Gilman [GIL 95] began their experiment with Bourne and Tompkins’s line but found that their negative control had turned into a positive control. The S49 cyc− indeed possessed the cyclase, but in a deactivated form. The intermediate transducer compound was actually in the control sample. At the same time, using forskolin, Pfeuffer’s team found a

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protein of 42 kDA that selectively associates with GTP. In 1980, Allyn Howlett, from Gilman’s team showed that the Gpp(NH)p leads to alterations of Pfeuffer’s protein, compatible with a dissociation phenomenon in several subunits. The hardest part was the purification that Gilman entrusted to Paul Sterweis and John Northup. Indeed, they purified a protein called Gs (s for stimulatory) presenting two α and β subunits. The γ subunit was not discovered until 1984. That same year, Citri and Schramm demonstrated that the G protein is in fact the true functional carrier of the transduction information.

A second transduction pathway controlled by the G proteins was subsequently discovered. This was the phospholipase PKC C/DAG + IP3/ calcium/PKC cascade that is described below.

7.3.5. Connection between transduction and genic expression

The final connection between transduction and cell response would be established in 1986 thanks to the work of both Robert Weinberg’s team and Howard Goodman’s team, who independently demonstrated that two signaling pathways, that of cyclic AMP/PKA and of calcium/PKC, culminate in the activation of genes via a protein, cyclic AMP response element B (CREB), phosphorylated by the activated protein kinases A and C. A page in cell biology had been turned. The overall picture of signaling pathways associated with seven domain receptors was, of course, going to become more dense and diverse, but the outline had been drawn.

This discovery would remain one of the hardest won in the history of modern biology. Its consequences in terms of applications were important, with its success rewarded in 1994 by a new Nobel Prize attributed to Rodbell and Gilman.

Since then, most functional cell assays that have been developed, particularly in the sector of new drug research, were based on these fundamental discoveries. At the end of the 1990s, the measurement of cyclic AMP production became the preeminent functional live cell assay. Radioactive methods were progressively replaced by approaches using image analysis on living cells [FUR 96] (Figure 7.1) soon combined with fluorescence in the context of high information content screening (HCS) or replaced by fluorescence or bioluminescence in the context of high-throughput screening (HTS) programs.


Figure 7.1. First cyclic AMP functional live cell assay developed in 1993 at INSERM (U 361, Paris). The results show the kinetic effect (injection at T = 0) of cyclic AMP (a, T = -30 min; b, T = 0 ; c, T = 30 min; d, T = 60 min; e, T = 90 min; f, T = 120 min) based on morphological change analysis (phenotypic approach). Model of primary human ovarian cells derived from cumulus oophorus. Image taken from [FUR 96] (photo C. Furger and M. Pouchelet)

7.4. The transduction pathways used in the context of live cell assays

Before turning to address live cell assays commercialized for high-throughput applications, it is worth noting the current state of knowledge concerning the main transduction pathways. Today, known signaling networks are extremely complex. They are also interconnected in a most impressive manner. A simple Google image search for the keywords “Pathways of Intracellular Signal Transduction” gives page after page an idea of the mass of pathways identified and decrypted. A brief glimpse is provided in Figure 7.2.

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We will limit the present treatment to canonical pathways applied to the most widespread cell assays applicable to seven domain receptors.

7.4.1. First level of regulation – activation of the transduction pathway

In terms of seven domain receptors, the transduction system comprises five basic elements: (1) the receptor/ligand couple, (2) the heterotrimeric G protein, (3) the system of second messenger synthesis and degradation, (4) the protein kinase activated by the second messenger and (5) the protein phosphorylated by the protein kinase that triggers the cell response (gene expression, cytoskeleton mobilization). All of these elements are exploited in the context of high-throughput cell assays.

Succinctly, the G protein comprises three subunits (α, β and γ). It possesses GTPase activity on the α subunit. In its form activated by the ligand-receptor couple, it exchanges GDP for GTP. Gα dissociates itself from Gβγ and, according to the function it is carrying, activates or inhibits the second messenger production enzyme. Gα form a family of proteins classed according to the transduction pathway that they govern. Gαs and Gαi/o respectively activate or inhibit adenylyl cyclase and thereby the cyclic AMP pathway.

The third sub-family of Gα proteins, called Gαq/11 actually activate the pathway known as PKC, independently of cyclic AMP, as revealed [BER 84] in the early 1980s. In fact, Gαq/11 activates Cβ phospholipase that catalyzes the formation of diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). On liberation, the latter associates with a specific receptor that directs Ca2+

efflux from the endoplasmic reticulum to the cytosol. As for the DAG, it activates the protein kinase C (PKC). A schematic description of this transduction pathway and the types of cell assay available for measuring its activation is presented in Figure 8.1 of Chapter 8.

A fourth subfamily of Gα proteins was subsequently discovered, known as Gα12/13, which activates the RhoGEF factor associated with the mobilization of the RhoA small G protein.


Figure 7.2. Screen capture taken from Google Images giving a brief overview of currently available intracellular transduction pathways

Overall, second messengers, such as cyclic AMP or DAG, in turn activate a protein kinase (in this case A or C), which by means of phophorylations on a (or on several) transcription factor(s), typically promote genic expression. However, this is not always required and cell response can also result in more direct and rapid action by the phosphorylation of functional proteins like phospholamban for instance and calcium channels in the case of smooth muscle relaxation.

7.4.2. Second level of regulation – desensitization and recycling

When a ligand remains on its interaction site, this triggers the receptor’s phosphorylation by kinases called GRKs. The phosphorylated receptor is then recognized by (it is said to recruit) the β-arrestins, which results in the blocking of the transduction pathways, the desensitization of the receptor and ultimately in its recycling by vesicular internalization (endosomes). The interest of β-arrestins for live cell assays is in the universality of their interactions with all the seven domain receptors, which makes them an instrument of choice for functional approaches. For the sake of completeness,

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it should be noted that β-arrestins can also play the role of transduction proteins. It was long believed that they are only involved in receptor inactivation, but today it is known that they also intervene in the G protein-independent transduction. They are also found to be involved in the activation of the major ERK1/2 pathway, whether it is associated with seven domain receptors or with receptor tyrosine kinases (RTKs), which are addressed below.

7.4.3. Third level of regulation – allosteric modulation

Classic tests for ligand binding detection have long been performed by competition with radioactive ligands. This approach had the advantage of being well adapted to miniaturization and throughput. However, its reliability was based on the concept of a single regulation site. Today, it is well known that things are by no means so straightforward.

Seven domain receptors are allosteric proteins and not simple switches that work on an on/off mode. Consequently, in addition to the endogenic ligand interaction site, known here as the orthosteric site, there is a distinct regulation site, known as the allosteric site, capable of binding other modulators in a context of cooperation between sites. It is the nature of such allosteric disturbances to bias the interpretation of ligand presence detected by competition at the orthosteric site. In fact, it is known today that these cooperativities attest to multiple (called iso-energetic) receptor conformations that modify the interaction with intracellular transduction pathways, resulting in specific responses. In the living cell, these varied conformations are interchangeable and coexist, forming a population of functionally different receptors. By selectively binding to certain configurations, a ligand can alter the relative proportion of this population.

We should also note that a compound selected on the orthosteric site risks, if the endogenic ligand is shared with other receptor subtypes, as is often the case, being not very selective of the considered subtype. However, the allosteric site, removed from this role, will not have undergone the same selection pressure and will provide superior structural specificity.

8

Applications in New Drug Discovery

New drug discovery constitutes the leading industrial market for cell assays. Despite the confidentiality surrounding research, today we have access to numerous aspects of the issue together with a certain perspective on the nature of programs launched by the pharmaceutical industry. Live cell assays are deployed routinely and often in a central role at different steps in research such as target identification, their validation, high-throughput screening, lead compound optimization, etc. They are also increasingly found in toxicology and in pharmacokinetics (ADME).

8.1. High-throughput screening, the leading market sector for cell assays

The aim of screening is to identify biologically active molecules and potential drug candidates. This stage of research starts with the availability of a library of molecules, typically in the hands of a manufacturer. These collections are frequently impressive, counting several million different chemical species in certain big pharma groups.

For a very long time, the selection of new molecular species for therapeutic purposes was principally made from isolated tissues whose acquisition and handling remained problematic and whose interpretation was made difficult by a lack of reproducibility and reliability. The screening process undertaken in the 1970s, initially using biochemical tests, aimed to introduce a higher throughput in a more standardized context. Screening came of age at the end of the 1980s with the arrival of combinatorial



chemistry technologies allowing for the fast and inexpensive production of structurally different new compounds. At the same time, the advent of genomics and the discovery that the genome possesses vast classes of potential therapeutic targets, such as seven transmembrane domain receptors, for example, was a source of optimism and enthusiasm, creating a fertile terrain for investment in miniaturization, process automatization and the development of new test methodologies.

Before long, high-throughput supports had become a requirement, particularly 384 well plates, which help minimize volume and thereby the real cost of trials. Today, industry leaders can routinely screen more than 100,000 molecules each day and launch up to 50 screening campaigns per year. These campaigns have a very low return, as expected, since the process consists in testing molecular diversity. Consequently, the chance of extracting a serious drug candidate is estimated at around one in 10,000.

The process of high-throughput screening is frequently dogged by criticism. One of the main issues is with its success rate, particularly the number of drugs derived from screening to be successfully commercialized. This is often adjudged to be very low. Detractors accuse systematic screening of the use of chemical libraries with low diversity or, conversely, of using molecules of any type, and essentially of not proceeding in a rational fashion, or at least of proceeding tangentially to scientific dogma, which prescribes following a rational process, step by step, according to hypotheses or knowledge acquired. The actors of the domain respond in unison [MAC 11] that screening is not irrational but intellectually neutral, and that the quality of molecular libraries, while initially low, has significantly improved, incorporating new preference criteria such as log P (hydrophobicity level) and molecular mass decrease, or even taking more rational structural motifs into account, similar to those identified in previous successes, for example.

After more than 20 years of this approach, the question of its success is still unresolved. On the one hand, the average time required for drug development (13.5 years) means that there is not yet enough distance for a clear view. On the other hand, it can be argued that market-derived data do not have a positive outlook. One retrospective analysis [RAS 11] showed that new drugs approved by the Food and Drug Administration (FDA) are stable at around 18/year through the 1982–2010 period for a rate of new therapeutic targets of 4/year, a quantity that remains very low. It follows that

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the impact of screening has been modest at best. This apparent failure of what initially seemed a winning strategy can also be explained by the fact that most classic therapeutic targets and most readily treatable pathologies are currently well provided for in terms of effective drugs and that the industry has had to engage with particularly abstruse pathologies during this time.

The debate over the success of screening extends to the consequences of certain strategic choices at play during this period. Advances in molecular biology from 1985 together with the sequencing of the human genome around 2000 have essentially provided the industry with a great number of potential therapeutic targets. This has permitted a shift from an old-fashioned vision based on the illness (also called phenotypic) to a modern vision centered on the target of the future drug. With only 25 years of perspective (around twice the time required to generate a new drug), the debate is fierce as to whether this shift was pertinent or not [EDE 14].

There are various studies indicating that the phenotypic approach, which is at present undergoing a renaissance of sorts thanks to the support of stem cell models in particular (see section 10.1), is the more productive. This path casts doubt on the linear vision of new drug discovery based on target identification, development of adapted tests, screening, selection of compounds of interest, followed by their validation then their progression to preclinical trials.

Be as it may, the phenotypic approach is of particular interest for the positioning of cell assays. Effectively, it prescribes setting aside the putative target and instead engaging more global information in connection with the cell effects of the target pathology. In terms of assays, this is expressed by measuring signals closest to an integrated intracellular response by selecting models closest to the physiopathological reality. This is indeed a growing sector for high-throughput cell approaches, introduction of which is now booming.

8.1.1. The role of cell assays in screening programs

The assays used for high-throughput screening are essentially of in vitro origin. They can be split into two clearly distinct categories [AN 09]: biochemical assays and live cell assays. The former are the oldest. Above all, they allow for the measurement of interactions between molecules and their


potential target but also of enzymatic activities or protein–protein interactions. They are typically performed in a homogeneous medium, which endows them with the significant advantage of minimizing the substantial level of variation inherent in more sophisticated biological tests. For these reasons, they are still used. However, they have limits, which needs to be worked around keeping in view the modern demand for information and pertinence. First, not all of the therapeutic targets identified readily lend themselves to the process of purification or indeed behave in a benevolent manner in a homogeneous medium. Second, ligand bonding to a target does not presume, at least in the case of receptors, the modulation that it will bring to the underlying cell function. And finally, the complexity of the action mechanisms employed by the industry often involves multiprotein complexes whose regulation is difficult to reproduce with biochemical approaches.

As we will see, the cell approach provides answers to these limitations to a great extent. This is why today cell assays represent between 50 and 60% of the total number of tests performed during the screening phase.

For this reason, of all academic, industrial or regulatory activities, high-throughput screening is the single biggest consumer of live cell assays. And by far! The most recent study [RAS 11] has counted 435 therapeutic targets in the human genome with at least one active drug. The receptor family, both membrane and nuclear, is the most common, with 193 targets (or 44% of total) followed by enzymes, 97 targets (29%), and transporter family proteins, 67 targets (15%).

As to the diversity within the receptor family, around 10% are collected at nucleus level and 90% at the plasma membrane. The various membrane receptor subclasses each show significant structural homogeneity but react to a tremendous variety of stimulants such as hormones, photons, odorant molecules, growth factors, neurotransmitters and amino acids. The flow of information between the plasma membrane and the cell response, often announcing a genic expression, is made up of a long chain of events offering many tools with which to measure the effect of candidate-drugs with live cell assays. The discovery of these receptors and of cell signaling associated with them (see Chapter 7) constitutes one of the most important chapters in the history of modern biology. It is the basis for most of the live cell assays used today for industrial applications.


8.1.2. The contribution of functional cell assays

The complexity of seven domain receptor regulation leads to a first conclusion. Without the contribution of assays integrating information at the living cell level, there would be no rational means of researching new active principles with therapeutic potential with this category of targets. This is clearly an area in which the functional cell assay comes into its own. Hence the considerable effort that was made in decrypting the intracellular signaling pathways involved in order to match receptor activation to cell functions of physiopathological interest.

