A Microfluidic Culture Model of the Human Reproductive Tract: Screening of Female Reproductive Toxic Chemicals

Shuo Xiao, PhD Assistant Professor Reproductive Health & Toxicology Laboratory Department of Environmental Health Sciences Arnold School of Public Health, University of South Carolina sxiao@mailbox.sc.edu

Conflict of Interest Statement

This is no conflict of interest for the work I present today.

Female Reproductive System

Menstrual cycle

Ovary

Infertility IVF

Vagina Cervix Uterus

Ovary

Fallopian tube

Follicles

Female Reproductive Tract and Pregnancy Fertilization

Ovulation

Embryo development & transport

Embryo implantation

Primordial follicle

Primary follicle Secondary

follicle Antral follicle

Corpus Luteum

Hormone Regulation of Female Reproductive System

• Highly regulated by pituitary hormones follicle-stimulating hormone (FSH) and luteinizing hormone (LH), and ovarian hormones Estrogen and Progesterone.

• Targets for endocrine disrupting chemicals (EDCs), pharmaceutical chemicals …

https://www.woodrufflab.org

Summary of Female Reproductive Toxicity Testing In vivo animal testing as a standard to test chemicals’ female

reproductive toxicity Organ weight, morphology, and histology Vaginal smear and serum hormone levels Ovulation and oocyte production Fertility, pregnancy, litter size, multiple generational reproduction testing …

In vivo models are time-consuming, costly, and harmful to animals Lack of in vitro models for testing fallopian tube, uterus, and cervix In vitro 2D ovarian cell/follicle cultures do not phenocopy the

physicochemical microenvironment and 3D tissue specific architecture

Guidelines for Reproductive Toxicity Risk Assessment

I. Using microfluidic system to establish the ex-vivo 28-day menstrual cycle hormone control and female reproductive tract on a chip II. Screening of female reproductive toxic chemicals

Outline

- Establish

- Discover

Organ-on-a-chip using Microfluidic Technology

Brain chip

Vessel chip

Lung chip

Heart chip

Clinical Research Only in Males

• Early testing of chemical toxicity in female is more difficult than in males because: − Existence of menstrual cycle and hormone changes − Lake of awareness of the importance of gender as a biological variable − Ethical issues associated with women who have potential or current pregnancy − Drugs have been withdrawn from the market for greater healthy risk in women

Considering gender/sex as a biological variable in all

NIH-funded research since 2015!

Hypothesis

I. Develop ovarian functions in the microfluidic system to mimic human 28-day menstrual cycle hormone control and ovarian function II. Establish ex-vivo female reproductive tract (FRT) on a chip by integrating and inter-connecting multiple reproductive tissues

Microfluidic Platform (MPS) Design

Fluidic interface

Actuator interface

Tissue Modules Media

donor

Passed media reservoir

Tissue module

• Materials: can be sterilized, non-toxic to cells/follicles, do not bind hormones; • Pumping pattern: computer program controlled.

Xiao et al, Nat Commu, 2017

Ovary Chip Based on Microfluidic Culture

• Sampling: easy handling and loading, collect the most recent passed media without disturbing a running culture.

Media donor

Passed media reservoir

Tissue module

Xiao et al, Nat Commu, 2017

• Apply the 3D in vitro follicle growth (IVFG) model in the microfluidic system, which mimics full ovarian cycle (follicular phase, ovulation, luteal phase)

Alginate encapsulation

Follicle growth in vitro In vitro maturation Ovulation

Live birth Embryo development In vitro fertilization Embryo transfer

Xiao et al, Repro, 2015 Xu et al, Biomaterials, 2006

Ovary Chip Based on Microfluidic Culture

Follicle Maturation & Ovulation in Microfluidic Culture

• Microfluidic platform supports mouse ovarian follicle development and oocyte maturation.

α-tubulin F-actin DAPI day 0 day 14 MII oocyte

day 0 day 14 In vitro ovulation α-tubulin F-actin DAPI

Xiao et al, Nat Commu, 2017

Follicle Luteinization upon hCG Stimulation

• The in vitro luteinization of follicular cells is similar to corpus luteum formation in vivo (cell hypertrophy and differentiation from granulosa cell to luteal cell).

Before hCG 48h hCG

16h 14 days

10 μm

0 10 20 30 40 50 60 70

Before hCG

24h after hCG

48h after hCG

14 days after hCG

nucl

eus

/1 m

m3

* *

Error bar: Standard deviation; *p

Ovarian Hormone Secretion in Microfluidic Culture

• Microfluidic platform recapitulates human 28-day menstrual cycle hormone control.

0

10

20

30

40

50

60

70

80

0 5000

10000 15000 20000 25000 30000 35000 40000 45000

-14 -13 -12 -11 -10 -9 -8 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Estrodiol Progesterone

Follicular phase Luteal phase

Est

radi

ol (n

M)

Pro

gest

eron

e (n

M)

day

Error bar: Standard deviation

Xiao et al, Nat Commu, 2017

Is the Female Reproductive Chip Possible ?

