The Organovo NovoGen™ Bioprinting platform©Copyright 2015, Organovo Holdings Inc. 3D bioprinted...

©Copyright 2015, Organovo Holdings Inc.

Deborah Nguyen, Ph.D. Senior Director of R&D, Tissue Applications

[email protected]

© Copyright 2015, Organovo Holdings, Inc. This report is solely for the use of intended audience. No part

of it may be circulated, quoted, or reproduced for distribution outside the organization without prior

written approval from Organovo Holdings, Inc.

The Organovo NovoGen™ Bioprinting platform: Changing the shape of drug discovery research


• Devices intended for

external use

– Exoskeleton (3D Systems

/ Ekso Bionics)

– Invisalign (Align

technology)

Additive manufacturing (3D printing) enters the life sciences arena

• Devices utilized in invasive medical procedures

– Surgical tools (ex: 3D printed drill guides used in the

installation of dental implants – Guide3D and StrataSys)

– Customized (ex: skull implants – Oxford Performance

Materials)

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• 2003-2004: Modification of ink-jet and deposition printers to dispense living cells in

defined patterns (Boland et al., Clemson University; Forgacs et al., U of Missouri)

• 2015: 216 publications containing the words ‘bioprint’, ‘bioprinted’, or ‘bioprinting’ in the

title, abstract, or subject heading; 77 publications in 2014-2015 alone

Ultimate goal >> create more physiologically relevant systems

Reiffel et al., PLoS One, 2013 Xu et al., Biofabrication, 2013

Additive manufacturing enters the cellular arena

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Recapitulating native physiology requires an appreciation of context

Cell Tissue Organism

Hepatocyte Liver Human

Cells reside in heterogeneous and architecturally structured environments

3


Demonstrated benefits

– Extended cell survival and/or function

– Improved morphologic features

(polarity, intracellular organization)

– Preserved signaling cascades

mediated by cell-cell interactions

(integrins, growth factors)

– Better prediction of human drug

effects

Context provided by multi-cellular 3D models

enables new insights in vitro

Schmeichel & Bissell. J Cell Sci. 2003 Jun 15;116(Pt 12):2377-88.

Takasato et al, Nature, 2015; epub Oct 7

Toxicity in mature proximal tubule cells

ECAD

CASP3

LTL

DAPI

Proper polarity of epithelial cells

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Development

Positional relevance of

mesenchyme and epithelium

during morphogenesis

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Context also provided by spatial relationships

Kim et al., Anat Rec 2009 292:123

Rei

del

et

al.,

20

11

Mo

l Cel

l Pro

teo

mic

s 1

0:2

46

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Homeostasis

Layer-by-layer metabolic

support for photoreceptors in

the retina

Pathogenesis

Disruption of epithelial /

vascular functions during

fibrosis


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The Foundation

Organovo’s proprietary NovoGen MMX™

Bioprinting Platform builds 3D tissues through

the automated, spatially-controlled deposition of

living cells in order to better mimic native tissue

structure and function.

Photo Credit: Melissa Jacobs (San Diego Business Journal)

(NYSE MKT: ONVO)


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Proprietary platform for 3D tissue generation

% CELLS

The successful development of a 3D tissue

requires optimization of each key parameter

Up to 100%

cellular


Potential advantages of bioprinting purely

cellular tissues

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• Avoid the use of non-native scaffolding that can alter cellular biology

• Able to achieve tissue-relevant cellular density

• Architecturally correct tissues with native cell types in their proper locations

• Intercellular interactions drive native tissue biology to form fine structures

(distinct layers, microvasculature) and intracellular connections

Spheroids Cell-Seeded

Scaffolds Bioprinted

Tissues

True 3D; >200m in x, y, and z axes ✔ ✔ ✔

Multiple tissue-specific cell types ✔ ✔ ✔

Tissue-like cellular density ✔ limited ✔

Spatially-controlled cell compartments ✗ limited ✔

In-vivo like tissue micro-architecture ✗ ✗ ✔


Areas of in vitro focus

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ExVive 3D™ human liver models for preclinical

testing and disease modeling

• Fully human, multicellular structure

• Compartmentalized architecture

• Viable and functional for at least 6 weeks,

enabling low dose, chronic testing of

compounds

• Able to model drug effects biochemically,

transcriptionally, and histologically

©Copyright 2014 Organovo, Inc. © copyright 2015 Organovo, Inc.

