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Development of an Integrated Ex Vivo Female Reproductive Tract:
Drug Discovery and Testing for the Future
Teresa K. Woodruff, PI presenting on behalf of The FemKUBE Team
UH2/UH3-Funded Research Program: National Center for Advancing Translational Science;
Office of Research on Women’s Health; the Eunice Kennedy Shriver National Institute of Child Health and Human Development; and the
National Institute of Environmental Health Science
Northwestern University Reproductive Sciences
FemKUBE: Engineering Reproductive Solutions
Draper Lab High-Value Engineering
Northwestern University Reproductive Sciences
Draper Lab High-Value Engineering
FemKUBE: Engineering Reproductive Solutions
The Team FemKUBE Technology Team: Jonathan Coppeta1*, Brett Isenberg1, Jeffrey T. Borenstein1, Hunter Rogers2 FemKUBE Biology Teams: EstroKUBE: Teresa K. Woodruff2,*, Shuo Xiao2 TubeKUBE: Joanna Burdette3*, Jie Zhu2, Alexandra Rashedi2 UteroKUBE: J.Julie Kim2*, Susan Olalekan2 EndocervixKUBE: J. Julie Kim2*, Thomas Hope2 EctocervixKUBE: Spiro Getsios4*, Kelly McKinnon2, Paul Hoover4 iPSC Development: Monica M. Laronda2; Hanna Valli2 Gynecology Tissue Core: Mary Ellen Pavone2*, Saurabh Malpani2
Gynecology Histology Core: Chanel Arnold-Murray2
Pharmacokinetics: Michael Avram5 Project Management: Elizabeth C. Sefton2
1Charles Stark Draper Laboratory, Cambridge, MA; 2Department of Obstetrics and Gynecology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611; 3Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago, IL 60607; 4Department of Dermatology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611; 5Department of Anesthesiology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611; *sub-project leader
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Reproductive Tract Toolbox: Earlier Discovery and Improved Safety
Contraceptive design and targets Environmental health toxin ID Vaccine development Infectious disease drug screening Cancer drug development Gestational drug development Early development/testing of drugs -Reduce sex bias in pipeline -Precision approach to drug development -Increase safety of drugs for both sexes Sex-specific drug metabolism
Woodruff. PNAS, 2014. Kim, Woodruff. Nature, 2010. Eddie et al. Experimental Biology and Medicine, 2014.
Laronda et al. Stem Cell Research and Therapy, 2014.
Northwestern University Reproductive Sciences
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Reproductive Tract Toolbox: Goals of Project
• Model the human female reproductive tractex vivo for basic, translational, and clinicalresearch
• Deploy dynamic fluid managementtechnology to support endocrine feedbackloops
• FemKUBETM: Using ovary as a physiologicsource of female steroids integratefallopian tube, uterus, and cervix, torecapitulate 28 day menstrual cycle
• EVATARTM: Integrate liver withreproductive tract tissues for steroid andcompound metabolism
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Endocrine Changes During the Menstrual Cycle
15 5 10 20 25 0
Func
tiona
lis
Bas
alis
Cervical mucous
LH E P FSH
Pituitary Hormones
Ovarian Hormones
Hor
mon
es
Fallo
pian
Tu
be
Ute
rine
Endo
met
rium
Va
gina
l Ep
ithel
ium
O
varia
n Fo
llicl
es
Menstrual Cycle (Days)
Menstruation
30
Basal Stratified
Cornified
Folliclular Phase Luteal Phase Ovulatory Phase
Hormone cycle used to validate FEMKUBE Follicular Phase: 0.1nM E2 Days 1-7 1nM E2 Days 8-14 Luteal Phase: 1nM E2+10nM P4 Days 15-21 0.1nM E2+50nM P4 Days 22-28 Reproductive Tract Integration: • Hormones produced by
ovarian follicle to provide hormones to TubeKUBE, UteroKUBE, and CervixKUBE
• Provide secreted factors • Effects of compounds on
endocrine function
Laronda et al. Stem Cell Research and Therapy, 2014
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Motivation: The reproductive tract is an integrated system with functional hallmarks that can be modeled in vitro
Primate Reproductive Tract Microfluidic Platform Toolbox
Benchmark 1: Advancing hardware design Benchmark 2: Improving microfluidic follicle culture Benchmark 3: Advances in tissue engineering Benchmark 4: Initial integration Benchmark 5: Early stage interaction
