Anthony Bahinski, Ph.D., MBA, FAHA

Lead Senior Staff Scientist

Advanced Technology Team,

Wyss Institute for Biologically Inspired Engineering at Harvard University

National Academy of Sciences, Tissue Chip Workshop

Washington, DC

July 21, 2014

Human Organs-on-Chips

Biomimetic Microsystems• Engineer microchips containing living human cells that

reconstitute organ-level functions for drug screening,

diagnostic, toxicology, and therapeutic applications

• ACCELERATE drug development & REPLACE animal testing

Biomimetic

Spleen

Don IngberKit Parker

George Whitesides

Ali Khademhosseini

Dave Weitz

Human Breathing Lung-on-a-Chip

Underlying Microsystem Challenge

Goal is to replicate human ORGAN-LEVEL functions in vitro,

but what defines an Organ?

ORGANS ARE:

• Composed of 2 or more tissues that exhibit unique functions when

they are interfaced

• Perfused by blood flowing through endothelium-lined vessels

• Controlled by chemical and molecular factors produced by

constituent cells or delivered through the vasculature

• Regulated by mechanical forces (e.g., due to motion, breathing,

peristalsis) and blood flow

• Structured to secrete or transport factors in specific directions

• Infiltrated with immune cells during inflammatory responses

• Physiologically coupled to other organs via factors transmitted in

blood flowing through linking vessels

We use Microengineering to:

• Recreate Tissue-Tissue Interface

- Analyze transport, absorption, transport, permeability, conductivity

• Provide Mechanical Cues necessary for relevant physiology

- Fluid flow, cyclical mechanical strain, Air-Liquid Interface, directional clearance

• Precisely Orient Cells for High-Resolution Real-Time Imaging

- Enables analysis of molecular and cellular mechanisms at critical tissue boundaries

• Place cells in separate channels to study different cell populations

- Harvest cells or medium independently for molecular genetic analysis

- Sample oriented (e.g., lumenal) secretions in real-time

• Control Fluid Flow through Microfluidic Channels

- Supports long-term culture

- Enables pharmacokinetic analysis

- Permits co-culture of Microbiome

• Create Endothelium-Lined Vascular Channels

- Permits real-time analysis of recruitment of circulating immune cells

- Potentially can use of blood or plasma to feed organ chips

- Enables physiological vascular coupling between different organ chips

A Human Breathing Lung-on-a-Chip(Dan Huh, Wyss Institute; Huh et al., Science 2010)

www.nucleusinc.com

Alveoli

Paton & Byron, Nat. Rev. Drug Discov. 2007

Air

Blood

BIODESIGN PRINCIPLES:• Tissue-Tissue Interface

• Dynamic Flow

• Cyclic Breathing Movements

Capillary endothelial cells

VE cadherin

Occludin

Endothelium

Epithelium

Alveolar epithelial cells

Capillary endothelial cells

Lee & Downey, Am J Respir Crit Care Med 2001

Lung inflammation

Pro-inflammatory cytokines

Endothelial activation

(express surface ICAM-1)

Leukocyte adhesion

Diapedesis & transmigration

Infiltration into alveoliPathogensAlveolus

Capillary lumen

EpitheliumTNF

Endothelium

Inflammatory Protein

White Blood

Cells (WBCs)

Bacteria

Mimicking the Immune Response

WBC Transmigrating WBC Killing Pathogens

Control Inflamed

Inflammatory Protein

White Blood

Cells

(WBCs)

Vascular leakage syndrome

Pulmonary edema

Interleukin-2 (IL-2)

Kidney cancerMelanoma

Chemo

Human Disease Model:Chemotherapy-Induced Pulmonary Edema

(Huh et al., Sci. Trans. Med. 2012)

Air

Liquid

IL-2

Membrane

Day 0

LiquidDay 4

Liquid

IL-2

Meniscus

Day 2 Day 3

AirLiq

uid

Liq

uid

IL-2

Human Disease Model:

Pulmonary Edema-on-a-Chip

(work of Dan Huh)

Day 0

Pro-thrombin

Fluorescent fibrinogen

IL-2Day 4

Fluid

Air

Fibrin

Alveolar epithelium

Fibrin clots

Alveolar Fibrin Deposition On-Chip

IL-2

Flow

FITC-inulin

Lun

gC

apill

ary

Modulating Lung Barrier Permeability with IL-2

Vascular Leakage Model

IL-2

Flow

FITC-inulin

Lun

gC

apill

ary

Epi

Endo

Control (10% strain)