24-tissue array

Hepatocytes Stellates EC

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Histological characterization of shows key

features of native liver

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DAPI: Blue

CD31: Green

Tissue-like cellular density

25 µm 50 µm

CD31

Desmin 20 µm

Albumin

ECad

50 µm

Hepatocyte tight junctions

Formation of

microvasculature Retention of desmin

positive stellates


Viability is sustained in 3D bioprinted liver

• Increase in tissue

ATP levels over time

supports ongoing

tissue viability

• Parallel sustained

production of albumin

over time

• Similar phenotypes

seen in tissues

comprising different

donor hepatocytes

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25 μ


Liver functions are sustained in 3D bioprinted liver:

CYP3A4 metabolism

13

25 μ

CYP3A4-mediated metabolism of midazolam increased over time and

was inducible in response to Rifampicin

Formation of hydroxymidazolam CYP3A4 mRNA (qPCR)


• Cytokines can be induced with LPS in exVive3D Liver tissues without

Kupffer Cells, suggesting other cell types (i.e., hepatic stellate cells) can

contribute to the inflammatory microenvironment

• Kupffer cells (KC) can be incorporated into exVive3D Liver tissues, and

enhance basal and LPS-induced cytokine production

Inflammatory Responses can be modeled in

3D Liver Tissues

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3D Liver tissues respond selectively to liver

toxicant

• Known hepatotoxicant Troglitazone (Trog) exhibited greater toxicity at 15x Cmax compared to non-toxic control Pioglitazone (highlighted boxes).

• Troglitazone treated tissues have higher frequency of TUNEL positive cells (arrowheads) compared to either Vehicle or Pioglitazone treated tissues

Viability (ATP)

Vehicle Trog

TUNEL

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Evidence of Troglitazone metabolites

detected in 3D Liver tissues

A

B

C

D

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exVive3D™ Liver tissues as a human surrogate for the

study of xenobiotic induced liver fibrosis

Advantages:

• Fully human

• Comprises hepatocytes, endothelial cells, and quiescent stellate cells ( )

• Enables assessment of tissue-level responses (fibrosis, remodeling, resolution)

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Assessing the tissue response to fibrogenic

compounds in exVive3D human liver

exVive3D Liver tissues

7-Day Exposure

MTX 1.0 µM

Vehicle 0.1% DMSO MTX 0.1 µM

TGF-β1 0.1 ng/mL

ENDPOINTS • LDH • ALBUMIN • HISTOLOGY • CYTOKINE PROFILE • GENE EXPRESSION

PROFILE

• Daily dosing with prototypical fibrotic agents: Methotrexate (MTX), TGFβ

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exVive3D liver tissues display hallmarks of

fibrosis after 7 days of toxicant exposure

TGF-β1 0.1 ng/mL Vehicle 0.1% DMSO 1.0 µM MTX

• At 7 days, collagen has accumulated in

the tissues (blue) but hepatocellular

function is preserved (no change in

albumin, LDH)

• Histological findings supported by gene

expression analysis

Clinical Sample (MTX) Osuga et al, Int J Clin Exp Pathol 2015;8(2):1961-1966

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Modeling the kidney proximal tubule

• The proximal tubule is a key target of renal toxicity

• Can we generate a 3D model of the proximal tubule to model

– Toxicity?

– Fibrosis?

– Transport?