The intimate understanding of the regulation mechanisms of seven domain receptors [ROS 09] has led to the development of cell assays that are increasingly close to the physiological reality, at least in its expression at the cell level. For example, taking the receptors’ allosteric reality into account has allowed for molecules to be screened according to the criteria of their binding to the allosteric site and their capacity to modulate the action of the endogenic ligand to which it is bonded by cooperativity [SEB 12]. This is essential for the pharmaceutical industry, which hopes to use it in identifying new classes of modulators whose action site preserves the endogenic ligand’s freedom of interaction, according to a reality closer to physiopathology. Some of these campaigns have been met with a certain success, with two compounds already on the market, cinacalcet, the first hyperparathyroidal treatment and maravirac, an anti-HIV drug.

8.1.3. Exploitation of transduction pathways

In order to meet the needs of high-throughput screening, the ideal functional cell assay must be robust, simple, miniaturizable on 384- or 1,536-well plates and above all adapted to automation and robotization [MAR 12]. Several visions are possible according to placement near the receptor or far from it at the end of the transduction chain. The former case offers greater specificity and limits false positives, but the signal is weak due to the membrane receptors being so sparse (in the range of several hundreds of copies per cell) on the cell surface. Functional information is also limited. Inversely, measuring an event at the end of transduction chain allows for major signal amplification, integrating more functional information but limiting the specificity and inevitably increasing the risk of false positives. Of course, the answer can be found between these two extremes. The different modes of functional measurement of this sequence of events are presented in Figure 8.1.


Figure 8.1. Transduction pathways coupled with seven transmembrane domain receptors and associated live cell approaches: (1) binding of ligand on the receptor, (2) activation of the transduction pathway, (3) desensitization of

the receptor and (4) internalization by endocytosis

Several parameters need to be taken into account. First, a seven domain receptor can be combined to several signalization pathways (PKA and PKC, for example). The possibility of oligomerization, a physiological phenomenon that has come to light in recent years, can also shift the message from one pathway to another.

Receptors are also subject to the phenomenon known as functional selectivity. This is a fundamental discovery of recent years. Now, receptors are described according to numerous isoenergetic conformations that can be stabilized by different ligands. Indeed, these conformations are attached to other transduction pathways than those typically described (see section 7.4). This selectivity in signaling is an essential crossroads that determines the orientation of the cell response, in particular the gene panel and the responses associated to it. The allosteric modulators are a typical example of actors working in the selection of receptor signaling pathways. These compounds are now known as biased agonists [KEN 09]. These advances have cast doubt on our understanding of the physiological and pathological transduction pathways, and establishing a functional test by targeting a given pathway can prove risky.


Here we will categorize functional cell assays according to the position of the event measured in the transduction chain, while bearing in mind the limits of this vision due to the transduction chain not being linear but punctuated with parallel pathways, feedback loops, etc. Cell assays will be described successively as proximal (around the receptor), intermediate (second messengers) and distal (integrated response) according to the nature of the events measured.

8.2. Measurements in the immediate environment of receptors

8.2.1. Assays on receptors

The functional oligomerization of seven domain receptors and its importance in pharmacology represents one of the major discoveries of the 2000s in this sector. This typically concerns identical (homodimerization), but can also concern different receptors (heterodimerization). Each situation can lead to the activation of a specific transduction pathway that can be studied by the technologies presented below. Different assays also allow for the measurement of functional dimerization directly at receptor level [ZHA 12]. While FRET and BRET have been widely used for research purposes, their use in ligand screening is problematic as the dimers can form within intracellular membranes, generating signals independent of ligand action.

The trick consists of using exclusively extracellular markers that as a consequence cannot associate with the receptor unless exposed to the plasma membrane. Launched in 2008, the Tag-lite assay sold by Cisbio is well adapted to this situation. It combines two approaches. Both the SNAP-tag and CLIP-tag systems conceived in the United States by the NEB company together with TR-FRET (time-resolved FRET) belonging to Cisbio and whose cyclic AMP application is described in detail in the next section. The receptor is first SNAP or CLIP tagged on the N-terminal segment exposed to the cell exterior. Each of these two tags binds to a specific TR-FRET partner (rare-earth cryptates for the energy donor, fluorescent marker for the acceptor) added into the culture medium. Only the association of receptors in dimers produces a TR-FRET signal (Figure 8.2).


Figure 8.2. TR-FRET approach for the measuring dimerization of seven domain receptors

8.2.2. β-arrestin activity assays

β-arrestin is a cytosolic protein whose dual action on seven domain receptors has recently come to light. The protein has been known for a long time for its universal role in uncoupling G proteins from the receptor, and addressing it toward clathrin equipped endocytosis vesicles. More recently, it was (or has been) associated with the activation of independent G protein transduction pathways such as ERK1/2, c-Src and AKT, a pathway identified in the context of the discovery of biased agonists. Indeed, β-arrestin can now be considered as a transduction protein in its own right.

The relatively universal nature of the β-arrestin bond to seven domain receptors soon led the industry to use this information in the identification of new functional compounds by high-throughput screening.


TransFluor, developed in the United States at the Duke University Medical Center, then commercialized in 1999 by Norak Biosciences, was the first functional assay based on the recruitment of β-arrestin to the plasma membrane [ZHA 12]. The principle is presented in Figure 8.3. It monitors the intracellular redistribution of a chimeric GFP/β-arrestin protein. The recruitment of the protein by receptors to the plasma membrane alters the fluorescence pattern, which goes from diffuse and cytosolic, to far more distinct…, membrane and vesicular. Measurement requires an image analysis device and is integrated in the high content screening (HCS) approach performed on tools such as ArrayScan (Cellomics/Thermo Fisher), Acumen, IN Cell analyzer (GE Health Care Life Sciences) or ImageXpress (Molecular Devices). Thanks to these tools, the Transfluor assay can be integrated into a process of multiplex analysis, which allows for the accumulation of several levels of information on the same cell sample (or even of the same cell) tested. Nonetheless, these approaches require monolayer adherent cell cultures with comfortable cytoplasm versus nucleus area ratios.

β-arrestin translocation is also used, in particular by the Transfluor assay, as the preferred method for the receptor deorphanization stage, a stratagem adopted by the pharmaceutics industry to expand their portfolio of therapeutic targets. Indeed, of approximately 700 genes coding for seven domain receptors identified during human genome sequencing, there remainded in 2012 around one hundred still orphan in terms of ligand association.

Image analysis limitations can be circumvented by non-visual approaches. Typically, this involves the classic BRET and reporter gene technologies together with enzyme fragment complementation (EFC) from DiscoverX, known as PathHunter, whose variant measuring cyclic AMP will be presented below. Here, β-arrestin is fused with an inactive mutant of β-galactosidase [EGL 07]. The target receptor itself is fused with a small fragment (ProLink) extracted from the segment of β-galactosidase deleted on the other construction. The receptor–β-arrestin interaction then reconstitutes β-galactosidase, which becomes functional again, transforming its substrate into a chemiluminescent product (Figure 8.3). For this approach, the commercial strategy consists of providing users with cell lines, which express the receptor/β-gal fragment and β-arrestin/deleted β-gal constructions conjointly.


Figure 8.3. Four main modes of β-arrestin measurement

Here we should note that the PathHunter approach also allows for the measurement of heterodimerization of seven domain receptors. In this case, only one of the two receptors is fused to the β-gal complementary fragment. The assay is based on the principle that β-arrestin is only recruited after the functional receptor dimerization.

BRET technology has been available since 2002. Here, the β-arrestin is fused with the Renilla luciferase and the receptor with a GFP (or inversely) (Figure 8.3). The BRET couples were subsequently optimized.

Tango (InvitroGen) falls into the category of reporter gene assays. Here, the reporter is activated by proteolysis. β-arrestin is fused to the TEV protease while the receptor is fused to a TEV specific cleavage site, which is


fused to the transcription factor Gal-PV16. In this way, β-arrestin recruitment to the receptor environment ensures the liberation of the Gal-PV16 factor that enters into the nucleus and activates the transcription of the β-lactamase gene (Figure 8.3). This can then cleave a specific substrate, which contains two FRET partners, separated for the occasion.

Of course, these last technologies are complex and suppliers have had to produce appropriate cell systems, comprising the stable expression of FRET or BRET partners together with that of modified therapeutic targets. These are particularly significant and expensive works of cellular and molecular engineering, which demonstrate the tremendous economic stakes that these functional assay approaches represent (or represented) for the pharmaceutics industry.

8.3. Measuring cyclic AMP

These assays are applicable on all receptors coupled with Gs and Gi proteins. Adenylyl cyclase, the cyclic AMP production enzyme, is controlled by the receptor-mediated activation of Gαs or Gαi/o subunits. The cyclase’s positive activation by the Gαs is easily detected, although the more common negative activation by the Gαi/o proves more challenging and typically requires the upstream addition of both forskolin, to activate the cyclic AMP synthesis, and phosphodiesterase inhibitors such as IBMX or Rolipram to limit the nucleotide’s degradation.

8.3.1. Classic cyclic AMP assays on cellular lysates

There are four classic cyclic AMP measurement systems in widespread use [ZHA 12]. All of them use a reagent to trigger cell lysis, which ensures access to intracellular cyclic AMP. Each of them has its advantages and disadvantages. They are principally based on the competition between endogenous cyclic AMP and a measurable biomarker. They do not allow for analysis of kinetics.

The first is the Alpha Screen assay commercialized since 2001 by the American company PerkinElmer [EGL 08]. It is performed in a homogeneous medium after cell lysis (Figure 8.4). It contains two beads, one donor and one acceptor. The donor contains a photo-sensitizing agent, phtalocyanine. When this is irradiated at 680 nm, it excites the dioxygen, which turns into its singlet form (1O2). The system benefits from excellent amplification as each


donor bead can produce up to 60,000 singlet oxygen molecules for each excitation. Moreover, the distance between the beads is compatible with the short lifespan (4 ms) of singlet oxygen, which corresponds to a covered distance of around 200 nm. Once produced, the 1O2 relaxes by exciting the acceptor bead. This is composed of three chemical colorants, thioxene, anthracene and rubrene. Initially, the 1O2 reacts with the thioxene, producing photons transferred to the anthracene then to rubrene, which emits between 520 and 620 nm. Consequently, this is a chemiluminescent signal.

Figure 8.4. Principle of cyclic AMP measurement on cell lysates. Example of AlphaScreen assay. (1) Energy produced by the donor bead is transmitted to

the acceptor bead by singlet oxygen; (2) the presence of endogenous (non-biotinylated) cyclic AMP repels the beads and turns the signal off


In case of cyclic AMP measurement, the system functions by competition. The donor bead is coated on the surface with streptavidin molecules that possess a strong affinity for biotin. The acceptor bead is coated with anti-cyclic AMP antibodies. The whole is held together by the presence of biotinylated cyclic AMP, which is recognized by both beads. Consequently, the system is functional. The assay measures the competition between endogenous and biotinylated cyclic AMP present on the antibody. The presence of endogenous cyclic AMP that does not possess an affinity for streptavidin keeps the two beads away and switches off the signal. The assay requires the use of phosphodiesterase inhibitors and forskolin, when using targets associated with the Gαi/o system.

The AlphaScreen system has recently become AlphaLISA in which the anthracene and rubrene have been replaced by europium chelate, a compound excited at 340 nm by the conversion of thioxene to diketone following the relaxation of the 1O2. The light emitted by the europium, an element of the rare earth family, is intense and characterized by a very sharp emission peak around 615 nm and a particularly long lifespan in the excited state (on the order of a microsecond). This results in a reduction of interference with the sample’s fluorescent compounds, the wavelength emission of which is typically more energetic and lifespan in the excited state shorter (in the order of a nanosecond).

The second type of test is the enzyme fragment complementation (EFC) assay or HitHunter commercialized by the Californian company DiscoverX and launched in 2003. This is an antibody competition assay between cyclic AMP and a version of cyclic AMP conjugated with an fragment (known as ED) isolated from β-galactosidase and required for its enzymatic function. As long as the concentration of cyclic AMP remains low, the conjugated version (cyclic AMP/ED) is bonded to the antibodies and cannot reconstitute the functional enzyme. If the concentration of cyclic AMP increases, the balance is shifted and cyclic AMP/ED is available to complement the enzyme and produce a luminescent signal proportional to the quantity of cyclic AMP (for the EFC principle, see Figure 8.3). The assay is performed in a homogeneous medium. All the ingredients (cyclic AMP/ED, lysis solution, anti-cyclic AMP antibodies) are added to the cell culture. The conjugation enzyme is added after incubation.

The third type of assay uses the technology called homogeneous time-resolved fluorescence (HTRF) sold by the French company Cisbio. The


technique uses FRET (see section 3.1) between a donor with long excited state lifespan such as the europium cryptate coupled to an anti-cyclic AMP antibody and a phycobiliprotein (the XL665, a pigment purified from red algae) or, more recently, the d2 acceptor (identified after screening 15,000 compounds, more stable and much smaller than the precedent). At the heart of this technology is the complex formed by the lanthanide rare earths (europium or terbium) bundled into a cryptate macrocycle, which allows for an extremely long lifespan in the order of a millisecond [DEG 09, GRÉ 00].

In principle, this is also an assay based on the competition between endogenous cyclic AMP and cyclic AMP coupled to XL665. Furthermore, the acquisition of the time-resolved fluorescence signal allows us to avoid the recurrent problems of parasite fluorescence associated with classic FRET technologies. Note that Perkin-Elmer also commercializes a competing system called LANCE that uses europium chelate and Alexa fluor as FRET couple.

The fourth type of assay uses exposure to polarized light. This is commercialized by the main companies focusing on this sector. In principle, the light emitted by a complex formed of a specific cyclic AMP antibody coupled to a fluorescent cyclic AMP is polarized because the formation of the complex limits its rotation in space. When the fluorescent cyclic AMP is displaced by the presence of endogenous cyclic AMP, it regains its freedom of rotation, losing its polarization property. This system is sold in the form of kits by several companies.

8.3.2. Cyclic AMP assays on live culture cells

A new generation of technologies now measures the presence of cyclic AMP in live cultured cells [HIL 10]. This opens up the possibility to kinetic monitoring. Intracellular chimeric biosensors have been created that integrate cyclic AMP-binding domains such as those present on the regulatory subunit of the protein kinase A (PKA) or Epac (section 7.3).