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Benchmark 1: Improved Hardware Design and Implementation
• Deployed tailored microfluidic systems and culture modules from Draper Lab to Northwestern University
• Culture modules accommodate tissue spheroids and transwells • 1X-cultures individual tissues (Solo-MFPTM) • 2X-cultures two interacting tissues (Duet-MFPTM) • 5X-cultures up to five interacting tissues (Quintet-MFPTM)
• Solo-MFPTM (follicle, ovary, fallopian) • Duet-MFPTM (follicle-fallopian; ovary-fallopian) • Quintet-MFPTM (ovary; EvatarTM) • Symphony-MFPTM (10-organ EvatarTM)
Jeff Borenstein, Ph.D., Laboratory Technical Staff, [email protected], (617) 258-1686 Jonathan Coppeta, Ph.D., Technical Director MPS Program, [email protected], (617) 258-4170
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Benchmark 1: Microfluidic Suite That Is Dimple, Versatile, and Flexible
• Flexibility – Plug-and-play format with specialized organ-specific modules – Software programmable flow rates and interaction schemes
• Significant advance in microfluidics with new pumping technology – Scalable to accommodate increased complexity and/or higher number of replicates – Automated programmable function for less handling
• Easy-to-use – Plug in and operate – Open format, single-ribbon cable connection, no vacuum lines to incubator
• Portable (12 x 16 x 6 cm) – Enables simultaneous operation – Adapts to your laboratory similar to well-plates – Amenable for use in high-containment facilities (BL3 or higher)
• PDMS-free construction – insignificant sorption • Long-term stable operation (4 week tissue cultures) • Five patents pending on Direct Drive and organs-on-chip technology
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Benchmark 1: Direct Drive Microfluidic Pump Fluidic movement via electromagnetic actuators
unpowered: the spring keeps the actuator head pressed against the membrane – closed
with power: the head is drawn to the electromagnet, opening the valve
ON Inlet valve
OFF Outlet valve
ON Pump
chamber Intake cycle
OFF Inlet valve
ON Outlet valve
OFF Pump
chamber Dispense cycle
V
V
V V P
actuators in series create a microfluidic pump reconfigurable to enable 4 (or more)-way pumps
Advantages over pneumatics – Scalability – Degrees of freedom – Portability – Ease of use – Cost
patents pending
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Benchmark 1: Chronic System Pump Reliability Data Intraplate reliability
Measurement of 20 individual pumps over 2-week period Open loop “dead reckoning” control Tighter Cv possible by calibration or closed loop operation with flow sensors
Pump reliability actuator and pump membrane accelerated testing 50 million cycles without failure => equivalent to approximately 1 year of operation Six systems manufactured with zero failures Deployed system to NU, 3 months of experimentation without failure
0.000.100.200.300.400.500.600.700.800.90
0 5 10 15 20
Pum
p St
roke
Vol
ume
(µL)
Time (Days)
Average= 0.58 µL/hr; Cv= +/- 20%
0
20
40
60
80
100
120
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Flow
Rat
e (u
L/h)
Day
5X Flow Rate
Average= 92.5 µL/hr; Cv= +/- 4.5%
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Benchmark 1: Connectivity • Scalable platform system to enable
– Multiple human tissue models with programmable interaction schemes – Higher number of replicates in smaller footprint
Metabolism
Elimination
Pituitary
Ectocervix Liver
Follicle Fallopian
Uterus
Acceptor
P P P
P
P P
P
P
P
P
P
P
P
P
P P
P P
P
Media B P
Endocrine Feedback
patents pending
Gonadal Physiology 101
Ovary Follicle
Woodruff, D’Agostino, Schwartz, and Mayo Science, 239:1296 (1988)
Embryo
Alak, Smith, Woodruff, Stouffer, and Wolf Fert. Steril.66:646 (1996))
Encapsulated In Vitro Follicle Growth (eIVFG)
Isolated follicles
Ovarian tissue
Shea and Woodruff, 2000-present
For a list of available reagents and tools visit the OC-SHARES program: All methods and material sources can be found at:
http://oncofertility.northwestern.edu/oncofertility-consortium-scientific-help-agreement-research-endeavors-shares-program
oncofertility.northwestern.edu