Occludin

VE-cadherin

t = 3 days

IL-2 & 10% strain

Tissue Barrier Integrity

Ventilation

(FITC-inulin, IL-2)

Perfusion

IL-2

Flow

FITC-inulin

Lun

gC

apill

ary

Bronchoalveolarlavage (BAL)

Results Confirmed in Whole Lung

Lung-on-a-Chip

IL-2 & Drug A

Flow

FITC-inulin

Lun

gC

apill

ary

DRUG A

Predicting Drug Efficacy

DRUG A

IL2 + TRPV4

Inhibitor

IL2 alone

(Huh et al., Sci. Trans. Med. 2012)

TRPV4 Inhibitor

TRPV4 Inhibitor

(With GlaxoSmithKline)

Biomimetic Microsystems Technology Pipeline

Other Organ-on-Chip Devices in Development:

– Lung alveolus-on-chip

– Heart-on-chip

– Small Airway-on-chip

– Gut-on-chip

– Bone Marrow-on-chip

– Kidney-on-chip

– Liver-on-chip

– Skin-on-chip

– Muscle-on-chip

– Blood Brain Barrier-on-Chip

– Eye-on-chip

Peristaltic Human Gut-on-a-Chip(Kim et al., Lab on a Chip 2012 & Integrative Biology 2013)

Human Intestine Microfluidic Platform

Human Gut Epithelium (Caco-2 cell monolayer in Microfluidic)

24 hr after seeding + Peristaltic-like motions

PNAS, 2007, 104:10295

Intestinal Villi

Lumen

Capillary

Gut Chip

Gut-on-a-Chip Mimics

Normal Gut Morphology

Bar, 20 μm

Transwell

Gut Chip

58 h 130 h 170 h

Formation of Intestinal Villi in the Gut-on-a-Chip(Caco-2 cells/ 0.01 dyne/cm2 Flow + 10% Cyclic Strain/0.15 Hz)

58 h 130 h 170 h

Crypt

Formation of Intestinal Villi in the Gut-on-a-Chip(Caco-2 cells/ 0.01 dyne/cm2 Flow + 10% Cyclic Strain/0.15 Hz)

Restoration of Basal Proliferative Crypts(0-2 hr EdU pulse-chase labeling for DNA synthesis)

Drug MetabolismBarrier Function Differentiation

Restoration of Intestinal Physiology

Mucus Production

Co-culture of Epithelial Cells with Human Gut Microbes

Microcolony of Human Gut MicorbesStrategy of Co-culture

Increased Barrier Function

Villi

Villi

Villi

Probiotic Co-culture

(> 10 days)

Villi

Villi

Villi

Villi

Bacteria

VSL#3 1. Bifidobacterium breve2. B. longum3. B. infantis4. Lactobacillus acidophilus5. L. plantarum6. L. paracasei7. L. bulgaricus8. Streptococcus thermophilus

Microenvironment-Dependent Changes in Gene Expression Profiles

Bacteria

in Gut-on-a-Chip (+Shear, +Strain)

Gut-on-a-Chip(+Shear, +Strain)

Transwell(Static)

• GEDI: Gene Expression Dynamics Inspector

• ~23,000 genes

Applications

Crohn’s Disease on a Chip Drug Transport on a Chip

PDMS

1 mm

8 Weeks

Bone-inducing

Material (DMP+BMPs)

3 mm

Bone Marrow-on-a-Chip(work of Yusuke Torisawa and Katie Spina

Nature Methods – 2014 May 4 [Epub ahead of print].)