Vagina Cervix

Uterus

Ovary

Fallopian tube

Follicle

Hormones drive 28 days menstrual cycle Female reproductive tract (FRT)

• Female reproductive organs are connected with each other to support reproductive functions: 28-day menstrual cycle and pregnancy.

Ex vivo Female Reproductive Tract in Microfluidic system

Follicle Ovary Fallopian

Fallopian tube Uterus

Uterine cervix

• Integrating and inter-connect multiple female reproductive tissues in vitro based on the microfluidic platform.

Fallopian tube Uterus Uterine Cervix Ovary

Liver

Xiao et al, Nat Commu, 2017

Integrated Female Reproductive Organs-EVATAR

Universal culture media

Liver spheroids

Ovary/follicles

Fallopian tube

Uterine endometrium

Uterine cervix

Xiao et al, Nat Commu, 2017

Fallopian Tube Tissue Remains Viable in MPS

• Microfluidic culture supported human fallopian tube viability, and cilia beating is controlled by dynamic hormone secretion patterns. Xiao et al, Nat Commu, 2017

Epithelium H&E Cilia beating

High E2

Low E2

OVGP1

OVGP1: oviductal glycoprotein

day 0

day 7

day 0

day 7

day 0

day 7

Recellularized Endometrium Remain viable in MPS

Xiao et al, Nat Commu, 2017 Olalekanet & Kim, BOR, 2017

• Decellularization: removing cells from tissue but keeping extracellular matrix (ECM) scaffold; • Recellulzrization: Reseeding cells into the decellularized scaffold.

Recelled Endo-H&E Decelled Endo-H&E Control Endo-H&E

Recelled Endo-Ki67 Recelled Endo-ER Recelled Endo-PR

Endo: uterine endometrium ER: estrogen receptor PR: progesterone receptor Decelled: decellularization Recelled: recellularization

Microfluidic Culture Supports Ectocervix in Response to Hormone from Upstream Ovary Chip

H&E

Ki67

day -7 day 0 with E2 peak

Xiao et al, Nat Commu, 2017 K McKinnon & S Getsios, unpublished

Microfluidic Culture Supports Liver Microtissues

• Microfluidic culture supported human liver spheroid viability and albumin production throughout 28-day culture period.

Hormone Secretion in Integrated Microfluidic Culture

• Downstream tissues consumed ovarian secreted hormones and/or integrated tissues changed ovarian hormone expression patterns!

0 10 20 30 40 50 60 70 80

0

10000

20000

30000

40000

50000

-14 -12 -10 -8 -6 -4 -2 0 2 4 6 8 10 12 14

Estrodiol Progesterone

Est

radi

ol (p

g / m

l)

Pro

gest

eron

e (n

g / m

l)

Ovary only in MPS

0

1

2

3

4

5

6

7

0

100

200

300

400

500

-14 -12 -10 -8 -6 -4 -2 0 2 4 6 8 10 12 14

Estradiol Progesterone

Est

radi

ol (p

g / m

l)

Pro

gest

eron

e (n

g / m

l)

Multiple tissues in MPS

I. Using microfluidic system to establish the ex-vivo 28-day menstrual cycle hormone control and female reproductive tract on a chip II. Screening of female reproductive toxic chemicals

Outline

- Establish

- Discover

Ovarian Toxicity of Doxorubicin (DOX)

• DOX is widely used for chemotherapy; WHO recommended • DOX significantly changed the follicle ovarian steroid hormone expression

patterns in the microfluidic cultures.

*p

Ovarian Toxicity of Doxorubicin

• DOX dose-dependently inhibited follicle growth, survival, and hormone secretion, • DOX has LC50 at 75.48 nM, which is relevant or even lower than the human exposure levels.

Xiao et al, Tox Sci, 2017

Dia

met

er (µ

m)

day

Follicle growth

Follicle survival

Sur

viva

l rat

e (%

) day 0

0.2 0.4 0.6 0.8

1

0 2 4 6 8

0 nM 2 nM 20 nM 200 nM *

150 200 250 300 350 400

0 2 4 6 8

0 nM 2 nM 20 nM 200 nM

*

y = 0.4197x + 18.773

0

50

100

150

0 50 100 150 200

Sur

viva

l rat

e (%

)

nM

LC50 of DOX

0 5

10 15 20 25

2 4 8

0 nM 2 nM 20 nM 200 nM

Est

radi

ol (p

g/m

l)

day

Estradiol secretion

*

Ovarian Toxicity of Doxorubicin

• DOX at low level exposure of 20 nM didn’t significantly affect follicle growth and survival, • However, DOX significantly increased the percentage of oocytes with abnormal spindle morphology

and chromosome misalignment. Xiao et al, Tox Sci, 2017

0 20 40 60 80

100 120

0 2 20 100 200

MII

perc

enta

ge (%

)

