An integrated in vitro model

of perfused tumor and

cardiac tissue

Steven C. George, M.D., Ph.D.Edwards Lifesciences Professor

Director, Edwards Lifesciences Center for Advanced Cardiovascular Technology

Department of Biomedical Engineering

University of California, Irvine

March 7, 2014

CARDIOPULMONARY TRANSPORT AND

TISSUE REMODELING

CollaboratorsZhongping Chen, PhDBernard Choi, PhDKaren Christman, PhD (UCSD)Bruce Conklin, MD (Gladstone)Stanley Galant, MD (CHOC)Enrico Gratton, PhDChris Hughes, PhDMasahiro Inoue, MD (Osaka)Abe Lee, PhDLeslie Lock, PhDBruce Tromberg, PhDMarian Waterman, PhD

Professional StaffAnna Aledia, BS

Linda McCarthy, MSChristina Tu, MS (Lock)

Post-Doctoral ResearchersMonica Moya (2009-present)Venktesh Shirure (2013-present)Chris Heylman (2013-present)Agua Sobrino (2013-present) (Hughes advisor)Xiaolin Wang (2013-present) (Lee Advisor)

Graduate StudentsLei Tian (2009-present)Nick Merna (2010-present)Seema Ehsan (2010-present)David Tran (2010-present)Luis Alonzo (2010-present)Yosuke Kurokawa (2013-present)Sandra Lam (2013-present)Rupsa Datta (2012-present) (Gratton)Jean Wang (2012-present) (Christman)

FundingR01-HL067954-08, UH2-TR00048, R01-CA170879, RC1-ES018361-01, R21-HL104203, R01-HL070645-06P41 Centers LAMMP (Tromberg) and LFD (Gratton), NSF Predoctoral Fellowships, NIH Pre- and Post-doctoral Fellowships, CIRM training grant

http://georgelab.eng.uci.edu/

Overall Strategy

fibroblast hiPS cellreprogramming differentiation

selection

extracellular matrix tumor spheroid endothelial cell cardiomyocyte fibroblastspheroid

1 cm

microfabrication

Integrated micro-organ systems

Integrated micro-organ system #2(perfused tumor spheroids)

Integrated micro-organ system #3 (perfused tumor and cardiomyocyte spheroids)

Integrated micro-organ system #1 (perfused cardiomyocyte spheroids)

arteriole arteriole arteriole

TS

SC/PC SC/PC

venule venule

venule

Microtissue with Perfused Human

Capillaries

Biology-directed

High-throughput design

Real-time monitoring

Dynamic model of “tissue

microenvironment”

1 mm

“Challenge Grant” RC1-ES018361-01

Soft Lithography

1 cm 5 mm 0.5 mm

z

y

Perfusion of 21 day Network(1 μm fluorescent beads)

Physiologic velocity: 0- 4000 μm/s (mean 690)

100μm

Moya, M. et al. Tiss. Engr. Part C, 19(9): 730-737, 2013.

arteriole

SC/PC

venule

Micro-organ #3 (cardiomyocyte spheroids in cardiac ECM)

SC/PC

venule

arteriole

CS

Perfused hiPS-derived vesselsin cardiac ECM

Network in Cardiac ECM and

ECs from human iPS (6 mos)

A

B C

D E F

A

B C

D E F

A

B C

D E F

>96%(passage 3)

Differentiation protocol from J. Thomson

Human iPS (WTC-11 – B. Conklin)

Human iPS-EC Vessel Network in Fibrin

(12 mos)

ECFC-ECs

hiPS-ECs CD31

CD31

0

10

20

30

40

50

60

hiPS-Ecs ECFC-Ecs

% V

esse

l A

rea

hiPS-ECS ECFC-ECs

0

5

10

15

20

25

30

hiPS-Ecs ECFC-Ecs

Dia

met

er (µ

m)

hiPS-ECS ECFC-ECs

Human iPS-EC Vessel Network in cECM

(18 mos)

Day 14 CD31 (EC)

100 μm

Perfused/Functional Human iPS-EC

Vessel Network in cECM (24 mos)

TS

SC/PC

venule

arteriole

Micro-organ #3 (tumor spheroid with microvessels)

Micro-organ #1 (perfused microvessels in cardiac ECM)

SC/PC

venule

arteriole

Micro-organ #2 (cardiomyocyte spheroids in cardiac ECM)

SC/PC

venule

arteriole

CS

Before Thrombin After Thrombin

100 μm100 μm

70 kDa FTIC Dextran

hiPS-CM spheroids with hiPS-

EC network

TS

SC/PC

venule

arteriole

Micro-organ #2 (tumor spheroid with microvessels)

Micro-organ #1 (perfused microvessels in cardiac ECM)

SC/PC

venule

arteriole

Micro-organ #3 (cardiomyocyte spheroids in cardiac ECM)

SC/PC

venule

arteriole

CS

Human iPS-CM with physiologic drug

response

-DAPI-cTnT

-DAPI-cTnT

Glucose Lactate

79.5%cTnT+ 91.6%cTnT+

A B

C D

A

B C

D E F

Human iPS (WTC-11 – B. Conklin)