The first of these constructions, published in 2000, consisted of using a FRET couple (see section 3.1, particularly Figure 3.3(1)) in which each of the two R and C subunits of the PKA are associated with a GFP [ZAC 00]. Since


cyclic AMP works by dissociating the two subunits, a reduction in FRET signals the activation of the transduction pathway.

In recent years, various FRET strategies have been optimized. Constructions containing Epac for instance, have been flanked by FRET partners of the GFP family, such as CFP and YFP. Intramolecular FRET signal measures an absence of cyclic AMP (see Figure 3.3.(2)) due to a favorable conformation placing the two GFPs at a distance close to the Förster radius range. Conversely, the signal is lost after conformational changes induced by cyclic AMP binding.

Complex optimizations have resulted in new solutions that measure cyclic AMP on live cells at physiological concentration (0.1–100 µM) with very favorable signal-to-noise ratios. This is the case of the construction bearing the fine name of mTurquoiseΔ-Epac(CD, ΔDEP)-cp173-Venus-Venus (or more simply TEPACVV), which uses mTurquoise GFP, whose quantic yield is very high [KLA 11]. Very similar strategies based on the BRET between the Renilla luciferase and the YFP have also been investigated with success.

The cAMP Glosensor system sold by the American company Promega is certainly the most complete, at least in industrial terms. The principle, published in 2011, resumes that of the activation of Photinus pyralis luciferase seen in section 3.1 but with one significant modification. The enzyme has been rebuilt so that its sequence incorporates the cyclic AMP-binding domain of the PKA regulatory subunit (RIIβB). Consequently, the resulting chimeric protein possesses a strong affinity for cyclic AMP. Moreover, the addition of this cyclic AMP-sensitive sequence maintains the enzyme in an inactive conformation. Binding of cyclic AMP then modifies the chimeric enzyme conformation, restoring its enzymatic activity (Figure 8.5).

This approach improves the signal dynamics and the sensitivity. It also seems to resolve the problems encountered with other technologies, such as the lack of linearity between measurement and the real concentration of cyclic AMP. Indeed, the supplier stipulates that the detection of activity connected to Gαi/o does not require the addition of forskolin. Another advantage of working on living cells in a non-destructive format is that the test is readily integrated in a multiplex approach. Furthermore, it allows for


the study of shifts between signaling pathways in the context of allosteric modulators or biased agonists (see section 7.4).

Figure 8.5. GloSensor system for the functional evaluation of the cyclic AMP pathway on living cells

Despite being very simple in appearance, the protocol nonetheless requires the transient transfection of cells with a plasmid (pGloSensor™-22F cAMP) containing the luciferase/modified RIIβB gene. Obtaining stable clones is no simple matter and using a transient transfection can cause problems of heterogeneity in the population of cells analyzed. A novel approach published in 2014, called Candles, proposes to circumvent this problem by the stable transfection of a generic line with one of the available sensors (GloSensor 22F, TEPACVV). This line can then be cocultured with the line (or primary cells) expressing the target of interest and benefit from the cytosol–cytosol transfer of the sensor by Gap junctions established between the cells [TRE 14].


8.4. Measurement of the PKC pathway and discrimination of the PKA/PKC pathways

8.4.1. IP3 measurement tests

As we have seen above, the PKC pathway is characterized by the presence of IP3 associated with the activation of Gαq/11 proteins. IP3 is a second messenger that controls calcium ion efflux to the cytosol. As with all second messengers, it is quickly neutralized by enzymatic hydrolysis and transformed to IP2 then to IP1, ultimately becoming simple inositol.

In terms of industrial assays, the technologies are often adapted from those used in the measurement of cyclic AMP [ZHA 12]. The presence of IP3 can be measured by the AlphaScreen (Perkin-Elmer) method and by fluorescence polarization (DiscoverX). IP1 accumulation, in correlation with the activation of the IP3 pathway, can be measured by the IP-One HTRF assay commercialized by CisBio. This approach blocks the degradation of inositol phosphates in the form of IP1, which accumulates following the addition of lithium chloride. The assay is available for ultrahigh-throughput in the 1,536 well plate format. The information approaches that of calcium assays.

8.4.2. Assays for the measurement of Ca2+

The measurement of intracellular calcium is particularly valued in screening campaigns on seven domain receptors. There are various reasons explaining its popularity. Above all, it is a major transduction pathway associated, like IP3, to the activation of Gαq/11 proteins. Moreover, the transduction pathways associated to other G proteins can be artificially diverted to the calcium pathway by two approaches that are widely exploited for industrial purposes.

The first consists of overexpressing the so-called universal Gα15 or Gα16, proteins from the Gαq family that have the dual role of being coupled to the calcium pathway and activated by most of the seven domain receptors. Consequently, their overexpression tricks the receptor and commutes its response to the calcium pathway [COW 99].

The other option, published in 1999 for uses in high-throughput screening, consists of creating Gαqi5 or Gαqo5 chimeric proteins, composed of Gαq

containing the five terminal amino acids of Gαi or Gαo. This modification


allows for receptors physiologically coupled to the production of cyclic AMP to shift toward the calcium pathway.

It follows that cell lines expressing these either natural or chimeric G proteins offer universal models demonstrating the functions of any seven domain receptors. They have also found an applicative niche of their own in the phase called receptor deorphanization, whose transduction pathways remain elusive. These lines offer a solution for the completion of this stage, which represents a major challenge for big pharma industrial property.

In terms of calcium detection technologies, they are described in section 4.6. Their use in high-throughput screening is already longstanding. They make use of classic fluorescent markers like fluo4 and calcium sensitive recombinant biosensors, the foremost of which is aequorin. The arrival of systems such as FLIPR toward the end of the 1990s allowed for the miniaturization of these protocols on 384 and 1,536 well plates.

8.4.3. Discrimination between the cyclic AMP and IP3/Ca2+ pathways by label-free methods

It should be noted here that both the cyclic AMP and IP3/Ca2+ transduction pathways can be discriminated without too much difficulty by label-free methods described in sections 3.2 and 3.3. The CellKey (MDS Analytical Technologies) system based on impedance measurement reveals specific profiles according to the G protein mobilized. The receptors activating the Gi provoke an increase in impedance. On the other hand, those activating the Gq provoke a transient reduction followed by a massive increase in signal strength, while those coupled to Gs provoke a clear reduction [SCO 10]. This impedance signature offers a particularly powerful screening tool. Things remain quite unclear in mechanistic terms, but some authors suggest that these profile differences could be linked to regulation at cytoskeletal protein level. Microtubule organization and especially actin filament modulation via Rho family GTPases are suspected.

Optics-based label-free measurement, presented as an alternative to the impedance approach, offers some very different but equally discriminating profiles. With the Bind (SRU Biosystems) system, the activation of receptors coupled to Gi and Gq presents a rapid kinetic profile with a maximum at 5 min, then decreasing, while receptors coupled to Gs present a slower and more sustained profile. The EPIC (Corning Inc.) system also allows for the discrimination of Gs, Gq and Gi pathways, but the profiles obtained depend on


the cell line used, which does not alter the quality of results obtained but complicates the adjustment of the test before beginning with screening.

More surprisingly, the xCELLigence (Roche Applied Science/ACEA Biosciences) impedance-based system presents profiles comparable to those obtained by optical systems. It could be that the results obtained by the various label-free technologies reveal similar intracellular events.

Evidently, the pleiotropic aspect of the transduction pathways attached to seven domain receptors lends itself particularly well to label-free approaches. Unlike classic methods that measure a precise pathway, the label-free detection mode does not describe any pathway in particular. In pharmacological terms, this approach also allows for the separation of the agonist and antagonist statuses from the inverse agonist status. Investigations are currently ongoing to verify the status of biased agonists.

While the position of label-free approaches in high-throughput screening is not in doubt, several limits should be noted nonetheless. The first is the cost, both of the system and of the consumables, most of the time dedicated and proprietary. False negatives can also appear when the contribution of two pathways is equal and antagonizes the signal level. The signals obtained are typically the result of different functional signals, including those connected to receptor tyrosine kinases (RTK, see next section), which then requires quite meticulous functional pharmacological work to characterize the transduction pathways implicated [NOS].

As to the benefits, there is notably improved overall sensitivity compared with classic methods, allowing for more comfortable work, particularly when using lines that express the targets in an endogenous manner. This strategy is often prioritized due to it closely approaching physiological or physiopathological conditions and above all providing a means to avoid constructions subject to rights restrictions. Label-free methods also help escape from cell component autofluorescence or quenching problems, which can disturb the signal in more classic approaches.

8.5. Measurement of distal signals

The last option for functional measurement of seven domain receptors consists of placing the read-out at the end of the transduction pathway, to be more precise, at the level of the gene transcription factors to which they are attached. These are the reporter gene approaches presented in section 3.1 here


applied for high-throughput screening. Notably, they use the activation of N-FAT, CRE and SRE response elements associated with the calcium, cyclic AMP and Rho pathways, respectively [CHE 10]. Their sensitivity allows for the detection of allosteric modulators for instance. Their availability on 3,456 well plates indicates their rare level of standardization for cell assays. Nonetheless, several prominent drawbacks can be noted, such as a tendency to produce too many false positives due to the large distance, in terms of biochemical cascade reactions, between the activation of the receptor and the observed signal. Luciferase is preferred as reporter gene due to its signal dynamic, its low signal-to-noise ratio and the linearity of its response.

8.6. Cell assays concerning other therapeutic targets

8.6.1. Measurement on ion channels

Ligand-gated ion channels represent the second group of receptor-type therapeutic targets by number. Fifteen of the 100 best-selling drugs in the world work through (or via) this sort of target. The main applications are cardiac arrhythmia, hypertension and neurological illnesses. Here too the economic stakes are colossal, still more so since, as with seven domain receptors, human genome sequencing has brought around 400 ion channels to light, many of which were unknown, representing potential targets for new therapeutic applications. Historically, the first demonstration of a link between an ion channel dysfunction, CFTR in this instance, and a pathology, cystic fibrosis, dates to 1989.

In terms of technologies, electrophysiology has long remained the benchmark method in this area, although throughput problems have led researchers, following a brief incursion into radioactive approaches, to functional cell assays, at least for the classic screening step. As recently as the 1990s, ionic efflux and influx assays were based on radioactive sensors such as 45Ca2+ or 22Na+. The sensor used for potassium efflux was 86Rb+.

In addition, the ion channels were long considered as somewhat unattractive targets for the implementation of high-throughput screening, connected in large part to frustration with the low returns in the selection of new compounds of interest. This is certainly due to the fact that compound libraries were more or less focused on screening for seven domain receptors, receptor kinases or enzymes structurally far removed from ion channels. This undertaking has often been compared in publications to searching for a needle in a haystack.


The arrival of functional fluorescent cell assays in the 2000s was a game changer. Stable cell lines expressing the channels were made available, together with fluorescent markers that could be used to monitor changes in the membrane potential. This combination proved to be fruitful, particularly when it came to identifying sodium or potassium modulators or ligands for channels working in this interaction mode.

As for calcium channels, the markers described in section 4.6 were used successfully and are still in use, as can be observed in the results of a screening campaign on cells expressing the GluK1 glutamate receptor using the fluorescent Fluo-4 performed by Merck in 2015. The process consisted of screening a library of millions of compounds on 1,534 well plates using a FLIPR Tetra device. Of these, 6,100 compounds were selected for more in-depth dose–response studies. Finally, high-throughput electrophysiological patch clamp studies showed that 1,000 of these compounds presented a dose-dependent inhibition with EC50 inferior to 12 µM. This example [SOL 15] demonstrates current expertise in ultra-high throughput cell assays and its routine use in the pharmaceutics industry.

Kits using DIBAC fluorescence quenching and the new generation of markers such as FLIPR membrane potential (FMP) allow for work on classic fluorescence readers. FMP kits also ensure the measurement of events on shorter kinetics (several dozen seconds) all while maintaining sensitivity. The FRET technology called VIPR (Aurora/Vertex), presented in section 4.6, has also been successfully used with various types of ion channels [FAL 02]. One drawback is its need for a proprietary VIPR device such as GENios Pro (Tecan) or FLIPR Tetra (MDS Analytical Technologies). Its main advantage stems from its highly accurate kinetic measurement (in the range of a second).

It should be noted that patch clamp technologies have today caught up with their rivals in terms of throughput. The transition between traditional glass electrode use and the approach called planar patch clamp based on a matrix, has been made progressively. The IonWorks Quattro (Molecular Devices) system, for instance, performs the measurement of electric currents on parallel cell populations, which significantly tightens the results’ statistic value while minimizing variability. The cells, whose confluent state must be carefully adjusted, grow on a medium pierced with micropores of 1–2 µm in diameter, which communicate with a subjacent chamber of very low volume representing the intracellular milieu. These pores ensure a low resistance passage favored by the presence of antibiotics of the amphotericin B family that facilitate the formation of membrane pores. This approach is conceived above all to address the case of voltage-dependent ion channels, typically


installed after stable (or possibly transient) transfection on HEK293 or CHO cells. The number of compatible cell lines seems very limited.

8.6.2. Measurements on receptor tyrosine kinases (RTK)

There are 58 known RTKs in humans, of which 22 are classed as therapeutic targets. As such, they represent 11.4% of the total class of receptor targets. Mutations within the receptor or disorders in their associated transduction pathways have been linked to many pathologies, most notably cancer, diabetes and inflammation. The research for molecules that attenuate or abolish RTK activity has led to numerous high-throughput screening campaigns using cell assays. According to a study published in 2010, a dozen small molecules and antibodies directed against RTKs were approved between 1998 and 2007 in the United States in the cancer treatment area alone.

The structure of RTKs appears to be much simpler than that of their seven domain receptor neighbors. RTKs essentially comprise an extracellular domain, a single transmembrane domain organized in α-helix and a cytoplasmic domain containing the kinase activity. In functional terms, RTKs are receptors that bind ligands, and kinases which phosphorylate proteins, the first element of the transduction cascade. Since the first works by Yarden and Schlessinger in 1987, it has been known that the association of the natural ligand (or possibly a drug) provokes receptor dimerization, a key step in the transduction of the message to the cell interior. The most well-known RTKs are the epidermal growth factor (EGF), NGF, FGF, VEGF or PDGF receptors, which function in this mode. On the other hand, insulin or IGF1 receptors form an RTK subfamily that distinguishes itself by a dimeric structure preexisting the ligand binding where dimers are associated covalently at the extracellular segment level by disulfide bridges.

