A Microfluidic Culture Model of the Human Reproductive Tract: Screening of Female ... ·...
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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 [email protected]
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Conflict of Interest Statement
This is no conflict of interest for the work I present today.
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Female Reproductive System
Menstrual cycle
Ovary
Infertility IVF
Vagina Cervix Uterus
Ovary
Fallopian tube
Follicles
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Female Reproductive Tract and Pregnancy Fertilization
Ovulation
Embryo development & transport
Embryo implantation
Primordial follicle
Primary follicle Secondary
follicle Antral follicle
Corpus Luteum
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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
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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
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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
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Organ-on-a-chip using Microfluidic Technology
Brain chip
Vessel chip
Lung chip
Heart chip
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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!
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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
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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
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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
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• 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
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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
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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
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Error bar: Standard deviation; *p
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Ovarian Hormone Secretion in Microfluidic Culture
• Microfluidic platform recapitulates human 28-day menstrual cycle hormone control.
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10000 15000 20000 25000 30000 35000 40000 45000
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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
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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.
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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
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Integrated Female Reproductive Organs-EVATAR
Universal culture media
Liver spheroids
Ovary/follicles
Fallopian tube
Uterine endometrium
Uterine cervix
Xiao et al, Nat Commu, 2017
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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
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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
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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
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Microfluidic Culture Supports Liver Microtissues
• Microfluidic culture supported human liver spheroid viability and albumin production throughout 28-day culture period.
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Hormone Secretion in Integrated Microfluidic Culture
• Downstream tissues consumed ovarian secreted hormones and/or integrated tissues changed ovarian hormone expression patterns!
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Estrodiol Progesterone
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g / m
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Pro
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Ovary only in MPS
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Estradiol Progesterone
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g / m
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Pro
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g / m
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Multiple tissues in MPS
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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
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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
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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
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0 nM 2 nM 20 nM 200 nM *
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0 nM 2 nM 20 nM 200 nM
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y = 0.4197x + 18.773
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LC50 of DOX
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Est
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Estradiol secretion
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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
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MII
perc
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DOX (nM) DOX (nM)
Spi
ndle
& c
hrom
osom
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norm
ality
(%)
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Oocyte MII percentage Spindle and chromosome morphology
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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
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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.
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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