Grantee Research Project Results
Final Report: Mammosphere Bioreactor For Life-Stage Specific Toxicology
EPA Grant Number: R835736C001Subproject: 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)
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.
Summary/Accomplishments (Outputs/Outcomes):
Conclusions:
During the project, 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 a series of mammary gland OCMs (MG-OCM) that a) are self-contained with six culture chambers and on-board computer-controlled planar micro pumps and valves; b) are compatible with conventional and high throughput / high content screening microscopes; c) contain larger culture chambers to minimize effects of stiffer PDMS on cell matrix during mammary development; d) are openable for tissue extraction and processing; e) are compatible with closed system loading of custom proteolytic Beacon loading for measurements of the mammoshere proteolytic activity; f) could be modified to include humidified gas delivery system within MG-OCM for proper pH and oxygen balancing of the delivered media and reduction of evaporative effects on the cells.
- We have developed protocols and validation approaches for a) testing Rotary Planar Peristaltic Micropumps (RPPMs) and Rotary Planar valves (RPV); b) testing compound interactions with bioreactor materials, such as PDMS using both UV-VIS and FT-ATR approaches; c) sample collection for mass-spectrometry assessment of metabolic activity; d) imaging protocols and have developed automated tool for mammosphere size analysis in MetaXpress software used to control image acquisition with ImageXpress microscope; e) labeling of MCF – family of mammary epithelial cells with Caspase-3 and histone 2B nuclear marker lenti-viruses from our collaborators from UP as biosensors to monitor apoptosis and proliferation.
- MG-OCM have been shown to work: a) with a single- channel pump coupled to passive microfluidic splitter network; b) with a single- channel pump coupled to and RPV or MX777-605 6 position / 7 port valve in time-division multiplexing mode; or c) with a single six-channel RPPM pump. The latter is especially important for effluent collection when cells in each chamber within the cartridge are cultured under unique conditions.
- We have screened numerous compounds for their interactions with PDMS as a function of time, PDMS composition, annealing status, and surface treatments and have generated a mathematical description of toxicant/bioreactor interaction. These compound-PDMS assessments are useful to establish effective / actual drug concentration levels within the MG-OCM.
- As part of our medium through-put assessment of putative toxicant effect on mammo-sphere formation, we have applied 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.
- We have completed experiments for assessment of toxicant effect on mammosphere formation over time using ER+ and ER- cell lines labeled with lentiviral biosensors.
- We have use mass-spectrometry to established metabolic secretion baseline signature for Mammary Gland OCM
- 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.
Journal Articles on this Report : 1 Displayed | Download in RIS Format
Other subproject views: | All 19 publications | 3 publications in selected types | All 3 journal articles |
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Other center views: | All 169 publications | 57 publications in selected types | All 56 journal articles |
Type | Citation | ||
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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) |
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:
Vanderbilt-Pittsburgh Resource for Organotypic Models for Predictive Toxicology (VPROMPT) Exit
Progress and Final Reports:
Original AbstractMain 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
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.
Project Research Results
- 2018 Progress Report
- 2017 Progress Report
- 2016 Progress Report
- 2015 Progress Report
- Original Abstract
3 journal articles for this subproject
Main Center: R835736
169 publications for this center
56 journal articles for this center