It was long believed that it was the ligand’s association with two RTK monomers that favored dimer formation and stabilized it in a conformation favorable to the activation of the intracellular kinase activity. This mode seems to be restrained to certain RTKs in which the ligand, which is dimeric, brings the isolated monomers physically together. Other modes in which the ligand brings no direct contribution are also considered. Finally, there are many intermediary modes in which the ligand only partly intervenes [LEM 10].

The mechanisms by which the ligand–dimer complex activates the receptor’s kinase activity are also highly diverse. Of still greater interest to cell assays is that the activated receptor draws numerous transduction


proteins into its immediate environment. The cascade of functional events begins, with rare exceptions, by autophosphorylations of the receptor itself. This happens in trans (that is, between the two monomers of the dimer) and can play an activation role on kinase activity itself. Above all, it leads to the phosphorylation of very specific tyrosines that are transformed to recognition sites for the assembly of transduction molecules. Autophosphorylations occur in a highly precise order that ensures both the removal of kinase self-inhibition and the recruitment of cytoplasmic proteins. These proteins are characterized by specific recognition domains called SH2 (Src homology-2) and phospho tyrosine-binding (PTB). Many proteins are recruited in this recognition mode, the most well-known of which are FRS2, IRS1 (associated with the insulin receptor) and Gab1.

The activated receptor can then be considered as a signaling hub, which by recruiting numerous adapter proteins, starts multiple transduction pathways. Unlike the seven domain receptors, the interaction logic between partners around RTK is not established by recognition at the level of the proteins themselves but at a certain number of protein domains shared between many proteins. The best model to illustrate this is the bricks used in a set of LEGO. It seems that it is the articulation of these domains that governs the transmission of the message from the activated receptor. These domains interact in the direct environment of the receptor, such as the SH2, for example, that binds to the receptor’s phosphotyrosines, or at a distance, like the PH domain, which binds to the membrane phosphoinositides such as PI(3,4,5)P3.

It is clear that the question of transduction pathways subjacent to RTK activation remains unresolved despite the great many studies that have been performed. It appears that the vision of linear pathways formed of biochemical cascades resulting in the cell response is simplistic. The signaling network is highly interconnected and subject to many feedback loops, with the receptor acting as an information transmission hub.

In the case of the EGF receptor, at least six major interconnected pathways have been identified: the canonical Grb2/SOS/Ras/Raf/MAP-K pathway, the PLCγ/IP3/DAG/PKC pathway, the STAT pathway, the FAK pathway, the Grb2/PI3K/AKT/PDK1 pathway and the Vav2/CDC42/MEKK1 pathway. However, a 2005 study indicated that this same EGF receptor acts on a signaling network of 322 components containing no less than 211 biochemical reactions. To complete this picture, it should be made clear that there can also be cross interactions between RTKs and seven domain receptors.


The two main functional sites of RTKs were regularly explored in connection to cancerous pathologies. Today, various cell screening strategies allow for the selection of small molecules that inhibit intracellular kinase activity, or alternatively, new ligand antagonist antibodies acting on the extracellular binding site.

In order to develop functional cell assays, it is therefore important to stay as close as possible to the receptor activation, upstream from the complex interconnection and feedback phases that govern the cell response. This task is helped by the numerous domain protein movements in the vicinity of the receptor or the plasma membrane. This is the case of the Akt protein that binds via its PH domain to two phospholipids of the plasma membrane, PI(3,4)P2 and PI(3,4,5)P3. Connection with the RTKs comes from their capacity to activate the PI3-kinase enzyme that produces the two aforementioned phospholipids. Once recruited to the membrane, Akt is itself phosphorylated at two different sites by PDKs that themselves bear a PH domain and are recruited to the plasma membrane. PDK-dependent Akt phosphorylation, in turn, activates its own kinase function, allowing phosphorylation of various proteins implicated in the control of cell growth, particularly in apoptosis.

In terms of high-throughput technologies, they vary little from those described for seven domain receptors. Among others: Alpha Screen, TR-FRET, ELISA, fluorescence polarization, flow cytometry approaches together with the measurement of kinase-dependent ATP consumption. Distant signals such as the activation of the SRE response element associated with the raf/MAP Kinase pathway are also exploited by the reporter gene approach [CHE 10]. Since RTKs are also associated with cell morphology and cytoskeleton mobilization, label-free technologies, which have been used since 2006 on the EGF receptor, have demonstrated their pertinence with sensitivity thresholds that allow to work with cells expressing endogenous receptors [ATI 06].

While the progression RTK/transduction pathways/cell response remains hypothetical most of the time, the causal link between these pathways and certain major physiological or physiopathological responses have been established. One such case for example is the role of the PI3 kinase/Akt pathway in controlling proliferation/apoptosis. Several drug research programs directly target these pathways, independently of any specific RTK, in a phenotypic approach. Functional cell assays are accordingly the natural tools to be used for these problems. For example, it has been demonstrated that the PI3 kinase/Akt pathway is deregulated in more than 50% of cancers


and major screening initiatives have been undertaken to find inhibitors of this pathway in connection with leukemia. Most of the time these projects remain confidential but we can cite the screening of 45,000 compounds during a campaign researching Akt translocation inhibitors with the help of assays similar the β-arrestin described above [LUN 05] (Figure 8.3, translocation).

If there is a domain in which the classic immortalized cell lines can be used with a maximum of pertinence, it is in oncology. For the past 12 years, access to ultrahigh throughput has allowed us to couple the issue of RTK inhibitor identification with that of existing tumor typology. Screening programs have been undertaken in order to match each RTK inhibitor identified to the types of tumor in which it is found to be active. This is an approach known as profiling. For example, a large American public–private program [MCD 07] has set up a screening facility using 500 cell models of solid tumors in parallel taken from the ATCC (USA), the DSMZ (Germany), the JHSF (Japan) and the ECACC (Europe)! The cellular effect is measured by means of antibodies targeting different phosphorylated transduction proteins following RTK activation. The sensitivity of each line is assessed by a viability assay coupled to genomic analysis on Affimetrix biochips. This example shows the extraordinary potential of high-throughput platforms with regards to the generation of live cell assay data.

8.7. Pharmacokinetics (ADME) in vitro

Pharmacokinetics covers the steps of development concerning the outcome of the drug once administered in the body. The four main steps are absorption, distribution, metabolism and elimination of the active ingredient. These steps are collectively known as ADME. Pharmacokinetics can be resumed by the aphorism of “what the body does to the drug”, which begs the question of the pertinence of cell assays in this highly integrated process. In fact, the interest of introducing a dose of in vitro in this branch of pharmacology stems from the hope of obtaining information on the outcome of molecules of interest far upstream to the regulatory preclinical trials. This is a way of benefiting from the high-throughput capacity of in vitro assays to reduce the time and cost of the future ADME study, together with the failure rate of candidate drugs during later phases.

As a consequence, ADME applications of cell assays are limited to the assessment of several functions that are expressed by well-established cell pathways. These correspond to a young but quickly expanding market sector and draw on highly innovative strategies such as the use of stem cells. It is


also of note that each supplier of ADME products or services has developed its own in vitro cell approaches. These, however, are merely variations on several larger themes.

8.7.1. M for metabolism

The best-represented subsector of ADME is that of M (metabolism). This stage consists of identifying and monitoring the outcome within the body of various potential metabolites for candidate drugs. The main metabolization sites are the liver, the intestines and the kidney. Metabolization by the liver is the most studied. This is largely dependent on an extremely numerous and diverse family of proteins collectively called cytochromes P450. In the liver, these proteins work like mono-oxygenases, that is, that they participate in the transfer of one oxygen atom from dioxygen to organic cell substrates. In the ADME context, the substrate is the candidate drug itself. This operation also requires the transfer of electrons, which is done by the NADH or the NADPH.

The cytochrome P450 activity is localized in cell sub-compartments, namely the mitochondria and a fraction called microsomes. The latter can be isolated from the cell and purified. For a long time, it has constituted the basic material for studying the activity of cytochromes P450, particularly in the context of ADME. The most famous pool of cytochrome P450 activity, and also the easiest to obtain, is the S9 fraction, akin to microsomes, which is prepared by centrifugation of cellular homogenate at 9,000g.

The use of cell assays based on hepatocytes derived from human donors is a means of going further in metabolic analysis in the context of ADME. These models are now widely employed in place of the microsomes or the S9 fraction for two key ADME studies, metabolic stability and metabolite identification. It should also be noted that in reality the metabolism declines in two phases, phase I (oxidation) dependent on cytochromes P450, which aims to minimize the direct interaction of compounds with intracellular targets, and phase II (conjugation), which implicates UDP-glucoronosyl-transferase (UGT) among others, which aims to detoxify the compounds by making them water soluble. Indeed, these enzymatic activities are present in all of the models employed, whether they are microsomes, the S9 fraction or cell models. Measurement of P450 activity on a cell model can be performed, for instance, by the P450-Glo system (Promega), which uses pro-luciferin as substrate of the cytochrome P450 subtype considered in order to produce luciferin…, substrate of luciferase. Light production reveals the P450 activity induced by the tested substance.


The induction of the main cytochromes P450 (in this case, 1A2, 3A4, 2B6) in the presence of the drug candidate constitutes another piece of important information made available by means of cells. The measurement used by the industry is a cytochrome P450 messenger RNA quantification performed on hepatocytes from donors. As for the cell models, the choice of using human donors is pertinent but also means that the approach is hard to standardize. Moreover, these models present supply problems together with phenotypic instability.

An alternative strategy consists of using the human stem (bipotent progenitor to be precise) cell line HepaRG capable of differentiating in culture to hepatocytes. Of tumor origin and used in oncology, this model offers the double advantage of being easily standardizable and perfectly competent in terms of its metabolic activity dependent on essential cytochromes P450 (1A2, 2A6, 2B6, 2C8/9, 2C19, 2D6, 2E1 and 3A4). Furthermore, its human origins offer advantages over the use of S9 fractions whose human versions remain expensive and difficult to obtain.

Like other strategies mentioned below for the influx/efflux functions, knock-out cell models are also increasingly used to characterize the P450 cytochromes associated with the metabolism of the assessed candidates.

8.7.2. A for absorption

The capacity of the candidate drug to be transported from the exterior to the interior of the cell and vice versa is one of the essential pieces of data of ADME that can be easily studied in cells. Cell influx/efflux is controlled for the efflux by ABC transporters and for the influx by various specialized organic anionic (OATP) and cationic (OCT) transporters. Furthermore, the efflux function is used ubiquitously to assess the loss of cell homeostasis (see section 4.5) and study the multiple resistance to drugs typically observed during certain chemotherapy treatments. The OATP influx function is particularly important for the transport of the candidate drugs into the liver and kidney.

The comparison between the Madin Darby canine kidney (MDCK) line and the MDCK-MDR line equipped with the human P-glycoprotein (P-gp) allows us for example to detect the presence of P-gp modulators. Other cell lines are available to assess membrane permeability and the influx/efflux functions such as Caco-2 cells (intestinal function), HepaRG cells (hepatic function), PTEC cells (renal function). The Sigma-Aldrich company has very


recently started to offer these same lines but in knock-out versions, that is, in which one or two of the transporter genes mentioned above has been invalidated [SAM 15]. This strategy allows for the identification of the transporter subtype(s) implicated in influx/efflux of the compound assessed.

Concerning the measurement approaches, the same classic technologies described in section 4.5 are used without further innovation.

Permeability assays, called A-B (or transcellular, or vectorial assays) form another original ADME approach [SZA 08]. This involves mimicking a simple epithelial cell barrier (Figure 8.6). The cells used here are cultivated in confluent monolayers and polarized with an apical (A) and a basolateral (B) pole. The function of ABC transporters is calculated according to the ratio of permeability from A to B and B to A. If the transporters are known to be localized on the apical pole, a flow ratio greater than 2 typically indicates the presence of a transport often mediated by the P-gp. Cell lines known to be polarized in this way and expressing the transporters on a precise pole are Caco-2, MDCK and the porcine LLC-PK1 cells. The Caco-2 cells, for instance, are known to establish polarized monolayers and present brush-like edges with characteristic intercellular junctions. The assay can be completed by liquid chromatographic and mass spectrometry measurements performed on both the A and B compartments.

Figure 8.6. Principle of permeability assays for cell influx/efflux evaluation


8.7.3. T for toxicity

The use of cell assays in toxicology is of particular interest for the pharmaceutical industry as it allows for the identification (and thereby the elimination!) of toxic compounds far upstream of costly and extremely selective preclinical trials.

The toxicity assessment of an active principle is a key step performed in parallel to ADME, often taking the acronym ADME/Tox. Toxicity is generally treated with the classic methods of toxicity assessment (genotoxicity, cytotoxicity) which have already been treated extensively. Often a multiplex approach is retained here, for which several cellular parameters are measured independently on each cell by image analysis systems with high throughput microscopy, an analysis known as high content screening (HCS).

Nonetheless, there are several specific studies, in particular cardiotoxicity, for which the toxicity criteria are very different according to the electric excitability of cardiac cells. Cardiac toxicity is expressed, for example, by disturbances of the action potentials that can lead to catastrophic reactions. The assessment of the function of hERG (human ether-a-go-go related gene) receptor, a voltage-dependent membrane potassium channel implicated in repolarization, is widely used during this stage. Most drugs that present side effects on cardiac rhythm are inhibitors of the hERG channel. Inhibition by a candidate drug provokes a long QT on the electrocardiogram that is expressed by a fatal ventricular arrhythmia called Torsade de Pointes, which is responsible for sudden death. An in vitro measurement of this activity has been recommended for the registration of pharmaceutics products since the 1990 International Conference on Harmonization (ICH). An FDA guideline was adopted in 2005. The cell assay developed for this study uses a cell line (for example, CHO or HEK293) equipped with the hERG channel. The measurement is performed by the high-throughput patch-clamp approach mentioned in section 8.6. It should also be noted that the murine equivalent to the human hERG channel is not expressed in the adult cardiac tissue of the mouse, which makes the model totally inappropriate for this type of study.

Other cardiac ion channels have since been added to the collection of cell assays performed on cardiac toxicity, such as the human Nav1.5 sodium channel, equally transfected to this effect in human cell lines. The same patch-clamp approach is used in their study.

