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
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
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
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)
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