Perfused

Microfluidic

Channels

Bone Marrow

Transplantation

In Vitro Blood Cell

Manufacturing

Stem Cell Niche Model

Bone-inducing

material

2 mm

eBM at 4 wk

Muscle side

Skin side

eBM at 8 wk

2 mm

PDMS Device

1 mm

Bone Marrow-on a Chip (after 8 Weeks Implantation)

50 mm100 mm

Trabecular & Cortical Bone

Bone Marrow Formation

500 mm 50 mm

eBM at 8 wk (H&E section)

Femur (mBM)

eBM is nearly identical to natural mBM

mBM

eBM

(8 wk)

cKit

Sc

a1

0.09

0.61

0.14

cKit

Sc

a1

0.08

0.59

0.30

Hematopoietic Stem and Progenitor Cell Distribution

Flow Cytometric Analysis

Dis

trib

uti

on

(%

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

Sca-1

cKit

CD34

CD135

LSK

mPB

(-RBC)

eBM

4wk

eBM

8wk

mBM

Lin-Sca1

Lin-cKit

Lin-Sca1+cKit

Lin-CD34

Lin-CD135

(HSCs)

mBM

eBM

(8 wk)

Flow Cytometric Analysis

CD45

Te

r11

9

35

58

CD45

Te

r119

33

56

Dis

trib

uti

on

(%

)

0

10

20

30

40

50

60

70

80

90

100

CD3

CD19

MacGr

Gr-1

Mac-1

Ter119

mPB eBM

4wk

eBM

8wk

mBM

CD3

CD19

Mac1+Gr1

Gr1

Mac1

Ter119

Differentiated Blood Cell Distribution

eBM is nearly identical to mBM

Dexter culture

mBM cells are culture on a

2D stromal feeder cell layer

In Vitro Culture

Viability

no significant difference0

10

20

30

40

50

60

70

80

90

100

Via

bilit

y (

%)

mBM (Dexter) eBM (on-chip)

Day4 Day4Day7 Day7Harvest Harvest

eBM

on-chip

0

1

2

3

4

5

6

HSCs and progenitors

Dis

trib

uti

on

(%

)

D4 D7Harvest D4 D7Harvest

mBM (Dexter) eBM (on-chip)

Lin-Sca1

Lin-cKit

Lin-Sca1+cKit

Lin-CD34

Lin-CD135

HSCs

Progenitors

Lin-Sca1+cKit

Long-term HSCs

0

0.1

0.2

Lin

- CD

150

+C

D48

-(%

)

D4 D7Harvest D4 D7Harvest

mBM (Dexter) eBM (on-chip)

αγγ

γ

Myelodysplasia

General term for haematologic

defects involving deficiencies

in the myeloid blood lineages,

which in fact manifest

dysfunction in multiple blood

lineages. Myelodysplasias are

sometimes referred to as

‘preleukaemias’ because they

can transform over time.

a generalized model for stem cell regulation. However,

in certain cases, direct evidence that this model is appli-

cable is still lacking. Nonetheless, it is widely accepted

that niches exist in most, if not all, tissues, and that they

provide basic cellular necessities, such as mechani-

cal support, trophic factors and hospitable physical

and chemical conditions, as well as stem cell-specific

self-renewal and differentiation cues (FIG. 2).

Several new and elegant techniques and model sys-

tems have been applied to the study of HSC develop-

ment, permitting an improved functional and anatomical

dissection of HSC interactions with the niche. In par-

ticular, real-time in vivo imaging has enabled the direct

visual ization of HSCs and their niches, providing key

insights into the origins, dynamics and physiological

regulation of the anatomical compartments in which

Figure 1 | Hierarchical model of haematopoiesis in the adult bone marrow. All haematopoietic cells ultimately

derive from a small population of haematopoietic stem cells (HSCs), which is separable into at least two subsets:

long-term reconstituting HSCs (LT-HSCs) and short-term reconstituting HSCs (ST-HSCs). LT-HSCs maintain self-renewal

and multi-lineage differentiation potential throughout life (represented by the bold arrow). ST-HSCs derive from LT-HSCs

and, although they maintain multipotency, they exhibit more-limited self-renewal potential. Further differentiation of

ST-HSCs generates multipotent progenitors (MPPs) and then oligopotent progenitors, which are marked with asterisks.

Haematopoietic progenitor cells lose their differentiation potential in a stepwise fashion until they eventually generate

all of the mature cells of the blood system (these are depicted at the bottom of the schematic). Several potentially

distinct subsets of MPPs have been described, but MPPs are shown here as a condensed population for simplicity.

