2017 Progress Report: Validating a fetal membrane on a chip model for characterizing reproductive toxicant exposure risks

EPA Grant Number: R835736C003
Subproject: this is subproject number 003 , established and managed by the Center Director under grant R835736
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).

Center: Vanderbilt Pittsburgh Resource for Organotypic Models for Predictive Toxicology
Center Director: Hutson, Michael Shane
Title: Validating a fetal membrane on a chip model for characterizing reproductive toxicant exposure risks
Investigators: Osteen, Kevin G. , Aronoff, David , Bruner-Tran, Kaylon L.
Institution: University of Pittsburgh Main Campus , Vanderbilt University
EPA Project Officer: Klieforth, Barbara I
Project Period: December 1, 2014 through November 30, 2018 (Extended to November 30, 2019)
Project Period Covered by this Report: December 1, 2016 through November 30,2017
RFA: Organotypic Culture Models for Predictive Toxicology Center (2013) RFA Text |  Recipients Lists
Research Category: Safer Chemicals , Health , Human Health

Objective:

Fetal membrane inflammation due to infection, known as chorioamnionitis (CAM), is a leading cause of preterm birth (PTB). Although numerous infectious agents have been associated with CAM; many women with microbial contamination of the amniotic cavity carry their pregnancies to term. Equally significant, women without an identifiable infection may also exhibit signs of CAM and deliver preterm. These clinical observations strongly suggest that, in addition to infection, host factors must also influence the risk for CAM-associated PTB. Supporting this concept, in a murine model, we identified a doubling in the occurrence of infection-mediated PTB in animals with a previous exposure to an environmental toxicant (2,3,7,8-tetrachlorodibenzo-p-dioxin or TCDD). This two-hit model of CAM is further supported by our in vitro studies using isolated human endometrial cells, which, following TCDD exposure, also exhibit an enhanced response to an inflammatory challenge related to an infection (i.e., LPS, IL-1β or TNF-α). Taken together, our data suggest the novel hypothesis that environmental toxicants prime the gravid uterus for exaggerated inflammatory responses to microbial invasion. More than 80,000 chemicals have been released into our environment since the Toxic Substances Control Act of 1976; however, few have undergone controlled experimental examination related to reproductive health. Therefore, we have developed unique platforms for the establishment and analysis of organ-on-chip systems amenable to medium throughput screening.

The Objective of Project 3 is to develop and validate an instrumented fetal membrane on a chip (IFMOC) system to specifically identify toxicants which negatively impact the maintenance of pregnancy.

Progress Summary:

IFMOC 3.0 Establishment. As established in the previous report, the IFMOC 3.0 device was successfully populated with endometrial fibroblasts (stroma) and human umbilical vein endothelial cells (HUVEC). In Year 3, we have utilized uterine microvascular endothelial cells (UtMVEC) co-cultured with endometrial stromal cells within the two-chambered device. We have successfully induced decidualization of human endometrial stromal cells cultured within the device and treated with estradiol (e, 1nM) and medroxyprogesterone acetate (MPA, 500nM). At the same time, we are able to demonstrate (Fig. 3.1) that decidualization is significantly inhibited in similar cultures additionally treated with TCDD (1nM) as measured via prolactin secretion. Additional studies conducted over the most recent funding year began to assess the impact of toxicant exposure on tight junction integrity. Using the two-chamber IFMOC platform, tight junction formation in amniotic epithelial cells was quantified by calculating the permeability coefficient using macromolecule diffusion (FITC-dextran). We found that exposure of cells to inflammatory stimuli provided evidence of a loss of amniotic epithelial barrier integrity (Fig. P3-2).

INSERT FIGURE P3 1 & 2 HERE

To ensure that the IFMOC 3.0 provides an accurate representation of the fetal membrane environment, ongoing studies continue to compare results obtained within the device to “gold standard” fetal membrane Transwell assay. These assays will be employed for the dose response curves studies for the 13 toxicants/chemicals outlined previously. Each dose response curve will inform the potential doses to be used for subsequent milestones. To date, fetal membrane Transwell assays have been performed using logarithmic serial dilutions (ranging from 10 pM to 10 mM) of bisphenol, estrone, mono butyl phthalate (MBP), and beno (a) pyrene (data not shown).

Validation and Exposure of IFMOC and Transwell Cultures. Using our established IFMOC 3.0 device (Fig. P3.4) we next examined the impact of a second hit relative to cellular functions that are critical to fetal membrane stability. As an example of the experimental protocols completed over the past year, as shown in Figure P3-5, amniotic epithelial cells growing in the device exhibit normal tight junctions between cells; however, treatment with LPS reduces the cell-cell integrity.

IF POSSIBLE, ADD Figures P3 3, 4 AND -5. HERE (ending on “Page 23 of 54”)

Further, dose-response curves for each selected toxicant are underway utilizing the fetal membrane transwell. To date, transwell dose response assays have been conducted utilizing serial dilutions (ranging from 10 pM to 10 mM) of bisphenol A, estrone, mono butyl phthalate (MBP), and beno (a) pyrene. Following 24 hrs treatment, conditioned media was collected and fresh media added back to all wells. Media contained the same toxicant/dose as well as LPS. All wells were terminated 4 hrs after LPS exposure and media and tissues collected. ELISAs for selected cytokines are underway utilizing conditioned media before and after LPS exposure allowing analysis of the impact of toxicant exposure with and without the secondary inflammatory challenge. Tissues were fixed and are being processed for immunohistochemical analysis.