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Benchmark 2: EstroKUBE Microfluidic Culture
FemKube Image Integration: Dino-Lite AM4815ZTL
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Benchmark 2: Solo-MPFTM 28-Day Reproductive Cycle
0
100
200
300
400
500
0
10
20
30
40
50
2 4 6 8 10 12 14 16 18 20 22 24 26 28
Estradiol
Progesterone
Follicular phase Luteal phase
Estr
adio
l (nM
)
Prog
este
rone
(nM
)
Day
>5 complete 28-day experiments 80 follicles per experiment Stable flow; hormone profiles
**PDMS-free system
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Ovulation in Microfluidic Paradigm
FemKUBE: Engineering Reproductive Solutions
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Benchmark 2: Ovulation in Solo-MPFTM
FemKUBE: Engineering Reproductive Solutions
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Solo-MPFTM: Drug Effects on Follicle Growth and Hormone Secretion
Control
DOX 200nM
d0
d4
d0 d4 d8
020406080
100120
0 2 4 6 8
Control2 nM20 nM200 nM
Follic
le s
urvi
val r
ate
(%)
Day
*
Follicle survival
0
500
1000
1500
2000
2500
2 4 6 8
0 nM2 nM20 nM200 nM
Day
Est
radi
ol (p
g/m
l)
*
Estradiol
Control
200 nM DOX
0
4000
8000
12000
16000
2 6 10 14 18 22 26
Control200 nM DOX
Day
Estradiol
* E
stra
diol
(pg/
ml)
02468
101214161820
2 6 10 14 18 22 26
Control200 nM DOX
Pro
gest
eron
e (n
g/m
l)
Progesterone
*
Day *p<0.05
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Quintet-MPFTM Supports Ovarian Follicle Growth
Day 0
Day 10
patents pending
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Quintet-MPFTM Supports 28-Day Endocrine Secretion
Est
radi
ol (p
g/m
l)
Pro
gest
eron
e (n
g/m
l)
hCG Follicular phase Luteal phase
0
10
20
30
40
50
60
70
0
500
1000
1500
2000
2500
3000
3500
1 2 3 4 5 6 7 9 10 11 12 14 15 16 17 18 19 20 21 23 24
Estradiol Progesterone
Day
Target flow rate: 100uL/hr; Average: 88.0uL/hr; CV:0.23
patents pending
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EVATARTM on Quintet-MPSTM
Liver Spheroids Ovary Pituitary Donor
Ectocervix
Uterus Fallopian Tube Acceptor (Sampling)
patents pending
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EVATARTM Quintet-MPSTM Playing Now
• Integrated multi-tissueexperiment ongoing– Ovary– Fallopian tube– Uterus– Cervix– Liver
• Endpoints– Histology– Secreted factors– Imaging
020406080
100120
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Flow
Rat
e (u
L/h)
Day
5X Flow Rate
Target flow rate: 100uL/hr; Average: 92.6uL/hr; CV:0.05
patents pending
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The Human Fallopian Epithelia Layers Can Be Cultured on up to 28 Days
Human Fallopian Epithelial layer Culture on membrane
H&E staining ERα DAPI PR DAPI OVGP1 DAPI Negative ctrl
Unc
ultu
red
14 d
ays
28 d
ays
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EVATARTM Quintet-MPSTM Fallopian Tube Functioning
patents pending
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CervixKUBE Secreted Factors
HGF IL-6 LIF MCP-1 MIP-1β TNF-α
GRO-α
IL-1α IL-1β IL-1RA PDGF-BB
Attracts macrophages/ NK
Epithelial-stromal interactions
Barrier function/ wound repair/ implantation
Pro-inflamatory Follicle ovulation
Sevim Yildiz Arslan
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Ectocervix Models Recapitulate Normal Cervix Morphology and Differentiation
Normal ectocervix tissue
Day 14 model
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LiverKube in the 5X MPS
SHBG Albumin
IGF-1
Estrogen Progesterone
Steroid hormones affect liver metabolism
Liver proteins affect sex hormone function
EstroKube
LiverKube
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Achieving Granulosa Cell Differentiation Through Mesoderm Derivation
SSEA
4 / N
ANO
G
OC
T4 /
BR
ACH
XX human iPSC colonies
Figure 25-21 What Is Life? A Guide To Biology © 2011 WH Freeman and Co. Mullerian Duct 1996-2015 Humpath.com – Human pathology; Hummitzsch et al. (2013) PLOS ONE
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h-iPSCs into Granulosa Cells
OC
T4 /
DAP
I undifferentiated
FOXL
2 / D
API
pre-granulosa
Diff
eren
tiatio
n C
ultu
re +
FSH
FS
HR
Summary: • Human iPSCs can differentiate into granulosa-like cells.
