Technology for high-fidelity podocyte cultivation

我们建议建立一个三维细胞培养系统的足细胞在体外。足细胞是肾小球中的主要细胞类型，其负责血液过滤和将废物清除到尿液中。目前，在体外研究这种细胞类型的能力有限，因为标准培养条件阻止足细胞获得天然细胞的典型结构和功能特性。在初步研究中，我们开发了一种“气泡”表面，包括球形地形特征，模仿肾小球足细胞培养的曲率。在这个项目中，我们将使用微加工，聚合物科学，生物化学，流体动力学和共培养技术来创建一个高度精细的足细胞细胞培养系统，目的是在体外产生分化的细胞。该项目的首要假设是，通过地形和生物化学线索控制细胞微环境将使足细胞能够在体外实现生理表型，如通过狭缝隔膜蛋白（包括nephrin）的存在和采用更接近体内足细胞的形态学特征所测量的。具体目标是：1。通过使用模型毒素，以地形线索作为评估nephrin调节的手段来构建培养系统; 2.利用生物工程技术促进足细胞表型成熟。在目标1中，我们将首先探索使用层粘连蛋白和胶原蛋白来开发限定的表面涂层，以促进足细胞附着在“气泡表面”上。将探索响应于模型毒素：阿霉素、高糖和嘌呤霉素氨基核苷（PAN）的nephrin表达的调节。在目标2中，我们将通过使用更小直径的球体，优化足细胞培养的“气泡”表面的曲率，以增加nephrin表达。然后，我们将创建transwell插入与“气泡”膜具有可调的渗透性共培养足细胞和内皮细胞。新的知识产权将被披露给多伦多大学，并根据现行的知识产权政策处理。通过拟议的研究，我们预计将培养至少1名博士后研究员，1名博士生，1名本科生和1名研究助理，与拟议项目的18个月时间线一致。HQP将在高度跨学科的环境中进行培训，使他们成为生物技术，制药和高科技工程行业的领导者。该项目将为足细胞的高保真3D培养提供第一个平台技术。这些结果将在GSK的密西索加研究中心和世界各地的发现研究中得到利用。最终，在加拿大，通过在发达的细胞培养平台上进行化合物筛选，为接受该平台使用培训的科学家提供新的工作岗位，以及制药行业的增长，将实现更高效，更快的发展过程，从而实现经济和社会效益。

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.