IFMOC-Liver Integration. Finally, to examine the impact of liver metabolism of selected toxicants on the fetal membrane, we have exchanged conditioned media with Project 4. Our tests showed that dilution of liver-conditioned media with fresh media is necessary to ensure cells receive appropriate nutrients and that the IFMOC cells are not adversely affected by the liver media diluted 50% with fresh media. Project 4 is currently collecting liver-conditioned media following exposure to TCDD and other selected toxicants for use in the IFMOC 3.0.

Future Activities:

Project 3 (Fetal Membranes), as planned, will validate the fetal membrane OCM along with human fetal membrane Transwell cultures on select agents, while also establishing the liver OCM (Project 4) upstream of our OCM in order to validate this integrated system with selected agents.


Journal Articles on this Report : 5 Displayed | Download in RIS Format

Other subproject views: All 21 publications 10 publications in selected types All 10 journal articles
Other center views: All 149 publications 39 publications in selected types All 39 journal articles
Type Citation Sub Project Document Sources
Journal Article Bruner-Tran KL, Gnecco J, Ding T, Glore DR, Pensabene V, Osteen KG. Exposure to the environmental endocrine disruptor TCDD and human reproductive dysfunction: translating lessons from murine models. Reproductive Toxicology 2017;68:59-71. R835736 (2015)
R835736 (2016)
R835736 (2017)
R835736 (2018)
R835736C003 (2016)
R835736C003 (2017)
R835736C003 (2018)
R826300 (Final)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
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  • Other: ScienceDirect-Full Text PDF
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  • Journal Article Ding T, Lambert LA, Aronoff DM, Osteen KG, Bruner-Tran KL. Sex-dependent influence of developmental toxicant exposure on group B Streptococcus -mediated preterm birth in a murine model. Reproductive Sciences 2018;25(5):662-673. R835736 (2017)
    R835736 (2018)
    R835736C003 (2017)
    R835736C003 (2018)
  • Abstract from PubMed
  • Abstract: Sage-Abstract
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  • Journal Article Gnecco JS, Anders AP, Cliffel D, Pensabene V, Rogers LM, Osteen K, Aronoff DM. Instrumenting a fetal membrane on a chip as emerging technology for preterm birth research. Current Pharmaceutical Design 2017;23(40):6115-6124. R835736 (2016)
    R835736 (2017)
    R835736 (2018)
    R835736C003 (2016)
    R835736C003 (2017)
    R835736C003 (2018)
  • Abstract from PubMed
  • Abstract: Bentham Science-Abstract
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  • Other: White Rose-Abstract
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  • Journal Article Gnecco JS, Pensabene V, Li DJ, Ding T, Hui EE, Bruner-Tran KL, Osteen KG. Compartmentalized culture of perivascular stroma and endothelial cells in a microfluidic model of the human endometrium. Annals of Biomedical Engineering 2017;45(7):1758-1769. R835736 (2015)
    R835736 (2016)
    R835736 (2018)
    R835736C003 (2016)
    R835736C003 (2017)
    R835736C003 (2018)
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  • Journal Article Hutson MS, Alexander PG, Allwardt V, Aronoff DM, Bruner-Tran KL, Cliffel DE, Davidson JM, Gough A, Markov DA, McCawley LJ, McKenzie JR, McLean JA, Osteen KG, Pensabene V, Samson PC, Senutovitch NK, Sherrod SD, Shotwell MS, Taylor DL, Tetz LM, Tuan RS, Vernetti LA, Wikswo JP. Organs-on-chips as bridges for predictive toxicology. Applied In Vitro Toxicology 2016;2(2):97-102. R835736 (2015)
    R835736 (2016)
    R835736 (2017)
    R835736 (2018)
    R835736C001 (2018)
    R835736C003 (2016)
    R835736C003 (2017)
    R835736C003 (2018)
  • Abstract: Mary Ann Liebert-Abstract
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  • Supplemental Keywords:

    Stroma, organs-on-a-chip, microfluidic, endometrium, toxicant exposure risks

    Relevant Websites:

    www.vanderbilt.edu/vprompt Exit

    Progress and Final Reports:

    Original Abstract
  • 2015 Progress Report
  • 2016 Progress Report
  • 2018 Progress Report

  • Main Center Abstract and Reports:

    R835736    Vanderbilt Pittsburgh Resource for Organotypic Models for Predictive Toxicology

    Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
    R835736C001 Mammosphere Bioreactor For Life-Stage Specific Toxicology
    R835736C002 Organotypic Culture Model to Analyze Developmental LimbMalformationsResulting from Toxicant/Teratogen Exposure
    R835736C003 Validating a fetal membrane on a chip model for characterizing reproductive toxicant exposure risks
    R835736C004 Organotypic Liver Model for Predictive Human Toxicology and Metabolism
    R835736C005 Systems Engineering & Analysis for Organotypic Culture Models