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Total Patients Consented R a c e o f th e C o n s e n te d p a t ie n t
T o ta l= 4 9 7
A s ia nB la c k / A fr ic a n A m e r ic a nW h iteM o re th a n o n e R a c eU n kno w nA m e ric a n In d ia n / A la s k a N a tiv e
E th n ic ity o f th e C o n s e n te d p a t ie n t
T o ta l= 4 9 7
H is p a n ic / L a t in oN o t H is p a n ic / L a tin oU n kno w n
T is s u e ty p e fo r e a c h a g e
T y p e o f t is s u e
Nu
mb
er
of
tis
su
es
F a llop ia
n Tu b e
Ov a r ie
s
E n d o me tr i
u m
Ce rv
ix
My o m
e tr iu m
0
1 0 0
2 0 0
3 0 0
1 8 -2 9 y /o
3 0 -3 9 y /o
4 0 -4 9 y /o
5 0 -5 9 y /o
6 0 + y /o
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Future Directions • Model back to back cycles • Model female reproductive tract tumors • Test endocrine disrupting compounds and
existing drugs for efficacy and/or safety • Develop high-throughput strategies • Work with FDA on validation • Work with pharma on integration within drug
discovery pipeline • On the horizon: DudeKUBE, PregoKUBE,
MyKUBE-CA
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Beyond the Female Reproductive Tract
• Disease modeling – Endometrial, ovarian, and cervical cancer – Leiomyoma, and endometriosis
• Pregnancy modeling: placenta and breast • Liver, skin, kidney, etc. • Osteochondral modeling • DudeKUBE, PregoKUBE, and
MyKUBE-CA
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Summary • Hardware/software ready for implementation • Individual reproductive tissues, tissue pairs,
and reproductive tract tissues will fit an unmet need for pharma – drug discovery, drug testing, and toxicology
• Female reproductive test models urgently needed – EvatarTM-SymphonyTM ready to deploy to meet this need
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University of Illinois, Chicago Joanna Burdette, PhD
Northwestern University Gynecologic Tissue Core Stacy Kujawa Saurabh Malpani, MS Mary Ellen Pavone, MD
Northwestern University – PK Tom Hope, PhD Mike Avram, PhD
Northwestern University Getsios Lab and SDRC Paul Hoover, PhD Spiro Getsios, PhD Northwestern University Kim Lab Susan Olalekan, PhD Sevim Yildiz Arslan, PhD J. Julie Kim, PhD University Virginia – Assay Lab Dan Hasenleder
Northwestern University Woodruff Lab and Office Monica Laronda, PhD Kelly McKinnon Shuo Xiao, PhD Peter Chen, MS Mingyang Jiang, MS Lu Bai, MS Cat Nguyen, MS Alex Rashedi Jie Zhu, MD Hanna Valli, PhD Chanel Arnold-Murray Beth Sefton, PhD Barb Cushing Leandra Stevenson Lauren Ataman Bryan Runkel Teresa Woodruff, PhD
Draper Lab Jonathan Coppeta, PhD Brett Isenberg, PhD Jeff Borenstein, PhD