Development of an Integrated Ex Vivo Female Reproductive ... · Reproductive Sciences . Draper Lab . High-Value Engineering . FemKUBE: Engineering Reproductive Solutions . Motivation:

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




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




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.




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




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




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




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

mailto:[email protected]

mailto:[email protected]

Northwestern University Reproductive Sc ces

Draper Lab High-Value Engineering ien


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




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




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%




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

http://oncofertility.northwestern.edu/






Benchmark 2: EstroKUBE Microfluidic Culture

FemKube Image Integration: Dino-Lite AM4815ZTL




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




Ovulation in Microfluidic Paradigm





Benchmark 2: Ovulation in Solo-MPFTM





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




Quintet-MPFTM Supports Ovarian Follicle Growth

Day 0

Day 10

patents pending




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




EVATARTM on Quintet-MPSTM

Liver Spheroids Ovary Pituitary Donor

Ectocervix

Uterus Fallopian Tube Acceptor (Sampling)

patents pending




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




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




EVATARTM Quintet-MPSTM Fallopian Tube Functioning

patents pending




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




Ectocervix Models Recapitulate Normal Cervix Morphology and Differentiation

Normal ectocervix tissue

Day 14 model




LiverKube in the 5X MPS

SHBG Albumin

IGF-1

Estrogen Progesterone

Steroid hormones affect liver metabolism

Liver proteins affect sex hormone function

EstroKube

LiverKube




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




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.




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




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




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




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




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

Documents

Development of an Integrated Ex Vivo Female Reproductive ... · Reproductive Sciences . Draper Lab . High-Value Engineering . FemKUBE: Engineering Reproductive Solutions . Motivation: