Description
- Abstract:
- Toxicity testing is undergoing a revolution, as the reliance on animal models has resulted in a large backlog of chemicals that have not been adequately safety tested. Animal models are expensive, time-intensive and do not accurately recapitulate human biology. As an alternative, the use of in vitro cell culture models is growing, as they are based on human cells and amenable to high-throughput screening approaches. Many of these in vitro models use cells grown in monolayer cultures, which respond differently to toxicants when compared to three-dimensional (3D) culture models. This thesis explored the use of 3D cultures of human MCF-7 breast carcinoma cells cultured using scaffold-free agarose hydrogels as a platform to assess estrogenic endocrine disrupting chemicals. To address the difficulties of imaging 3D microtissues, both traditional histopathological and optical clearing techniques were adapted to enable the visualization of the microtissues. Following these experiments, MCF-7 microtissues were characterized, and were found to contain a luminal space after 7 days in culture. Based on mRNA expression and localization of epithelial markers, MCF-7 microtissues are more differentiated than monolayer cultures. The response of MCF-7 microtissues and monolayer cultures to estradiol was explored, and demonstrated that at early time points (4 and 8 hours), MCF-7 monolayers and microtissues have a similar response to estradiol. The responses diverge at 24 hours, with 3D cultures showing dynamic changes in proliferation and cell adhesion. The effects of estrogenic compounds on MCF-7 microtissue morphology and gene expression were assessed. By using receptor-specific agonists, it was determined that ERα activation led to reduced lumen formation with increased proliferation, while ERβ activation did not alter morphology. The effects of two estrogenic endocrine disrupting chemicals bisphenol A (BPA) and genistein (GEN) were assessed. At high concentrations, BPA promoted lumen formation, while GEN did not alter lumen formation. BPA and GEN treatment altered gene expression and morphology in a compound-specific manner. This model provides evidence that supports the use of 3D MCF-7 microtissues to examine both gene expression and morphologic changes following exposure to estrogenic chemicals. This platform allows for the assessment of traditional histopathological endpoints in an in vitro system.
- Notes:
- Thesis (Ph.D. -- Brown University (2016)
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Citation
Vantangoli, Marguerite May,
"Assessment of Morphologic and Molecular Effects of Estrogen Exposure Using MCF-7 3D Microtissues"
(2016).
Pathobiology Theses and Dissertations.
Brown Digital Repository. Brown University Library.
https://doi.org/10.7301/Z0P26WJ5