Lastly, hiPSC stem cells (see section 10.1) differentiated into cardiomyocytes have recently been used for these same ends.

9

Impact on Health and the Environment

This chapter addresses live cell assays developed in direct connection with public health and the status of the environmental milieu. These two issues are connected and often use similar analysis tools.

Despite the wide variety of approaches available to researchers and, on occasion, to clinicians, few of them are live cell assays. And while many of the experimental approaches seen in Chapters 3 and 8 have been explored with a view to improving diagnostics, understanding pathology or for the production of biomonitoring tools for environmental quality assessment, only a few are actually used.

Military programs linked to toxicity are situated exactly at the crossroads of health problems and environmental issues. They aim to protect the health of soldiers in direct contact with a potentially hostile environment, possibly charged with contaminants introduced deliberately into food or drinking water. Accordingly, it seems appropriate to include these programs in this chapter.

If we are to remain faithful to our definition of cell assays, it is worth mentioning that a few approaches in connection with health or the environment have reached the level of development compatible with standardized, routine and high-throughput use. These approaches are described below.

9.1. Patient diagnosis

The use of patients’ cells in the diagnosis of pathology is an integral part of the history of 20th century medicine. For a long time, this was limited to the microscopic analysis of biopsy tissue (histological approach). By the



1950s, John Enders had observed, as an aside from his use of the HeLa model for the production of the poliomyelitis vaccine (see section 2.2), that cells infected in the culture change morphology. He then used the approach to diagnose the infection directly on patients’ cells. According to Hannah Landecker, it was this development that had at the time propelled cells into the biotechnology era [LAN 07].

The later development of cell analysis by flow cytometry opened new perspectives, particularly in hematology, using cells circulating in fluids such as blood. The main cells taken from patients to be used in cytometry are lymphocytes, monocytes, red blood cells and granulocytes. As university hospitals were generally well-equipped for cytometry, new cell analysis assays were developed in connection to many pathologies including leukemia, lymphomas, or autoimmune illnesses such as spondylitis linked to the HLA-B27 marker together with rare illnesses such as paroxysmal nocturnal hemoglobinuria or Bridges–Good syndrome. These processes were often initiated by hospital services themselves, becoming routine within the laboratory over many years, typically outside of formal procedure of validation or regulatory acceptance, even if initiatives are ongoing. These tools are nonetheless highly significant insofar as they assist clinicians in the diagnosis process.

However, few of these assays meet the criteria used in this work to define live cell assays (standardization, international recognition, throughput, commercialization, etc.). Only a few assays have reached a sufficient level of development. The first is the karyotype assay used in the detection of various syndromes and cancers. Several assays referred to as molecular diagnosis are also recognized and distributed throughout the world. These are IGRA assays for the diagnosis of tuberculosis and MAT assays for diagnosis of the presence of pyrogenic substances for medical material and in connection with septicemia risks. Lastly, several assays are also available for monitoring chemotherapy efficacy.

9.1.1. Cytogenetics

The karyotype is a cell assay that allows for the study of chromosomes. The karyotype is used in public health for diagnosis and screening purposes. Its guidelines concern the antenatal and postnatal periods, particularly where anomalies such as Down’s syndrome, Turner syndrome or others are suspected. The initial method, briefly described in section 2.6, based on contrasted dark-light bands after Giemsa coloration is still valid and in

Impact on Health and the Environment 199

routine use. Its resolution of around 5 megabases is sufficient for observing the classic trisomic (as in Down’s syndrome, for instance) or monosomic chromosomal rearrangements.

In screening applications, the human cells used can be lymphocytes, trophoblastic cells or skin fibroblasts. Culture time can vary from 72 h for the first to two weeks for the others. If the initial sample is taken from blood, treatment with phytohaemagglutinin allows for the stimulation of lymphocyte growth. At the end of the culture, dividing cells are held in the metaphase by the effect of colchicine, a classic inhibitor of tubulin polymerization and consequently of mitotic spindle formation. For observation purposes, the cell membranes are subject to osmotic (hypotonic) shock destabilizing them. Cells are then fixed by a mixture of alcohol and acetic acid. While being spread on the glass slide, these fragilized cells are lysed though retaining their chromosomes brought together. Observation is performed by image analysis under microscope. For applications in oncology, cells used for karyotyping are generally taken from blood, bone marrow or lymphatic biopsies, and occasionally from tumors. Guidelines for the assay concern hematologic tumors.

For inframicroscopic anomalies (below 5 megabases), locus-specific research, telomere rearrangement or interchromosomic exchanges, a cytogenetic molecular technique based on the in situ hybridization of fluorescent sensors called Fluorescence in situ Hybridization (FISH) is used. This type of hybridization allows for the targeted study of certain specific chromosomal regions. The guidelines concern the exploration of complex chromosomal rearrangements and the rapid diagnosis of microdeletion syndromes (Di George, Prader-Willi, Angelman, Williams-Beuren, to mention the most frequent).

The karyotype informative content can also be improved by painting the whole chromosome via a cohybridization of 24 sensors, one per chromosome type. This is referred to as spectral karyotyping, also known as the SKY technique. Here, each sensor is coupled to a specific combination of five fluorophores whose mixture, analyzed by interferometry, applies a unique color to each chromosome.

These technologies are starting to show their age, being increasingly replaced by comparative genomic hybridization (CGH) performed on chromosomes or on biochips.


9.1.2. Diagnosis of tuberculosis

With 8.8 million estimated cases in 2010, tuberculosis is one of the major infections that humanity must confront, particularly given that around a third of the global population are carriers of a latent tuberculosis infection (healthy carriers of Mycobacterium tuberculosis). One of the major problems in the diagnosis of tuberculosis is that there exists no simple method of quantifying the presence of antigens in vivo, as is the case for the HIV virus, for instance. Until recently, and for over a century, the tuberculin skin test remained the only diagnostic tool for the latent form. The skin test uses hypersensitivity, based on the recruitment of memory T lymphocytes at the site of intradermic injection of a mixture of more than 200 of the bacteria’s constitutive proteins. The skin reaction, measured after 48 or 72 h, announces prior exposure to the mycobacteria, or, alternatively, efficacy of the Bacillus Calmette–Guérin (BCG) vaccine.

The arrival of IGRA cell assays (interferon-γ released assays) changed these practices. Their specificity and sensitivity with regards to the latent form of tuberculosis are considered superior to those of the skin test [PAI 08]. Moreover, the latent form of tuberculosis is known for being very lightly charged with mycobacteria and can be missed in vivo. IGRA assays were developed following the works on M. tuberculosis genome in the 1990s, using the knowledge of chromosomic regions absent from the BCG in particular. This led to the identification of early secreted antigen-6 (ESAT-6) and culture filtrate protein-10 (CFP-10), which are coded by the region known as region of difference-1 (RD-1), whose deletion is known to attenuate the mycobacteria’s virulence. For example, RD-1 is absent from all strains of BCG used worldwide. Accordingly, the response of a T lymphocyte to these antigens signals the presence of a real infection by the virulent strain M. tuberculosis.

The two commercial cell assays approved by the FDA rely on the activation of the T lymphocytes by these two ESAT-6 and CFP-10 antigens. The first assay, QFT-GIT (Cellestis, Carnegie, Australia) was developed in 2007. It is comprised of a cocktail of the two aforementioned antigens with a third, TB7.7, which act to stimulate the T lymphocytes present in the blood sample tested. Cocktail and blood sample are put into contact overnight at 37°C. If the T lymphocytes have previously been in contact with M. tuberculosis, they react to the cocktail’s antigens, producing interferon γ (IFN-γ) measured by a classic ELISA approach where INF-γ is sandwiched


between an anti-IFN-γ antibody attached to the support and another anti-IFN-γ coupled to a colorimetric system.

The second IGRA assay approved by the FDA in 2008 is T-SPOT (Oxford Immunotec, Abingdon, UK). Venous blood is centrifuged in order to isolate peripheral mononuclear cells that are counted and redistributed on culture plates whose surface is covered in anti-IFN-γ antibodies, before being put in culture overnight in the presence of ESAT-6 and CFP-10 antigens. In positive cases where T lymphocytes are activated by the antigens, the IFN-γ produced is captured by the antibodies on the surface of the culture plate in the immediate vicinity of the T lymphocyte. The readout system uses a second antibody coupled to the alkaline phosphatase, producing an insoluble precipitate at the reaction spot. Accordingly, each spot represents an individual lymphocyte, with their number attesting to the abundance of T lymphocytes sensitized by M. tuberculosis.

These two assays were quickly adopted following various publications that showed their high levels of specificity (96–99% for QFT, 86–93% for T-Spot) and sensitivity (61–84% for QFT, 67–89% for T-Spot) for the diagnostics of latent form of tuberculosis [THI 14].

In terms of limitations, it should be noted that IGRA assays do not strictly distinguish between active and latent forms of the illness. Another negative point concerns the cost of the assays, which is still far greater than that of the skin test. This significantly limits the diffusion of these assays in countries with limited public health resources. Also, the risk of false-positives is not excluded as the ESAT-6 and CFP-10 antigens are not exclusive to M. tuberculosis but are expressed by four other nonpathologic strains of Mycobacterium from the environment. These strains are very rare, however, and do not present significant problems for IGRA assay validity.

Finally, while the application of IGRA assays is currently limited to diagnosis of the latent form of tuberculosis, many research teams are working on the capability of this approach for predicting the risk of a healthy carrier developing the pathology, and if so over how long.

9.1.3. Cell assay for the detection of pyrogenic substances

The term “pyrogenic” covers a series of toxins that act to increase body temperature. The most well-known of these are endotoxins, lipopolysaccharidic in nature, liberated during bacterial lysis (gram negative). They are associated


with septic shock. There are also nonendotoxin pyrogenic substances such as lipoteichoic acid (from gram-positive bacteria), and particles of yeast, of viruses or derived from the environment. Due to these major systemic toxic effects, apyrogenicity is an essential condition for medical material, particularly for that used in injections. Critical levels of pyrogenicity have been set by regulatory authorities, with various measurement approaches already validated. The ISO 10993-11 (2006) norm for the biological assessment of medical devices states in annex F that pyrogenic effects must be assessed for endotoxins by test LAL and for nonendotoxins by an in vivo testing on rabbits.

For the test on rabbits, the sample under assessment is injected into three animals. Their temperature is taken. The result is positive if the increase in temperature exceeds 1.15°C.

The limulus amebocyte lysate (LAL) assay is one of the oldest assays on the market. It follows an observation of Fred Bang in 1956. At the time, this American researcher was studying blood circulation on the model of the horseshoe crab (Limulus polyphemus). The species is nicknamed king crab due to the blue color of its blood, a peculiarity due to the presence of copper, rather than iron, in the heme of its blood cells specialized in oxygen transportation. One day, during an experiment, Fred Bang found one of his Limulus had turned into a gelatinous mass. He investigated the matter, finding that its death could be retraced to the presence of Vibrio cholerae bacteria. Curiously, the same effect was reproduced using dead Vibrio bacteria, then using other bacteria, both alive and dead, all gram-negative. It was later discovered that the mechanism involves Limulus lymph cells, amebocytes. These cells are covered in granules filled with a coagulation factor, coagulogen, sensitive to infinitesimal doses of endotoxin. The test was approved by the FDA in 1970. From then, it was routinely used for establishing the endotoxin levels in pharmaceutical products and medical material. It replaces the pyrogenic test on rabbits, though has not been approved for the diagnosis of septicaemia. The test kit includes a lyophilized Limulus amebocyte cell lysate. Due to the massive demand, the collection of Limulus blue lymph became tantamount to an industry with nearly half a million individuals caught in the sea each year. Ultimately, the rabbit test has indeed been replaced by an in vitro test, albeit one that consumes a colossal quantity of crabs. In response to the controversy, the Limulus are now reimplanted in their natural habitat having lost a third of their blood and with a chance of recovery estimated according to studies at between 60 and 85%.


Since 2010, a cell assay called MAT has been introduced into the market. This has the advantage of not threatening any animal species. It also allows for the assessment of endotoxin pyrogenic effects and, unlike the LAL assay, nonendotoxin effects with excellent sensitivity. The monocyte activation test (MAT) uses frozen human blood taken from donors. It is sold under the name PyroDetect System by the Millipore company. The working principle is different to that of LAL. The MAT assay mimics the innate immune response arising in the context of fever provoked by a pyrogenic effect. In humans, the immune response involves interleukins 1β and 6 and interferons α and γ. The MAT assay doses the interleukins 1β produced by the activated monocytes. In operational terms, the test sample is mixed with human blood on 96 well plates then left to incubate overnight. If pyrogens are present, then interleukins are produced, which are detected in the morning by means of ELISA based on the double recognition by antibodies, of which one is coupled to a horse radish peroxydase (HRP) activity that produces a colorant based on luminal [WUN 14].

9.1.4. Cell assays for predicting efficacy of chemotherapy

The problem of multidrug resistance (MDR) in chemotherapy exists since the outset of the discipline and has never been totally resolved despite the discovery of the ABC family of transporters, coded by genes of the mdr family, as seen in section 4.5. This assessment demonstrates, if it was needed, the extreme complexity of cancer biology. In any event, clinicians are confronted in their therapeutic choices with a major problem in so far as almost 50% of cancer cases present this type of resistance from the very beginning of the treatment, while a significant part of the remaining half develop resistance through the course of treatment.

In this context, a pretreatment diagnosis of the tumor status with respect to resistance to treatment on patient level would clearly improve the prognosis. For a long time, the only observable criterion of a treatment’s effect was the development of tumor size observed a posteriori. Three methods are now available on this therapeutic front [LIP 09]: the culture of cells taken from fresh tumors, serum biomarker tests (like the PSA antigen for prostate cancer or CA125 for ovarian cancer), and positron emission tomography (PET) that uses image capture to analyze the dynamic evolution profile of the tumor.

Only the culture of cells taken from fresh tumors is of interest to us here. The approach has existed since the 1950s and reached its pinnacle in the 1970s. Techniques for the cell preparation were standardized for many types


of cancers: colorectal, testicular, skin, lung, brain, ovary, breast, pancreas, etc. The procedure consists of transferring the biopsy to the laboratory, dividing it into small fragments and then totally dissociating it by enzymatic digestion (day 1). The cells are then placed in culture and incubated for 6 days with the therapeutic agent at concentrations judged appropriate to stimulate the treatment effect. Metabolic activity is measured on day 7 with the help of the classic methods described in Chapter 4, the most widespread, ATP-TCA, also called ATP-b (HAT 09), using ATP measurement by bioluminescence (see section 4.7). An absence of reduction in ATP production signals resistance to the tested drug [AND 95].

