Grantee Research Project Results

2016 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. , McIntyre, J. Oliver , Auner, Alex , Markov, Dmitry , Schafer, Emily , Bazilevich, Gloria , Fryman, Phillip
Current Investigators: McCawley, Lisa J. , Markov, Dmitry
Institution: Vanderbilt University
Current 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, 2015 through November 30,2016
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 this second year, we focused on protocol development and validation for medium throughput on-chip analysis using known toxicants of mammary development; the development of subchronic delivery of compounds and analysis of proteolytic activities within the OCM.

1. We have developed calibration protocols and validated Rotary Planar Peristaltic Micropumps (RPPMs) from Project 5 for the MG OCM. These custom-built pumps from Project 5 are in active use.
2. We have applied protocols developed last year using UV-VIS spectroscopy and attenuated total reflection (ATR)—FTIR spectroscopy to assess toxicant interaction with PDMS including saturation and on/off rates where measurable interaction was noted. These drug-PDMS assessments are useful to establish effective / actual drug concentration levels within the MG OCM.
3. As part of our medium throughput assessment of putative toxicant effect on mammo-sphere formation, we are applying Caspase-3 and histone 2B nuclear marker lenti-viruses from Project 4 as biosensors to monitor apoptosis and proliferation using MCF-10A, MCF-12A, and MCF-7 cell lines. We have generated stable cell lines with simultaneous expression of both lentiviral biosensor systems.
4. We have initiated validation experiments for assessment of toxicant effect on mammo-sphere formation over time using these cell lines labeled with lentiviral biosensors.
5. We have designed and fabricated a bioreactor for protease activity measures within a closed system.

Future Activities:

• We will continue collaboration with Project 5 on upgrading MG OCM to include smaller footprint valves and pumps necessary for sampling and coupling the liver OCM upstream of the MG OCM.
• Using measures on/off rates and saturation values, we will generate a mathematical description of toxicant/bioreactor interaction.
• Screen toxicants: validate MG OCM reactor system for medium throughput for stage specific and acute toxicity assessment using optical and electrochemical sensing.
• Move towards High Information Content profile analysis beginning with proteinase activity analysis of mammosphere formation using identified toxicant.
• Develop strategies for liver MG OCM coupling.

Journal Articles:

No journal articles submitted with this report: View all 19 publications for this subproject

Supplemental Keywords:

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

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

Final Report

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