Microfabricated platforms for epithelial cultures and cell-based assays

Temitope Raheemot Sodunke

doi:10.17918/etd-3467

Epithelial cells, the major target of adult cancers have served as in vitro culture models for cancer related research for many years. Despite the increased use of these models, their potential as a cell-based screening tool for therapeutics has been hampered by the lack of existing miniaturized platforms that can: (1) facilitate single cell analysis (2) support cell cultivation in a physiological context, and (3) enable parallelization for highthroughput screening. Recent advancements in microfabrication technology has led to the development of miniaturized systems capable of handling very small (microliter - nanoliter) volumes of fluid, biomaterials, and bioparticle solutions. The central goal of this thesis was to develop miniaturized technologies that transcend the limitations of conventional macroscopic two-dimensional culture systems, by approaching the native environment of cells in vivo through technologies that allow direct screening of cultivated cells with drugs and targeted therapeutics in an appropriate context. To this end, we have developed miniaturized cell culture platforms towards three distinct applications: breast cancer research, liver biology/pathology, and studies of hepatitis B viral replication. Pertaining to breast cancer research, we have developed new techniques to create micropatterns of the most popular biomatrix for 3D epithelial cell culture (Matrigel). Moreover, we demonstrated that the micropatterned matrix can support the 3D cultivation of normal and breast cancer cell lines with comparable phenotypes to standard 3D culture techniques. In addition, by combining this micropatterned Matrigel with microfluidic techniques, we were able to develop a new platform to study individual cancer cell migration and invasion. Unlike previous demonstrations of micro-scale 3D systems that rely on just coating surfaces with thin layers of matrices, we took an approach that fully recapitulates standard 3D culture assays, where cells are totally embedded or overlayed with ECM to mimic the 3D microenvironment necessary to establish tissue-specific functions as well as mechanical and structural signals. Our development of a microfabricated platform for studying breast cancer cells can not only recapitulate normal epithelial structure and function but can also allow for more detailed understanding of tissue dysfunction in disease states and provides a more realistic milieu for modeling therapeutic intervention. We have also developed a novel "mini-liver" culture system using a microfluidic platform that simulates the basic functional unit of the liver (the hepatic sinusoid). The microfluidic platform is composed of a layered co-culture of hepatocytes and sinusoidal endothelial cells (the two major cell types in the liver) in a microchannel that mimics the liver sinusoid. Unlike previous liver models that rely on the culture of hepatocytes alone or random co-culture of hepatocytes with other cell types, we have developed a more realistic model that recreates important aspects of the liver including the architecture, fluidic environment, and more importantly the cellular composition. Moreover, we demonstrated the utility of our microchannel-based platform for meaningful biological experiments; we developed a microfluidic based in vitro assay for studying hepatitis B viral replication in hepatocytes. This platform will find many useful applications infundamental liver biology research, viral-mediated liver cancer studies, as well as potential use as a novel pharmaceutical platform for epithelial-based screening of genes and targeted cancer therapeutics.

Microfabricated platforms for epithelial cultures and cell-based assays

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Microfabricated platforms for epithelial cultures and cell-based assays

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