However, several criticisms published most notably in the New England Journal of Medicine in the 1980s have moderated the use of these assays, highlighting their limited predictive ability, putting an end to their routine use. Improvements implemented since then have allowed us to draw a line between drug sensitivity and resistance. These cell assays are indeed unreliable for assessing sensitivity, however they are reliable for predicting resistance. The assays on fresh tumor cells are officially in clinical use today for cases of ovarian cancer as a selection means when several equivalent chemotherapy options are available [NCC]. They have also been validated by the Japanese Ministry of Health for monitoring advanced gastric cancers.

9.2. Military programs

Another aspect of public health concerns the health of soldiers, in the knowledge that ultimately these specific studies could contribute to the implementation of protection schemes for the wider population.

A small number of studies made public allow for the assessment of the role of cell assays in military procedures, particularly those addressing the issue of bioterrorism. The application areas that we have been able to identify concern:

– diagnostic assays;

– the research of inhibitors and neutralizing antibodies working specifically on agents known to be lethal, such as botulinum toxins, anthrax, toxins derived from Clostridium difficile, Shiga-type toxins or ricin, to cite only the best known examples;


– field assays concerning drinking water, not for bacterial pathogen agents (this problem has long been settled) but the presence of chemical toxins, in particular those connected with attempts of water poisoning.

Note that two processes are performed simultaneously. One specifically targets known toxins, the other, more generic in principle, aims to take advantage of cell models as a source of expression of the toxic effect independent of the nature or type of toxin responsible. Unfortunately, due to the fact that military research is typically confidential, by no means can this section claim to be in any way comprehensive.

9.2.1. Detection and screening of botulinum toxin inhibitors

Botulinum is an illness provoked by the neurotoxins produced by the Clostridium botulinum bacteria. They are known to be the most effective natural toxins on human beings (lethal dose approaching 0.1–1 ng/kg of body mass). They work by inhibiting synaptic neurotransmission leading to muscle paralysis and respiratory failure. Exposure can be accidental or associated with hostile action with the aim of poisoning, a major danger at population level, still more so given than toxins can remain stable and active for months.

It is by no means a frequent occurrence for a serious military problem such as bioterrorism to cross paths with a key issue of the cosmetics industry. But if there is an example that fully typifies the doctrine of the Swiss physician and researcher Paracelsus (1493–1541) whereby “the dose makes the poison” (see Figure 6.2), it is indeed that of the botulinum toxin. While already lethal at very low doses, at still lower doses, it proves to be an excellent therapeutic agent and an extremely effective muscle relaxant that is widely used in controlled topical application under the name of Botox.

Botulinum toxins are comprised of two subunits called heavy and light chains, connected by a covalent disulfide bridge. The introduction of the toxin in the neuron is performed by endocytosis. This is dependent on heavy chain domains that interact with the plasma membrane. Once in the endosome, the toxins dissociate themselves into free subunits. With the assistance of the heavy chain, the light chain rearranges itself and enters into an active proteolytic conformation. The resulting enzyme is a zinc metalloproteinase, which, by endopeptidase activity, specifically cleaves the SNARE protein, an essential control point of exocytosis of the synaptic neurotransmitters.


The botulinum serotype A protein is of particular interest to the military (and the cosmetics industry!). It is this protein that provokes the inhibition of acetylcholine liberation in the neuromuscular synapse, leading to the mechanism of flaccid paralysis that typifies botulism. In molecular terms, the effect is due to the toxin’s cleavage of a component of the SNARE family, SNAP25, which loses its 9 C-terminal amino acids going from SNAP25206 to SNAP25197 (Figure 9.1).

Figure 9.1. Action of the botulinum toxin at the neuro-muscular junction. (1) Acetylcholine release in the synaptic space requires the functional SNARE protein complex; (2) internalization of the botulinum protein (LC + HC) after binding with receptor SV2; (3) activation of LC (protease) by HC; (4) cleavage of SNAP25 by LC renders SNARE inactive, blocking acetylcholine release and muscular function

Before launching a high-throughput screening program for inhibitors of botulinum toxins, the United States military had the idea of developing a cell assay for measuring proteolytic activity using antibodies that only interact with the whole version of SNAP25 as opposed to the cleaved version [NUS 10].

Trial platforms were set up concurrently to these works in order to detect botulinum toxins in food samples, both for quality control and for field applications in potentially contaminated areas. Here too, the first tests to be implemented took mouse LD50s as a quantitative measurement following intravenous or intraperitoneal injection. Variation of results between laboratories was particularly high here, between 20 and 40%. One method of standardization published in 2003 nonetheless managed to reduce this disparity to 15%.


An intermediate method between in vivo and in vitro, known as ex vivo has been proposed in recent years. This is a model simulating the respiratory failure observed in vivo and based on an organ extract (diaphragm and phrenic nerve) excised from a balb/c mouse. With the nerve acting on the diaphragm’s contraction and relaxation, the electric current resulting from muscular activity is measured.

Finally, several cell assays were developed [PEL 13]. They can be grouped into several categories, immunologic, endopeptidase and functional and use neurons or neuronal lines. These new approaches represent real progress. The ELISA immunologic cell assays are more sensitive than the mice tests though do not distinguish between active and inactive toxins. Inversely, endopeptidase activity assays are functional but confronted with the problem of false-positives due to potential protease activity present in the sample. Functional assays on neurons are the most highly developed in that they cover all stages of intoxication associated with botulinum toxins. Here again, sensitivity is typically an improvement to that of animal tests. Several approaches were developed around 2010 such as SNARE cleavage measurement on cell lysate by Western blot (low throughput) or by ELISA. The classic fluorescence approaches such as FRET have also been explored in order to perform more functional measurements on living cells. A SNARE–FRET construction was performed following the caspase protease activity assays method seen in section 4.9, albeit with disappointing results compared with those of mice tests.

Here we should make reference to the history of Allergan, even if it is placed outside of the military context. In June 2011, the French company (headquarters in Irvine, California) that develops and supplies ona-botulinum-toxinA (type A botulinum toxin), better known by the name Botox, announced that the FDA had validated the regulatory use of its cell assay for the efficacy and stability of Botox. This assay replaced the mice test that had been in use until then. Allergan announced that it had spent 10 years and $65 million to achieve this result. Here we see the incredible amounts of effort required to convince regulatory authorities. The assay principle was published [FER 12] in November 2012. This is again a measurement of SNAP25 cleavage on the cell lysate taken by sandwiching the SNAP25197 peptide between an anti-SNAP25197 stuck to the support and an anti-SNAP25 antibody coupled to a Sulfo-tag chemiluminescent marker (method called ECL-ELISA). The success of this method depends in part on the use of a neuroblastoma line, SiMa, more sensitive to the toxin’s effect than the more typically used Neuro2A and PC12 lines.


9.2.2. Antibody-based toxin neutralization assays (TNA): application on anthrax and ricin

Bacillus anthracis is a spore bacterium responsible for a deadly pathology, anthrax. Here we again find the same causes and the same effects as for the botulinum toxin. Anthrax spores are toxic at very low doses, stable for several months and easily dispersed as aerosols, making them a fearsome weapon in bioterrorism. The picture is complicated here by the fact that diagnosis is difficult to establish with the first symptoms being nonspecific. The result is a particularly low survival rate from 10 to 55% depending on the situation [WHI 12].

In fact, the bacteria produces a couple of toxins called edema toxin (ET) and lethal toxin (LT) that operate in a sort of ménage à trois. In its intracellular active form, LT is composed of the lethal factor LF and the protection antigen PA. As for ET, it is composed of PA and edema factor EF. There follows a sequence of biochemical events. PA binds to its plasma membrane receptor, undergoes cleavage by the furin then polymerizes into a particular heptameric structure that recognizes the EF and LF factors and guides their entry into the cell. It is these last two factors that act when associated with PA.

EF is an adenylyl cyclase that, following the model seen in Chapter 7, produces cyclic AMP, the second messenger involved here in the expression of edema [TAN 09]. It is thought that the EF activity, dependent on the presence of calcium and of calmodulin, a protein cofactor, consists in facilitating the survival of the bacteria by inhibiting the immune response triggered by competent cells.

LF, on the other hand, is a zinc metalloprotease that, as with the botulinum toxin, works by cleaving the terminal segment of a kinase, here the MAPK, thus blocking the transduction pathway controlling cell proliferation.

Neutralization cell assays for the anthrax toxin have been developed during the last decade. They use the murine macrophage lines RAW264.7, J774A.1 or the more classic CHO cell line [NGU 10]. These models are preferred to human models that, strangely, are not sensitive to the toxins, at least in terms of cytotoxicity, which remains the criterion that is easiest to assess by means of the wide array of measurement approaches seen in Chapter 4. The CHO line itself was used specifically in the development of an ELISA chemiluminescence assay for the measurement of cyclic AMP on cell lysate.


Concerning ricin, a toxin derived from the Ricinus communis plant, an Israeli team developed in 2015 a new cell assay on HeLa capable of detecting its biological activity within a few hours [GAL 15]. Ricin is considered to be one of the most deadly toxins for human beings. It is a glycoprotein comprising two subunits connected between one another by a disulfide bridge. The B subunit is a lectin that favors the binding of the toxin on the plasma membrane and its entry into the cell. The A subunit is an N-glycosidase that deactivates the ribosomes, thus blocking protein production and entailing cell death soon after. In technical terms, the method developed resumes the fluorescence-based ones, described in section 3.1.

The assay is based on the activity exerted by ricin on HeLa cells stably expressing the ubiquitin (Ub-FL) degradation system coupled to luciferase. The assay measures the capacity of the ricin to act on protein synthesis via the modification of ubiquitination activity. The authors propose the high-throughput use of the assay in targeting neutralization antibodies and other ricin inhibitory molecules.

We should also mention current developments for the main toxins cited using the construction of new neutralization antibodies based on the famous VHH (heavy-chain only) antibodies [HER 15], which are camelid antibodies known for possessing only heavy chains (VH), conferring on them the capability of penetrating intracellular compartments.

9.2.3. Field measurement of water potability

This is certainly the label-free cell assay that has reached the highest levels of development outside of the pharmaceutical industry. The United States military has for many years been interested in the development of simple and reliable field device for the detection of toxins in drinking water. Until 2010, these technologies were essentially dependent on the analysis of specific agents. These approaches remained limited in the face of an increasing number of threats, particularly with regards to the use of unknown or improbable toxins.

In order to establish a cell assay capable of integrating these threats, four essential biological and technological barriers had to be overcome:

– finding a competent cell model in terms of toxicity measurement;

– ensuring the transfer and maintenance in culture of these models both autonomously and over the long-term;


– choosing a miniaturizable measurement technology compatible with portable instrumentation, readily available in the field at short notice;

– obtaining a rapid diagnosis, within a few hours at most. This is what seems to be in the process of being achieved according to reports published between 2012 and 2014.

The barriers concerning cell models have already been overcome thanks to the rainbow trout gill cell line called RTgill-W1. This line, well known to specialists in environmental toxicology since 1993, has several major advantages [BUR 14]: it is adherent to culture support, compatible to 96 well plates, robust and sensitive to many toxins at doses compatible with public health issues. Its high sensitivity is due to its origin, the gill, an organ soaked in water and serving as first point of contact with xenobiotics. Furthermore, it has a surprisingly long capacity for conservation at 4°C without requiring carbon dioxide. The RTgill-W1 cells can effectively remain dormant for several dozen weeks in their culture dish without requiring any attention from outside.

Another development came in 2012 with the introduction of the ECIS label-free impedance technology described in section 3.2. The desk top apparatus used in 2012 has since been the subject for miniaturization, integrating a fluidic biochip containing RTgill-W1 cells in confluent monolayers [BRE 12]. These layers grow on a support whose interface is equipped with electrodes. The device described in 2014, weighing around 1.4 kg and measuring 23 × 14 × 9 cm, can function on the mains or on battery. In their report, the authors recommend keeping the biochips filled with cells in the containers used for transporting temperature sensitive liquid material such as blood transfusion packs.

According to initial data [WID 15], the device detected eight out of nine toxins tested (ammoniac, arsenic, azide, copper, mercury, pentachlorophenol (PCP), toluene, methylparathion) after only 1 h of contact at doses below the Army’s human lethal concentration (AHLC) norm based on consumption of 15 L of water per day for a 70 kg adult. Of course, these preliminary experiments were performed by contaminating the water samples with toxins, which remains a simplistic approach. What’s more, the impedance measurements could be positive (copper for example) or negative (mercury for example) and thereby offset each other whenever two or more toxins are present in a field sample. Nevertheless, it remains that this apparently unique approach miniaturizing cell assays represents real progress, opening new perspectives for application, particularly for the measurement of environmental pollutants.


9.3. Pollution and quality of environment

The notion of ecotoxicity in the wider sense extends from the impact of pollutants on the ecological milieu to the measurement of toxicity with respect to human beings based on samples taken from the environment. These two disciplines appeal to very different methodologies. The study of disturbances to ecosystems typically appeals to notions of biomarkers, sentinel populations or species, which are outside the remit of this book. Cell approaches are only used occasionally and on the fringes of the issue in order to understand the toxicity mechanisms at work. However, monitoring pollution or toxicity for reasons of public health can involve simple and routine measures (biomonitoring) that can appeal to cell assays. This is the case in determining water potability, at least in terms of chemical contamination, as briefly described in the preceding section. Furthermore, certain cell lines initially created to satisfy ecological problems are currently used for public health reasons, such as the RTgill-W1 trout cells also mentioned above.

The complex nature of the test samples is something that characterizes the measurement of contamination present in the environment. Independently of the contaminant effect, any methods chosen must confront a significant matrix effect that can often hide the signal and generate false-positives.

Due to their natural sensitivity, living microorganisms are well suited to measure contaminants in a liquid, such as river water for instance. The advantage of this process compared with detecting specific contaminants is of once more integrating the toxic response at the microorganism level independently of the known or unknown nature of the contaminant(s).

Note that with regards to the environmental sector, the test standardization passes either through a validation and an OECD guideline or through a certification and an official ISO norm.

9.3.1. The MicroTox assay

Aliivibrio fischeri is a remarkable marine bacteria, known for being symbiotic with animal species such as certain fish or squid, in whose light-producing organs it lives. In exchange for a little food, it provides them with precious light that serves them in the darkness of the oceans to entice prey, attract a mate or intimidate potential aggressors. Its symbiosis with Eupryma scolopes, a squid living in the warm waters of the central Pacific is an


example of sophistication. The light emitted by A. fischeri is redirected thanks to, among others, the activity of the E. scolopes refectin protein present in the reflective plates of the cephalopod’s light-producing organs. The photons thus canalized are used by the squid to disguise its shadow as it moves above rocks, thereby escaping certain predators that use the contrast to spot their prey. It is also of note that A. fischeri, which is nonpathogenic for human beings, is a close relative in phylogenetic terms of Aliivibrio cholerae, and indeed retains the imprint of the latter’s cholera toxin as a pseudo-gene.

The benchmark MicroTox assay for determining aquatic toxicity makes elegant use of the natural bioluminescent property of A. fischeri. This assay was developed in 1979 on the premises of the Azur Company by Anthony Bulich, and then commercialized in the following years by Beckman Instruments. It was then presented as a simple, reproducible and, above all, fast approach for the measurement of water contamination. It is limited by low sensitivity to metals and a hypersensitivity to detergents. Disturbances to cell homeostasis according to the toxicity of the agent tested are announced by a reduction in metabolism and in photon emission whose measurement allows for the calculation of EC50. The MicroTox assay was accepted as the preferred method due to its very extensive range of detection (around 1,200 toxic agents already detected) and above all for its execution speed of between 15 min and 1 h. This success led to it being standardized then certified in 1998 by the norm ISO 11348-3 (revised in 2007). An automated and continuous MicroTox analysis approach for water toxicity was proposed in 2012. The authors demonstrated that the system works, but indicated the low EC50 sensitivity of the MicroTox assay since most EC50s remain superior to the pollutant concentrations found in water [LOP 12]. Moreover, they note a hypersensitivity to disinfectants such as chlorine, which is widely used.

9.3.2. Mobility of the Daphnia test

The main rival test, based on measuring the mobility of Daphnia magna, has a much longer history. It was developed in 1934 and was the subject of an ISO (no. 6341, revised in 2012) norm in 1996 and adoption by the OECD in the form of guideline no. 202 entitled: Daphnia sp. Acute immobilisation test, dated April 1984 and reviewed in April 2004. The test is applicable to the measurement of acute toxicity (24 h and 48 h). It is not, strictly speaking, a cell assay since daphnia are multicellular creatures. Its application domain is somewhat different to that of MicroTox. It is more sensitive to certain contaminants and less so to others. The measurement is an EC20 or an EC50 corresponding to the concentration at which 20% or 50% of the daphnia are


immobilized. The test is more complex to implement due to the requirement of cultivating the daphnia, which reproduce by parthenogenesis. The crustaceans are left in survival mode (in the dark, with no food) for typically 48 h with five individuals per recipient along with increasing doses of the assessment sample.

9.3.3. Fish embryo acute toxicity (FET) test (OECD guideline no. 236)

This approach was adopted by the OECD July 26, 2013, under guideline 236. This is a test for acute toxicity (1–4 days) performed on freshly fertilized zebrafish (Danio rerio) embryos. The observation of toxic effects is manual and microscopic: coagulation level of fertilized eggs, presence or absence of somite formation, detachment or not of caudal bud (tail) in the yolk sac and the presence or not of a heartbeat. As a consequence this is a low-throughput test.

The test is performed in 24 well plates, requiring a microscope and a controlled aquarium environment. In addition, the progenitor fish must be nurtured. Reproduction can take place in spawning tanks with males and females in a ratio of 2:1 put together before nightfall. According to the OECD guideline, artificial plants can be added to act as a spawning stimulus! Caged spawn traps are fitted to avoid predation of the eggs by adults. The first division occurs after 15 min followed by synchronous divisions until the 32 blastomere stage. Exposure to the test sample begins at latest by the 16 blastomere stage. 20 embryos spread between 20 wells are used for each sample concentration tested. The positive control used is 3,4-dichloroanilin at 4 mg/L.

Beyond the measurement of environmental toxicity performed on biological systems such as daphnia or zebrafish embryos, which may be considered in vitro, the OECD has adopted in its guidelines 201 and following various tests performed on adult individuals of numerous species: fish, birds, amphibians, bees and earthworms. And in terms of norms, we note one toxicity test making use of the multicellular fresh water rotifer Brachionus calyciflorus (French norm NF T 90-377). Many other approaches that either use unicellular prokaryotic or eukaryotic microorganisms have been employed in various contexts without ever attaining the status of standardized and recognized tests.


9.3.4. The DR CALUX assay

The CALUX approach was developed by the Biodetection Systems (BDS, Holland) company to measure the presence of dioxin or its derivatives in the environment and agribusiness. The cell function targeted is the aryl-hydrocarbon receptor (AhR) activated by various pollutants such as dioxins, PCBs, furans or HAP such as benzo(a)pyrene.

The Ah receptor is a transcription factor maintained inactive in cytosol in the form of an AhR/hsp90/AIP complex. The binding of a ligand such as dioxin liberates AhR from the hsp90 chaperone and its AIP co-chaperone. The AhR then passes into the cell nucleus and acts as transcription factor after association with a cofactor (or translocator) called ANRT. The AhR/ARNT complex then activates the dioxin response element (DRE) present upstream of several genes, the best known of which are CYP1A1, 1A2, 1B1 coding for P450 cytochromes (Figure 9.2).

Figure 9.2. Principle of DR CALUX assay to measure the presence of dioxin or its derivatives: (1) the ligand binds to cytosolic AhR; (2) AhR

activates transcription of luciferase gene leading to (3) expression of the luciferase; (4) cell is then lysed and luciferin added as read-out


The DR CALUX approach is based on the modified rat hepatoma H4IIE cell line, stably expressing the DRE coupled to the luciferase reporter gene [SCI 04]. The complex samples taken from agribusiness or the environment are treated upstream by sulphuric acid in order to eliminate fats in particular, followed by chromatographic fractionation. The bioluminescence read-out is performed on cell lysates. Variations of CALUX have been proposed that target more specific ligands.

9.3.5. Biomonitoring and field issues

One of the major stakes in environmental quality assessment is spatiotemporal monitoring. Beyond mere diagnosis, this allows for the acquisition of evolution profiles in the pollution of a particular area, searching for patterns and projecting prospective scenarios. For these programs to be realistic in terms of cost and organization, they ought to be performed as close as possible to the sample source. They should be performed outside of the comfortable environment of the cell biology laboratory (i.e. without culture facilities, thermoregulated incubator, storage in liquid nitrogen, heavy instrumentation, etc.).

In addition, measurement has to be fast (in the range of several hours) and robust. Manipulation has to be simple, and not reliant on complex operational or technical abilities. The measuring tool has to be light and portable in order to be deployed to the site. Given the recurrent need for data, above all, measurement has to be cheap. The use of cell assays (or of microorganisms) seems to meet several of these criteria, and various approaches are beginning to be developed accordingly.

One overview, published in June 2013, observed that up to that date no field detection tool was available on the market [MIC 13]. Since May 2014, a first portable tool (around 1 kg) dedicated to the assessment of a wide range of biocontaminants and usable on site has been commercialized by the British company Modern Water based on the model of the MicroTox bacterial assay described above.

However, two obstacles remain concerning the application of cell assays for field applications. The first concerns storage possibilities for cell models in the absence of freezing, the second, contaminants’ bioavailability.

The storage of bacteria is relatively easily done by lyophilization followed by rehydration at the time of the test. Many prokaryotes share this


property and are able to reduce their water content to extremely low levels as little as 2% over very long periods. In such cases, it is easy to conserve bacterial materials over very long periods in proximity to the testing location. However, this is by no means the case for eukaryote models. Among the few multicellular animals tolerant of desiccation are tardigrades (known for production of trehalose, a vitrification sugar), certain rotifers from fresh water zooplankton and some nematodes. Cell models of plant or mushroom (Saccharomyces cerevisiae, for instance) can also go through this sort of cycle without too much damage.

Despite several results announced by an Israeli team [NAT 09], the lyophilization of human cells, or more generally of mammal cells, has not been mastered. The consequences of lyophilization for the cell are extremely severe. The major deleterious factors are in connection to the cell membrane (lipid phase transition) and cytoskeleton, to which is added the damage to lysosomes or mitochondria at the time of rehydration. Desiccation is also accompanied by a reduction in metabolism, an increase in viscosity and salt concentration, protein denaturation, macromolecule aggregation. To complete the clinical picture, the creation of free radicals following the Fenton reaction is also observed, together with products from Maillard reactions… Nothing to feed the hope of finding a healthy cell at the end of the process. Indeed, cells live in water. And even if all of these steps were circumvented, the problem of a traditional culture environment would still be unresolved, in particular the need for an incubator thermoregulated at 37°C maintaining constant levels of CO2.

An interesting solution is to use the cells of vertebrate poikilotherms such as the RTgill-W1 rainbow trout line, mentioned earlier in the context of military programs, whose growth shows a certain flexibility in terms of temperature. Additionally, this type of cell can be kept without growth for several months at 4°C and has the advantage of not requiring any supplementary CO2 supply. These precious properties are shared by several other cell lines taken from geckos, iguanas or other fish such as the Japanese eel or the tilapia, which can be used as alternatives [CUR 13].

The second obstacle mentioned above concerns sample contaminant bioavailability. This problem is often dismissed by suppliers of measurement technologies or kits. Even in academic studies the conceptual proofs are often produced by contaminating a host milieu, typically water, with increasing doses of contaminants. These results are difficult to extrapolate to the reality in the field, where the sample is never simple but rather a complex matrix in which organic molecules jostle one another. In addition, this matrix itself can


exert a toxic effect due to it being the site of numerous contaminants complexing with the living and particle matter. Even a most simple matrix like tap water can be problematic as seen with the chlorine effect perturbating the MicroTox assay.

Yet by complexing, contaminants that are present in the sample often lose their reactivity, becoming invisible to the measurement. In other words, their water-solubilized portion is often low. They are not then bioavailable with regards to the cell model that is presented to them for toxicity analysis. This problem of bioavailability, that does not preoccupy chemists, is specific to cell approaches.

One last key point to consider is that of bioaccumulation and bioamplification. Certain contaminants, heavy metals for instance, bioaccumulate in the body of animals and plants exposed. This is particularly the case for aquatic species such as microorganisms, algae, crustaceans, fish and aquatic mammals. This bioaccumulation is then amplified by the fact that these species are predatory between one another. There follows a bioamplification of the contaminant levels between species from the beginning to the end of the trophic chain. For example, the methylmercury level ratio between contaminated water and the brain or the flesh of a piscivorous fish or cetacean can be of the order of one in a million. So it would be much more pertinent to analyze samples such as fish flesh, which is more representative of the real amount of contaminant that will reach the plates of consumers. Of course, the problem of bioavailability due to the complexation of contaminants in organic material, particularly in fats, must also be considered.

In conclusion, the use of cell assays for the assessment of aquatic environments is promising. Cell assays lend themselves well to miniaturization, indeed some models are compatible with use in the field, though their use, unlike that of chemical measurement methods, must account for the effect of matrix and bioavailability.

10

Outlook

10.1. Stem cells, an opportunity for the future of cell assays

By their capacity of differentiation to a quasi-infinite array of physiological cell models, stem cells open up an array of application horizons that have remained limited until recently by the cancerous phenotype of classic cell lines. By providing easily standardizable, human, highly differentiated and physiological (as opposed to tumorous) models, stem cells could someday eliminate the controversy linked to the predictability of in vitro versus in vivo approaches with regards to human effects. Furthermore, the capacity of stem cells to maintain their differentiated phenotype over the long term represents another major advantage for industrial applications.

Stem cells can quickly be categorized according to their origin in adult or embryonic tissue. The first embryonic stem cells were isolated and maintained in culture in 1981 from murine tissue and then in 1998 from human embryo blastocysts [THO 98].

On the regulatory side, human embryo stem cells’ potential for toxicity measurement was explored in the context of a European project called Embryonic Stem cell-based Novel Alternative Testing Strategies (ESNATS) (2008–2013, €11.9 million). This targeted four areas of research: reprotoxicity, neurotoxicity, toxicokinetics, while continuing the quest for the toxic signature (genomic and proteomic) of embryonic stem cells. The project ultimately selected a battery of five approaches (implemented over 6 weeks at a cost of €11,000) without, however, reaching any conclusion, despite a hundred scientific publications covering their effective applications in the regulatory sector [HTT].



In addition, the human embryonic origin gave rise to serious ethical problems that thankfully have been bypassed following the discovery of stem cells in adults, identified within different organs such as the central nervous system or the derma. The breakthrough in the area of stem cells unequivocally came with the 2006 discovery by Shinya Yamanaka (Nobel Prize in 2012) of adult murine somatic cells’ capacity to be reprogrammed in models presenting all the characteristics of embryonic stem cells [MES 12]. These cells were named induced pluripotent stem cells (iPSCs). From 2007, the same team and others produced human iPSCs from dermal fibroblasts [TAK 13].

However, the four transcription factors (Oct3/4, Sox2, Klf4 and c-Myc) required to reprogram the iPSCs had to that date only been expressed using viral vectors that proved problematic in that they could integrate transgenes in cell DNA, leading to genomic disorders. This would be resolved by different strategies such as the transfection of RNA messengers, micro-RNA, minicircle DNA or with non-integrating viral vectors [HAY 13].

Today the differentiation in culture of human iPSCs (hiPSCs) to neurons and muscle, cardiac, blood or other cells is beginning to be well controlled. They began to be used in the measurement of toxicity specific to organs or tissues, in particular of hepatic, cardiac and neuronal origin [SCO 13]. To limit the present treatment to the use of hiPSCs in terms of cell assays, we note various fields of potential application. The most basic concern high-throughput screening, which, thanks to these differentiated tissue models, work closer to the physiologic context than classic approaches using cancerous models by default.