50 μm

hiPS-CM, fibroblast, and EC in cECM

within the Microdevice

(with Bruce Conklin and Leslie Lock)100 μm

Human iPS-CM and vascular network

TS

SC/PC

venule

arteriole

Micro-organ #3 (tumor spheroid with microvessels)

Micro-organ #1 (perfused microvessels in cardiac ECM)

SC/PC

venule

arteriole

Micro-organ #2 (cardiomyocyte spheroids in cardiac ECM)

SC/PC

venule

arteriole

CS

cTnT – iPS-CM

CD31 - EC

100 μm

Drug response of human iPS-CM in

device

-12 -11 -10 -9 -8 -7 -6 -5

0

20

40

60

80

100

120

140

y = 22.3+(100-22.3)/(1+10^(m...

ErrorValue

0.27993-9.5032m3

NA857.98Chisq

NA0.84925R

Isoproterenol

-9 -8 -7 -6 -5 -4 -3

0

20

40

60

80

100

120

140y = 0+(100-0)/(1+10^(x-m3))

ErrorValue

0.28193-5.8969m3

NA1273.8Chisq

NA0.8671R

Propranolol

%M

ax R

esponse

Log [Concentration (M)]

Control

(52 bpm)

10-7 M

Isoproterenol

(160 bpm)

10-6 M

Propranolol

(76 bpm)

Log IC50=-5.9Log EC50=-9.1

In vivo therapeutic plasma concentration

Tumor spheroids with hiPS-EC

network

TS

SC/PC

venule

arteriole

Micro-organ #2 (tumor spheroid with microvessels)

Micro-organ #1 (perfused microvessels in cardiac ECM)

SC/PC

venule

arteriole

Micro-organ #3 (cardiomyocyte spheroids in cardiac ECM)

SC/PC

venule

arteriole

CS

• 7 days

• Fibrin

• CD31 (hiPS-EC)

• GFP (SW620)

200 μm

TS

SC/PC

venule

arteriole

Micro-organ #3 (tumor spheroid with microvessels)

Micro-organ #1 (perfused microvessels in cardiac ECM)

SC/PC

venule

arteriole

Micro-organ #2 (cardiomyocyte spheroids in cardiac ECM)

SC/PC

venule

arteriole

CS

Cancer Tissue-Originated Spheroids

(CTOS)

Professor Masahiro Inoue

Osaka Medical Center for Cancer and Cardiovascular Diseases Research Institute

Key Features:

Inexpensive and efficient methods of preparation

Easy to culture in vitro

Retains three dimensionality

Preserves biochemical and mechanical characteristics of original tumor

★ Significant potential for personalized therapy

original tumor CTOS tumor cell line

z

GFP (CTOS)CD 31 (EC)

Angiogenesis

model

Drug (5FU) sensitivity of human tumor

spheroids in device

0

1

2

3

4

5

-3 -2 -1 0 1 2 3 4 5 6

0

1

2

3

4

5

-3 -2 -1 0 1 2 3 4 5 6

days

Fold

in

cre

ase

(are

a)

Fold

incre

ase (

are

a)

SW620 Alone

SW620 + Fibroblasts + EC

3 days0 days

0 uM

500 uM

0 uM

500 uM

control

10 μM

100 μM

500 μM

100 μM500 μM

control

LD50 ≅ 500 μM

LD50 ≅ 500 μM

Perfused human tumor spheroids

(UH3)

• 10 days

• GFP SW620s

tumor spheroids

• Human iPS-ECs

• Fibrin

• 1 μm beads 200 μm

Commercialization

24

Licensing• License technology to pharma for drug discovery

• License technology for personalized drug screening

Service• Manufacture and sell microtissue platform

• Perform heart toxicity screens

• Perform compound library screens for anti-tumor drug

discovery

• Determine optimal combination personalized drug therapy

Discovery• Perform library screens “in house” and license lead

compounds

Personalized Anti-cancer Therapy1. Isolate cells from body 2. Create and expand iPS cells

3. Create EC, CM, Fib

4. Test drug

susceptibility

Stromal cells (fibroblasts)

Cancer cells

4. Add matrix and

create 3D array of

tissuesO2

tumor spheroid

TS

SC/PC

Integrated micro-organ system #2 (perfused tumor spheroids)

venule

arteriole

Integrated micro-organ system #3 (perfused cardiomyocyte spheroids)

SC/PC

venule

arteriole

Integrated micro-organ system #3 (perfused tumor and cardiomyocyte spheroids)

venule

arteriole

Conclusions and Future Work

Drug interaction with PDMS

Vessel remodeling (pruning, arterioles, venules)

Red blood cells, white blood cells, platelets

Reproducibility and validation

Questions/Future

Summary

Tumor and Cardiac “microenvironments” with perfused

human capillaries

Control of flow, dimensions, matrix, cell, and gradients

Patient-specific models (primary tumor, iPS cells)