DOX (nM) DOX (nM)

Spi

ndle

& c

hrom

osom

e ab

norm

ality

(%)

0 20 40 60 80

100 120

0 2 20

* *

* * * * * *

Oocyte MII percentage Spindle and chromosome morphology

Take Home Message

• Ovary chip and female reproductive tract chip • Introducing gender/sex to co-cultured cells/tissues; • DOX has dose-dependent ovarian toxicity, which increases the risk of infertility during chemotherapy; • Low but human-relevant exposure level of DOX disrupts oocyte meiotic maturation(avoid this window for

oocyte cryopreservation and donation, and pregnancy).

Vagina Cervix Uterus

Ovary

Fallopian tube

Follicles

Reference

1. Xiao, S., Zhang, J, Liu, M., Iwahata, H., Rogers, HB, Woodruff, TK. (2017) Doxorubicin has dose-dependent toxicity on mouse ovarian follicle development, hormone secretion, and oocyte maturation. Toxicological Sciences. Doi:10.1093/toxsci/kfx047.

2. Xiao, S., Coppeta, J., Rogers, H., Woodruff, TK. (2017) A microfluidic culture model of the human reproductive tract and 28-day menstrual cycle . Nature Communications. Nature Communications. Mar 28; 8:14584.

3. Laronda, MM., Rutz, AL., Xiao, S., Whelan, KA., Duncan, FD., Roth, EW., Woodruff, TK., Shah, R. (2017) A bioprosthetic ovary created using 3D printed microporous scaffolds restores ovarian function in sterilized mice. Nature Communications. May 16; 8:15261.

4. Xiao, S., Zhang, J., Romero MM., Smith KN., Shea, LD., Woodruff, TK. (2015) In vitro follicle growth supports human oocyte meiotic maturation. Scientific Reports. 5:17323.

5. Xiao, S., Duncan, FE., Bai, L., Nguyen, CT., Shea, LD., Woodruff, TK. (2015) Size-specific follicle selection improves mouse oocyte reproductive outcomes. Reproduction: Piirep-15-0175.

6. Olalekan, SA., Burdette, JE., Getsios, S., Woodruff, TK., Kim, JJ. Development of a novel human recellularized endometrium that responds to a 28-day hormone treatment. Biology of Reproduction. 95(5):971-81.

Acknowledgements Northwestern University Teresa Woodruff Lab Hunter Rogers, MS Jiyang Zhang, MS Mingjun Liu, BS Monica Laronda, PhD Kelly McKinnon Peter Chen, MS Mingyang Jiang, MS Lu Bai, MS Cat Nugyen, MS Alex Rashedi, BS Jie Zhu, MD Danijela Dokic, MD Beth Sefton, PhD Chanel Arnold-Murray

Northwestern University Kim Lab Susan Olalekan, PhD Sevim Yildiz Arslan, PhD J. Julie Kim, PhD

Draper Labs Brett Isenberg, PhD Jeff Borenstein, PhD Jonathan Coppeta, PhD

Financial supports: NIEHS/NCATS: UH3TR001207 Subcontract award to S Xiao (UH3TR001207) Arnold School of Public Health Research Fund

University of South Carolina Xiao Lab Yingzheng Wang, MS Mingjun Liu, MS Megan F. Kopp

A Microfluidic Culture Model of the Human Reproductive Tract: Screening of Female Reproductive Toxic ChemicalsConflict of Interest StatementFemale Reproductive SystemFemale Reproductive Tract and PregnancyHormone Regulation of Female Reproductive SystemSummary of Female Reproductive Toxicity TestingOutlineOrgan-on-a-chip using Microfluidic TechnologyClinical Research Only in MalesHypothesisMicrofluidic Platform (MPS) DesignOvary Chip Based on Microfluidic CultureSlide Number 13Follicle Maturation & Ovulation in Microfluidic CultureFollicle Luteinization upon hCG StimulationOvarian Hormone Secretion in Microfluidic CultureIs the Female Reproductive Chip Possible ?Ex vivo Female Reproductive Tract in Microfluidic systemIntegrated Female Reproductive Organs-EVATARFallopian Tube Tissue Remains Viable in MPSRecellularized Endometrium Remain viable in MPSMicrofluidic Culture Supports Ectocervix in Response to Hormone from Upstream Ovary ChipMicrofluidic Culture Supports Liver MicrotissuesHormone Secretion in Integrated Microfluidic CultureOutlineOvarian Toxicity of Doxorubicin (DOX)Ovarian Toxicity of DoxorubicinOvarian Toxicity of DoxorubicinTake Home MessageReferenceAcknowledgements