The phenotypic screening approach (pathology orientated) addressed in section 8.1 could also be developed significantly with the use of patient-derived hiPSCs according to the plan presented in Figure 10.1. Indeed, these hiPSCs have the peculiarity of conserving the genetic characteristics of the patient whose phenotypic expression can be assessed through comparison with the hiPSCs derived from healthy donors. These screening campaigns have recently begun for various major clinical problems such as neurological problems (Charcot’s disease, schizophrenia, Parkinson’s disease, Alzheimer’s disease), cardiac illnesses (long QT syndrome, diabetic, hypertrophic or dilated cardiomyopathy) or even hepatic, blood and skeletal pathologies [TAN 15]. Personalized medicine procedures based on these same culture protocols but now with the aim of assigning patients with the most effective drug for him or her, are also undergoing assessment

Outlook 221

[FER 13b]. In terms of high-throughput toxicity screening, cardiomyocyte and hepatocyte differentiated hiPS cells are beginning to be exploited.

Figure 10.1. Various possible ways of using human stem cells taken from adult somatic cells

How can we estimate the true pertinence of hiPSCs compared with more classic cell lines? A recent study [PEN 13] reassessed 44 compounds known to be neuroprotective in mice or classic models of human cells. The authors observed that on the one hand only 16 of these 44 compounds proved their efficacy on the hiPSC model and, on the other hand, that this difference was positively correlated with the performances demonstrated by these compounds during clinical trials. These observations and others make the use of hiPCS cells particularly attractive in the sector of new candidate drug identification [ENG 14].

Regenerative medicine forms another future area of application for hiPSCs, the culture of which is the only source of differentiated autologous cells. Further genetic modifications can be envisaged in order to correct (or generate when deleted) the mutations involved in various pathologies. However, this last application falls outside the remit of this work.


As no cell model is ever perfect, here we will note the limitations of hiPSCs. The generation of differentiated hiPSCs is a complex process that uses totally differentiated adult cells to dedifferentiate them in pluripotent stem cells before reprogramming them to a new type of differentiation. Yet these reprogramming methods have a particularly low yield (often lower than <0.1%!), high production costs, contamination sources with precursor cells not mastered and chances of genetic or epigenetic modifications through their long life in culture. At the end of the process, certain industrial actors advocate performing a karyotype analysis or more in-depth cytogenetic studies even going as far as sequencing the entire genome [ENG 14]. For these reasons, the number of cell types differentiated from hiPSCs remains limited to a few suppliers that have mastered these points. A recent study of hiPSC production by reprogramming fibroblasts presented a culture program spread over 12 weeks before obtaining mature neurons [KUM 12]. However, faster and more direct methods of neurogenesis also feature in the study.

In conclusion, stem cells are still far from becoming the norm for live cell assay applications. Their potential as more physiologic models is evident nonetheless and we can be assured of their capacity to ultimately replace many classic cell lines in a variety of areas.

Lastly, stem cells are naturally associated with current developments on organotypic models, whose outlook will be resumed in the following section. It could also be said that models such as hiPSCs represent the cornerstone in the construction of a future global solution.

10.2. Organs-on-a-chip

Even if progress can be expected due to adult stem cells, the glaring question that arises throughout this work has still not been resolved. Ultimately, how representative can cell assays really be in the role of assessing physiological and pathological aspects native to complex organisms such as human beings?

It is widely agreed that they are not very representative. This would appear to corroborate the words of the great French doctor and anatomo-pathologist Marie François Xavier Bichat (1771–1802), that “the tissue is the plastic principle of the living being and the final term in anatomical analysis” [CAN 52]. In their recent review article, Andries van der Meer and Albert van den Berg (University of Twente, Holland) went still further. They posit that cell approaches have reached an impasse [VAN 12]. These researchers

Outlook 223

present the problem in the following manner: there are two in vitro approach models, the simple model and the realistic model. For the former, the response to a stimulus is typically homogeneous and predictable though often of limited value vis-à-vis physiological reality. For the latter, the information level is closer to physiological reality but with the answer observed being more heterogeneous and less predictable (Figure 10.2). In sum, biological complexity creates noise in the measurement.

Figure 10.2. Issue of the complexity of in vitro models: comparison of simple and realistic models according to

van der Meer and van den Berg [VAN 12]

As a result, the challenge is to proceed from the simple to the realistic model while maintaining a homogeneous and predictable response. The first promising examples of this were reconstituted skin models from keratinocytes used to meet the requirements of the cosmetics industry (see section 5.6).

Several obstacles must be overcome in order to multiply the functions targeted and increase the realistic aspect. The geometric, mechanical and biochemical aspects have been studied in light of this. Microdevices have been conceived from microelectromechanical (MEM) systems, adapted here to the requirements of biology. Fluid mechanics was applied to resolve the problem of fluid control. And the cell itself brought its own contribution by showing its capacity to self-organize, leading to functional tissues [VAN 12].


Ultimately, the most attractive technology for drawing in vitro toward physiology and pathology can today be resumed in four short words: organ-on-a-chip. This refers to miniaturized devices that mimic the functions of individual organs and integrate microcompartments [MOR 12] connected by microfluidics and at times cell models applied in coculture. The first organ-on-a-chip, mimicking the liver, was published in 2008 by Salman Khetani and Sangeeta Bhatia of MIT (United States) [KHE 08]. It is a microdevice with several compartments using hepatocytes and fibroblasts in coculture, organized in a precise manner so as to capture the essential aspects of hepatic function. There followed organs-on-chips mimicking the gastrointestinal tract (2009), tumoral metastasis (2009), the lung (2010), the heart (2011) the blood brain barrier (2012), chemiotaxis (2012), angiogenesis (2012), vasculogenesis (2013) and blood vessels (2013) [SUN 14].

The challenge issued by van der Meer and van den Berg seems to have been settled in part. Organs-on-chips can now fulfil key functional aspects without diminishing measurement precision. In retrospect, it appears that the correct strategy leading to maintained robustness of the whole stems from the sophistication of the microdevices rather than the multiplication of cell types that inevitably reiterate the problem depicted in Figure 10.2. Certainly, the cell model is essential. It must recapitulate the intended function and be pertinent in physiological terms, but the whole is more easily optimized by manipulating MEM and fluidic systems that are more readily controlled [VAN 12].

10.2.1. Homo chippiens

Confronted by this success, today, a higher level of integration is being studied. This is no more, no less than the integration of different organ biochips between one another. We can call this body-on-a-chip or even human-on-a-chip.

John Wikswo, physiologist at the Vanderbilt University (Nashville, United States), coined the fine name Homo chippiens. The man on a chip is on the way. Nature announced in February 2015 the funding of a research program on the subject with an $18 million budget [REA 15]. The first applications address the needs of the military, but ultimately, we may envisage the use of biochips in pharmacokinetics, in clinical approaches or

Outlook 225

for the regulatory assessment of health risks, which requires more predictive models for human effects.

Based on the principle evoked above, biochip assembly philosophy relies on MEM and fluidic devices to limit their biological complexity. The microtools available are consequently, pumps, tubes, syringes, inert polymers, connections, reservoirs, taking physical parameters such as gravity or surface tension into account, as well as biological parameters such as feedback loops, exerted forces on capillaries or hydraulic impedance distribution. Here too, pluripotent stem cells could readily find application uses due to their capacity to differentiate in culture.

John Wikswo correctly points out that of course these models can never reproduce the workings of a human being. They keep living reality at the same distance as a roadmap to the realities of land. They are a representative dimension, no more, no less. Like a road map, if they are well conceived, humans-on-chips provide reliable, albeit extrapolated information (Figure 10.3).

In order to reach this objective and recapitulate human physiopathology as well as possible, the organs-on-chips must above all retain relative sizes in accordance with the real organs and be interconnected by fluid networks compatible with the reality of capillary exchanges at work in the body. This requires a change in scale, both mentally and physically. For example, this could require replacing a human of 70 kg by a smaller unit, a µHu worth 70 mg. Then at this scale researchers could try to identify the functions of essential organs in the overall system, such as the blood brain barrier or the molecular filtering at kidney level [WIK 13].

10.2.2. The contribution of PBPK models

The purpose of physiologically-based pharmaco-kinetics (PBPK) approaches, which are already widely employed throughout the industry in pharmacokinetics, is to use mathematical models to assess the quantity of drugs or toxic agents present in a subject at a given time [SUN 14]. These models already represent the body as a sum of components connected by blood flow. Consequently, the adjustment of inter biochip connection volumes using PBPK modeling should allow for near realistic simulation of metabolite exchanges between microorgans. The addition of models used in pharmacodynamics could also guide these adjustments.


Figure 10.3. Conceptual plan of a micro human (µHu) with four organs: (A) peristaltic ventricular assistant, (B) left heart, (C) lung, (D) right heart,

(E) liver, (F) peripheral circulation and (G) microchemical analyzer of metabolic activity (redrawn from John Wikswo review [MOR 12])

10.3. Conclusion

Twenty years of scientific development and technologic optimization have led to the integration of cell assays into the industrial landscape with the current global market evaluated at several billion dollars. The pharmaceutics industry is by far the main proponent.

Other application sectors are still emerging, in particular the regulatory assessment of health risks, which has been flirting with cell approaches for several decades. With toxicologists calling for a new toxicology based on knowledge, this traditionally reticent sector is undergoing a profound conceptual transformation.

Throughout this work, we have showed the extent to which the cell vision is reductionist by nature. This is both an advantage and a hindrance. The world of in vitro and, by definition, of cell assays has now been developing for 50 years with the nagging presence of this paradox: the cell comprises the essence of life, it is the simplest unit of life, but also life’s most simplistic entity.

Outlook 227

The description of the major cell assays in their application context gives a good indication both of their polyvalence and their capacity to resolve problems as diverse as the neutralization of botulinum toxins or the research for antagonists at the allosteric site of a seven-transmembrane domain receptor. This faculty for adaptation explains the multiplication of their uses, which are not limited to traditional sectors but concern an increasing number of sectors, notably in environmental management, which requires monitoring measurements in the field.

Furthermore, the capacity to create stem cells from adult human tissue certainly represents a tremendous advancement in modern cell biology. This new model, which could soon establish itself as a benchmark, clearly questions the pertinence of more classic models, immortalized or derived from human tumoral phenotypes.

Finally, while traditional 96-, 384- or 1,536-well culture plates still have many a day ahead of them, organotypic culture modes, associated with microdevices, which are more pertinent in terms of physiopathology and predictability of effects on human beings, are currently in development. As such, they may well represent the most promising future for live cell assays.

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Index

ΔΨ potential, 93

A, B, C

A-B, 194 AK, 82 AKT, 174, 191 Alamar Blue, 93 Alpha Screen, 177, 190 Ames, 43 Annexin V, 83, 102, ARE-NRF2, 127, 142 ATPlite, 94 Bind, 63 BrdU, 47 Brilliant Black, 92 C11-Bodipy, 79 Ca++-Aequorin, 91 Calcein-AM, 85–88, 93 Cameleon, 55, 90 cAMP Glosensor, 181 Candles, 182 Caspase-3, 55, 102 CAT, 75 CellKey, 62, 184

CellRox, 78 Cell Titer GLO, 94 CM, 78, 112, 113 CM-H2DCFDA, 78 Comet, 100, 101, 143, 147, 186 CRE, 100, 101,143, 147, 186 cytokine release, 108

D, E, F, G

DIBAC, 187 DR CALUX, 148, 214, 215 ECIS, 61, 210 EPIC, 63, 184 EpiDERM, 118, 120 EpiOcular, 115, 145 EpiSkin, 119, 120 EST-100, 118 FLUO-4, 89,187 fluorescein leakage, 125, 126, 142 FMP, 187 Gα15/16, 183 GAPDH, 82 GF-AFC, 93



H, K, L, M

H295R, 124, 125 H3-Thymidine, 47 h-CLAT, 146 hERG, 195 HitHunter, 179 Hoechst 33342, 87, 88 HTRF,179,183 Karyotype, 4, 34, 38, 45, 47, 198,

199, 222 LANCE, 180 LDH, 81 LUCS, 86, 88 LuSens, 146 MAT, 198, 203 MCB, 78 Micronucleus, 126, 143, 147 MicroTox, 211, 212, 215, 217 MitoSOX Red, 78 MTT, 39 MUSST, 146

N, P, Q, R

NAD(P)H, 40, 65, 67, 79 NFAT-RE, 90 NRU, 41, 42, 44, 75, 123, 141, 142,

145 P450-Glo, 192

PAM, 66 PathHunter, 175, 176 Phototoxicity, 103, 122, 123, 132 Protease, 55, 56, 70, 83, 93–95, 97,

98, 102, 176, 206–208 QFT-GIT, 200 Rhodamine, 87, 93, 123

S, T, V, X

SkinEthic, 115, 118, 145 RHE, 118

SNAP25, 206, 207 SNARE, 205–207 SRE, 186, 190 STE, 115, 127 STTA, 124 TA ERBG1Luc, 124 TagLite, 173 Tango, 176 TBARS, 79 TK, 45 TNA, 208, 209 Transfluor, 175 T-SPOT, 201 TUNEL, 100 VIPR, 91, 187 xCELLigence, 61, 185

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Cell assays include all methods of measurements on living cells.Confined for a long time to research laboratories, these emergingmethods have, in recent years, found industrial applications thatare increasingly varied and, from now on, regulatory.

Based on the recent explosion of knowledge in cell biology, themeasurement of living cells represents a new class of industry-oriented research tests, the applications of which continue tomultiply (pharmaceuticals, cosmetics, environment, etc.). Cellulartests are now being positioned as new tools at the interfacebetween chemical methods, which are often obsolete and notvery informative, and methods using animal models, which areexpensive, do not fit with human data and are widely discussedfrom an ethical perspective. Finally, the development of cellassays is currently being strengthened by their being put intoregulatory application, particularly in Europe through the REACH(Registration, Evaluation, Authorisation and Restriction ofChemicals) and cosmetic directives. This book is the firstsummary ever written on this emerging subject, coming freshfrom research laboratories.

Christophe Furger is a cell biologist, Doctor of Pierre et MarieCurie University (Sorbonne Universities, Paris, France) andDirector of Research and Development at L.E.D. He has beenworking since 1995 on the conception of cell assays. He currentlyheads a public/private research program in toxicity at theLAAS/CNRS lab (Toulouse, France).

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Live cell assays: from research to health and regulatory applications

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