Lineage-committed oligopotent progenitors derived from MPPs include the common lymphoid progenitor (CLP),

common myeloid progenitor (CMP), megakaryocyte-erythrocyte progenitor (MEP) and granulocyte-monocyte progenitor

(GMP) populations. HSC and progenitor populations can be discriminated by flow cytometry, using antibodies that

recognize unique combinations of cell surface markers. Some commonly used profiles for identifying these cells are

shown adjacent to the HSC and progenitor populations. Dotted arrows denote a proposed lineal connection. CD135, also

known as FLK2 and FLT3; IL-7R, interleukin-7 receptor; lin, lineage markers (which are a combination of markers found on

REVIEWS

644 | OCTOBER 2011 | VOLUM E 12 www.nature.com/reviews/molcellbio

© 2011 Macmillan Publishers Limited. All rights reserved

LT-HSC

ST-HSC

MEP CMP CLP

MPP

Composition of blood cells in cultured eBM maintains intact

whereas that in Dexter culture is completely different

Bone marrow-on-a-chip contains

a functional hematopoietic niche

In Vitro Culture

0.0

0.5

1.0

1.5

2.0D

istr

ibu

tio

n (

%)

with

cytokines

without

cytokines

D4 D7Harvest D4 D7

eBM

(on-chip)Lin-Sca1

Lin-cKit

Lin-Sca1+cKit

Lin-CD34

Lin-CD135

HSCs

Progenitors

Lin-Sca1+cKit

In Vitro Culture

Cytokines

SCF

IL-11

Flt-3

LDL

The microenvironment of eBM can function in

an autonomous fashion to support the hematopoietic cells

Without supplemental cytokines to support HSCs and progenitors

Bone Marrow Transplant

GFP mouseLethally irradiated

mouse (no GFP)GFP+ eBM

The cultured eBM cells engrafted and populated all blood lineages

Hematopoietic compartment of eBM retains fully functional

self-renewing, multi-potent HSCs

En

gra

ftm

en

t (G

FP

+%

)

6 wk 16 wk

mBM eBM D4

0

10

20

30

40

50

60

70

80

90

100

mBM

sBM

Post transplant

0

10

20

30

40

50

60

70

80

90

100

CD3

CD19

Mac1

Gr1D

istr

ibu

tio

n (

%)

in G

FP

+C

D4

5+

Cell

s

CD3

CD19

Mac1

Gr1

mBM mBMeBM

D4

eBM

D4

(6 wk) (16 wk)

(T cell)

(B cell)

(Myeloid)

0

0.5

1

1.5

2

2.5

0Gy 1Gy 4Gy

in vivo

eBM

Dexter

0

0.1

0.2

0.3

0.4

0.5

0Gy 1Gy 4Gy

in vivo

eBM

Dexter

Cell D

istr

ibu

tio

n (

%) in vivo

eBM

Dexter

HSCs (Lin-Sca1+cKit+) Progenitor Cells (Lin-CD34+)

Ce

ll D

istr

ibu

tio

n (

%)

Bone Marrow Chip Mimics Response to Radiation(Funded by FDA Medical Countermeasures Grant HHSF223201310079C)

Radiation Countermeasure

G-CSF (granulocyte colony-stimulating factor, 500 U/mL)

was added 1 day after exposure to g-radiation

0

0.2

0.4

0.6

0.8

1D

istr

ibu

tio

n (

%)

1 Gy

with

G-CSF

1 Gy 4 Gy

with

G-CSF

4 Gy

*

*

After 3 days in culture with G-CSFLin-CD34+

Lin-cKit+

HSCs

G-CSF induced proliferation of HSCs and hematopoietic progenitor

cells in the bone marrow chip in vitro, as previously reported in vivo

Organ-on-Chip Technology Pipeline

• Ongoing projects

– Lung Alveolus

– Lung Small Airway

– Heart

– Liver

– Small Intestine

– Large Intestine

– Kidney Proximal Tubule

– Kidney Glomerulus

– Bone marrow

– Skin

– Cancer

– Heart valve

– ……

Integrated Human Body-on-a-Chip

Organ-On-Chip

INTERROGATOR

Instrument

– Automated instrument

integrates multiple organs

– Designed for ease of use

“plug-and-play” approach

– Generate data to predict

human response

Universal

Chip Holder

DARPA Multiphysiological Systems Grant(D. Ingber, K. Parker, J. Wikswo & CFDRC/ G. Hamilton & D. Levner)

Enabling Human Trials Designs with Cells

From Different Genotypes on Chips?

wyss.harvard.edu