Technology for high-fidelity podocyte cultivation

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We propose to develop a 3D cell culture system for podocytes in vitro. Podocytes are the primary cell type in the kidney glomerulus that are responsible for blood filtration and waste removal into the urine. Currently, there is a limited ability to study this cell type in vitro because standard culture conditions prevent the podocytes from acquiring structural and functional properties that are typical of the native cell. In preliminary studies, we developed a "bubble" surface that included spherical topographical features that mimic the curvature of the glomerulus for podocyte cultivation. In this project, we will use techniques from microfabrication, polymer science, biochemistry, fluid dynamics, and co-culture to create a highly elaborate cell culture system for podocytes, with the aim of generating in vitro-differentiated cells. The overarching hypothesis of this project is that the control of cell microenvironment via topographical and biochemical cues will enable podocytes to achieve a physiological phenotype in vitro as measured by the presence of slit diaphragm proteins, including nephrin and the adoption of a morphological profile more redolent of in vivo podocytes. The Specific Aims are: 1. Validate the culture system with topographical cues as a means to assess nephrin regulation through the use of model toxins and 2. Enhance podocyte phenotypic maturation using bioengineering. In Aim 1, we will first explore the use of laminin and collagen to develop a defined surface coating to facilitate podocyte attachment on "bubble surface". Regulation of nephrin expression in response to model toxins: doxorubicine, high glucose and puromycin amino nucleoside (PAN) will be explored. In Aim 2, we will optimize the curvature of the "bubble" surface for podocyte culture with respect to increasing nephrin expression, by using smaller diameter spheres. We will then create transwell inserts with a "bubble" membrane with tuneable permeability for co-culture of podocytes and endothelial cells. New IP will be disclosed to the University of Toronto and treated according to the current IP policy. Through the proposed studies, we expect to train at least 1 Post-doctoral fellow, 1 PhD student, 1 undergraduate student and 1 research associate, consistent with an 18 month time-line of the proposed project. The HQP will train in a highly interdisciplinary environment required for them to become leaders in biotechnology, pharmaceutical and high-tech engineering industry. This project will provide the first ever platform technology for high fidelity 3D culture of podocytes. These results will be exploited at GSK's Mississauga Research Center and around the world in discovery studies. Ultimately, economic and social benefit will be realized, in Canada, by compound screening in the developed cell cultivation platform, new jobs for scientists that will be trained on the use of this platform and growth in the pharmaceutical industry that will experience a more efficient and faster development process.