Grantee Research Project Results

2018 Progress Report: Mammosphere Bioreactor For Life-Stage Specific Toxicology

EPA Grant Number: R835736C001
Subproject: this is subproject number 001 , 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: Mickey Leland National Urban Air Toxics Research Center (NUATRC)
Center Director: Beskid, Craig
Title: Mammosphere Bioreactor For Life-Stage Specific Toxicology
Investigators: McCawley, Lisa J. , Markov, Dmitry
Institution: University of Pittsburgh , Vanderbilt University
EPA Project Officer: Aja, Hayley
Project Period: December 1, 2014 through November 30, 2018 (Extended to November 30, 2019)
Project Period Covered by this Report: December 1, 2017 through November 30,2018
RFA: Organotypic Culture Models for Predictive Toxicology Center (2013) RFA Text | Recipients Lists
Research Category: Chemical Safety for Sustainability

Objective:

Taking advantage of unique collaborations afforded through VPROMPT, we will develop a microfluidic Mammary Gland organotypic culture model (MG OCM) for toxicant assessment, for monitoring dynamic toxicant-dependent changes to cellular functions and signaling cascades, and elucidation of Adverse Outcome Pathways. In vivo the mammary gland is particularly sensitive to toxicant exposures during different life stages co-incident with times of active tissue growth and remodeling. We propose to extend our previously developed, microfabricated Mammary Gland Thick Tissue Bioreactor (MG-TTB) to support the combination of 3D culture methods with controlled exposure to investigate chemicals for potential mammary gland toxicity. We will utilize a simple mammosphere organotyic culture system and additionally evaluate a tri-culture system (normal human mammary epithelial cells + human mammary fibroblasts + sub-cutaneous adipocytes) for a more accurate recapitulation of mammary gland biology and crosstalk between heterotypic cells. We will validate this system for use as a medium throughput toxicology screening as well as for high information content analysis of targeted toxicant-dependent alterations to mammary formation. We will exploit this system for in vitro evaluation of potential environmental toxicants for effects on mammary development using both 1) chronic exposure and 2) acute exposures co-incident with various stages of gland development. Furthermore, this system will be used for high content analysis of putative toxicants with a focus on key biomarkers. Our specific objectives to validate the microfluidic OCM for toxicant are as follows: (A) to develop and validate the predictive utility of self-contained, fully-automated MG OCM modules, (B) to investigate the effects of chemical exposures on MG OCMs for chemicals shown to reduce lactation index in only the F2 generation of multigenerational studies - strongly suggesting a role for life-stage specific exposure; and (C) to develop and validate toxicant assessment for compounds requiring metabolic activation using a paired Liver-OCM/MG-OCM.

Progress Summary:

For the past year, we focused on validation of medium through put on-chip analysis using known toxicants of mammary development; the development of subchronic delivery of compounds and the development of connectivity strategies with common medias laying the foundations of integration with the Liver OCM .

We have developed automated tool for mammosphere size analysis in MetaXpress software used to control image acquisition with ImageXpress microscope.
In order to simplify hardware setup and being able to sample each chamber individually within the single MG OCM we have evaluated performance of a multichannel peristaltic developed by project 5. This would allow us to replace a MX777-605 6 position / 7 port valve and pump combo with a single multichannel pump.
We have screened multiple compounds for their interactions with PDMS as a function of PDMS surface treatments and have generated a mathematical description of toxicant/bioreactor interaction.
We have completed pilot experiments for assessment of toxicant effect on mammosphere formation over time using ER+ and ER- cell lines labeled with lentiviral biosensors.
We have assessed functional coupling of Liver- to MG-OCM using conditioned media from Liver-OCM challenged with toxicant and evaluated experimental conditions that would allow for proper media mixing and delivery during direct Liver - MG OCM coupling.

Future Activities:

We will continue collaboration with Project 5 on upgrading MG OCM to include customized valves and pumps necessary for media mixing, sampling, and coupling the liver-OCM upstream of the MG-OCM.
Screen toxicants: validate MG OCM reactor system for medium through-put for stage specific and acute toxicity assessment using optical and electrochemical sensing.
Test direct Liver-MG-OCM coupling.

Journal Articles on this Report : 2 Displayed | Download in RIS Format

Publications Views
Other subproject views:	All 19 publications	3 publications in selected types	All 3 journal articles
Other center views:	All 169 publications	57 publications in selected types	All 56 journal articles

Publications
Type	Citation	Sub Project	Document Sources
Journal Article	Auner AW, Tasneem KM, Markov DA, McCawley LJ, Hutson MS. Chemical-PDMS Binding Kinetics and Implications for Bioavailability in microfluidic Devices. Lab on a Chip 2019;19(5):864-874	R835736C001 (2018) R835736C001 (Final) R835736C005 (2018)	Abstract from PubMed Abstract: Lab on a Chip - Abstract HTML Exit
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) R835736C001 (2018) R835736C003 (2016) R835736C003 (2017) R835736C003 (2018)	Abstract: Mary Ann Liebert-Abstract Exit

Supplemental Keywords:

Mammary development, mammary toxicology, organs on chip, mammary on chip, PDMS bioreactors, thick tissue bioreactor, PDMS interactions, organotypic cell culture.

Relevant Websites:

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

Progress and Final Reports:

Original Abstract

Main Center Abstract and Reports:

R835736 Mickey Leland National Urban Air Toxics Research Center (NUATRC)

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 DevelopmentalLimbMalformationsResulting 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

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The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.