• Design mimicking native architecture

– Basal interstitium

– Monolayer of polarized proximal tubule epithelial cells

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Histologic characteristics of renal model

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• Deposition of collagen to enable tissue cohesion (trichrome stain)

• Reproducible formation of microvascular structures by CD31+ HUVEC cells

• Tight junction formation between epithelial cells, evidenced by E-Cadherin

• Putative brush border formation by day 14 (arrows)

Trichrome CD31+ HUVEC

Ecad+ Epithelial cells

H&E (Day 14)

20um

CD31 TE7

ECad


Demonstration of renal epithelial function

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Glutathione (GSH) produced by the

liver is degraded by the GGT

enzyme in renal proximal tubule

epithelial cells (RPTEC)

L. H. Lash Vet Pathol 2010;48:408-419

RPTEC

3D Gen1 kidney proximal tubule

prototype demonstrates GGT

activity, which increases with

maturation



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How can bioprinting be used to better model

the tumor microenvironment?

• Existing technologies lack key disease features

– True tissue density

– Distinct cellular compartments

– Appropriate architecture without scaffolds

• 3D bioprinting addresses these challenges

– Native tissue biomechanical properties

– Assess effects on normal and tumor compartments in one well

– Flexibility of cellular inputs

– Incorporate primary tissue – personalized medicine

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Preliminary 3D breast cancer studies

show promise

• Cancer cells surrounded by bioprinted breast tissue stroma (fibroblasts, HUVEC, pre-adipocytes)

• Bioprinted tissues retain compartmentalization, and demonstrate interactions between cancer cells and stroma

• Formation of endothelial networks (CD31) and differentiation of adipocytes (Oil Red O) observed

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King et al., ASCB and AACR (December 2013, April 2014)

Oil Red O


Bioprinted human tumors can be applied to

studies of drug penetration / distribution

• Small, hydrophilic

compounds like methotrexate

(C) and fluorescent dyes (B)

penetrate deep into the

tissues

• Large, lipophilic compounds

like paclitaxel concentrate in

the outer regions of the tissue

(D)

King et al., AACR (April 2014)

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Bioprinted tumors can be used to assess

compartment-specific drug effects

• Differential sensitivity seen between component cells in 2D and 3D

tissues

• Histologically assess effects on stroma and cancer compartments

simultaneously

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Bioprinted Human Skin:

Demonstrating injury response to toxicants

• Tissues exhibit stratified epidermal architecture with distinct basal and

differentiated keratinocyte layers

• Tissues show reduced viability in response to known toxicants, SDS and Triton X

• IL-1α production spikes with exposure to SDS, further signifying injury response

P B S 5 % S D S 1 % T ri to n X

0

2 5

5 0

7 5

1 0 0

A la m a r B lu e 4 2 h r s P o s t T re a tm e n t

% V

iab

ilit

y2 .4 5 %

6 8 .7 5 %

0h

rs

15m

in

24h

rs

42h

rs

0h

rs

15m

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24h

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42h

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24h

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42h

rs

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1 0 0

1 2 5

1 5 0

IL -1

IL-1

(p

g/m

l)

P B S 5 % S D S 1 % T rito n

H&E

Layered morphology

Cornified

Granular /

spinous

Basal

Dermal

fibroblasts


3D bioprinted tissues enable better predictive,

physiologically relevant in vitro modeling

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• Successful 3D tissue modeling depends on the interplay of cellular inputs,

fabrication platform, and measurable outcomes

• The significance of bioprinting will depend ultimately on the translational and

clinical value of the human 3D tissues that are created.

• Toxicity/efficacy models fill the gaps between in vitro and in vivo preclinical research

• Disease models have applications across the pre-clinical drug discovery workflow

Target Discovery

Target Validation

Lead Optimization

Pre-Clinical non-GLP

Pre-Clinical GLP Clinical Trials

3D Human Tissue Models

In vitro In vivo

Ga

pProgram Costs ->

$ $$ $$ $$$ $$$$ $$$$$

Potential Applications for 3D Human Tissue Disease Modeling

Human 3D toxicity /

efficacy models

Human 3D disease models


Thanks to the team! ®

Edward L. LeCluyse, PhD

Leah M. Norona

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The Organovo NovoGen™ Bioprinting platform©Copyright 2015, Organovo Holdings Inc. 3D bioprinted...

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Transcript of The Organovo NovoGen™ Bioprinting platform©Copyright 2015, Organovo Holdings Inc. 3D bioprinted...