2016 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: Vanderbilt University , University of Pittsburgh Main Campus
Current 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, 2015 through November 30,2016
RFA: Organotypic Culture Models for Predictive Toxicology Center (2013) RFA Text |  Recipients Lists
Research Category: Safer Chemicals , Health , Human Health

Objective:

The objective of Project 3 is to develop and validate an instrumented fetal membrane on a chip (IFMOC) system in order to specifically identify toxicants that negatively impact the maintenance of pregnancy. Intrauterine infection during pregnancy, known as chorioamnionitis (CAM), is a leading cause of preterm birth (PTB) as a consequence of triggering fetal membrane inflammation that drives labor processes. Although numerous infectious agents can cause CAM, some women with microbial contamination of the amniotic cavity carry their pregnancy to term. Significantly, women without identifiable infection also may exhibit signs of CAM and deliver preterm.  Thus, we hypothesize that host factors likely influence the risk for CAM-associated PTB. Using a mouse model, we previously identified a doubling in the occurrence of spontaneous PTB when an infective agent represented a “second hit” following a previous exposure to an environmental toxicant (2,3,7,8-tetrachlorodibenzo-p-dioxin or TCDD). This two-hit hypothesis is supported by our in vitro models using isolated human endometrial cells, which, following TCDD exposure, exhibit an enhanced response to an inflammatory challenge related to infection (i.e., LPS, IL-1 or TNF-α). These 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. To meet this challenge, we have developed novel platforms for the establishment and analysis of organ-on-chip systems that are amenable to medium throughput screening.

Progress Summary:

Specific Aim 1: Define the temporal and concentration-dependent effects of TCDD on native fetal membrane immune responses to infection. We have successfully tested and identified experimental concentrations of lipopolysaccharides (LPS, 10 ng/ml) in combined use with TCDD (10 nM) for an exposure of 4 hours (LPS exposure) and 24 hours (TCDD exposure). In addition, we have selected and validated a minimum set of inflammatory biomarkers influenced by TCDD (IL-1 beta, TNF-alpha, IL-6 and IL-8) using the fetal membrane punch biopsies. We interrogated this selection using a cytometric bead array panel with further validation of each individual candidate using quantitative PCR and ELISA assays. Additionally, IL-1β production pathway correlated with activation of Nod-Like Receptor Family, Pryin Domain Containing-3 (NLRP3) and other components of the inflammasome complex. The greatest change was seen in the combination exposure of TCDD and LPS. For our fetal membrane transwell setup, we have analyzed responses to TCDD and/or LPS in decidual cells and amniotic epithelial cells via cytokine multiplex array using the BD™ Cytometric Bead Array (CBA). Both cell types were highly responsive to either stimulus and exhibited enhanced expression of inflammatory cytokines. Next, we assessed MMP and TIMP networks in the transwell model. We found that MMP7 is decreased in TCDD primed amniotic cells, while MMP8 is increased. MMP2 and MMP9, which are proteinases for collagen type IV (an extracellular component of the basement membrane), are dysregulated by TCDD exposure. Fluorescent collagen IV staining showed that extracellular matrix production is greatly decreased in TCDD exposed decidua. Most significantly, our observations using the transwell fetal membrane model revealed that sidedness of exposure was not a critical determinant of response in the context of toxicant screening. For this reason, we have concluded that the punch membrane biopsies are an appropriate model for the initial screening studies.  

Specific Aim 2: Implement an in vitro, microscaled, living, instrumented fetal membrane on-a-chip (IFMOC) that recapitulates the physiological properties of the human tissue. We have made significant progress in developing the IFMOC 3.0, initially utilizing isolated human endometrial stromal cells (HESC) and the human monocyte cell line, THP-1.  We have been using the IFMOC 3.0 device in two different configurations: 1) as a dual-chamber device using a 6 µm thin membrane with 10% porosity to separate the two chambers, and 2) as a single chamber device using no membrane. In this manner, we have compared the inflammatory cytokine response of cells to a challenge with LPS. Comparisons between devices without cells and with cells showed conclusively that the cells are able to form tight junctions and prevent diffusion of FITC-dextran across the membrane. We also tested co-cultures of HUVEC and monocytes for hormone-mediated changes of endothelial function and recorded a 50% increase in endothelial proliferation in E2+MPA [Medroxyprogesterone acetate (a synthetic progestin)] exposed cells in co-culture over mono-culture.  Finally, co-cultures of human amniotic epithelial cells (hAMEPI) with stromal decidual cells or trophoblasts were successfully maintained for 7 days inside two-chamber IFMOC devices.


Figure 1. Design and characterization of a microfluidic two chamber device: A transparent, semi-permeable membrane was used in the device to separate the cell types within two microfluidic chambers. The membranes are fabricated by using a photolithographic method with biocompatible resin.

Future Activities:

We now have exposed a HRT-8/SV-neo trophoblast monolayer to 10 nM TCDD for 48 hours inside a single chamber IFMOC device. The next step will be to repeat these studies in the presence of a secondary inflammatory challenge with LPS. Murine studies ongoing in our lab, but not funded by the EPA, demonstrated that prior exposure to TCDD significantly enhances the maternal inflammatory response to GBS. These studies will guide our introduction of a second stressor (i.e., GBS, LPS or second toxicant) into the IFMOC.


Journal Articles on this Report : 4 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
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  • Full-text: ScienceDirect-Full Text HTML
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  • Abstract: ScienceDirect-Abstract
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  • Other: ScienceDirect-Full Text PDF
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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
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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)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
  • Full-text: Springer-Full Text HTML
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  • Abstract: Springer-Abstract
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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

    Relevant Websites:

    Vanderbilt-Pittsburgh Resource for Organotypic Models for Predictive Toxicology (VPROMPT) Exit

    Progress and Final Reports:

    Original Abstract
  • 2015 Progress Report
  • 2017 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