Technology for high-fidelity podocyte cultivation

我们建议在体外建立足细胞的三维细胞培养系统。足细胞是肾小球中主要的细胞类型，负责血液过滤和废物排入尿液。目前，在体外研究这种细胞类型的能力有限，因为标准的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，我们开发了一个“气泡”表面，其中包括球形地形特征，模拟肾小球的曲率，用于足细胞培养。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复杂的足细胞细胞培养系统，目的是产生体外分化的细胞。该项目的首要假设是，通过地形和生化线索控制细胞微环境将使足细胞在体外实现生理表型，通过存在狭缝隔膜蛋白（包括肾素）和采用更具有体内足细胞气味的形态轮廓来测量。具体目标是：1。用地形线索验证培养系统，作为通过使用模型毒素评估肾素调节的手段。利用生物工程技术促进足细胞表型成熟。在Aim 1中，我们将首先探索使用层粘连蛋白和胶原蛋白来开发一种明确的表面涂层，以促进足细胞附着在“气泡表面”上。将探讨肾素表达在多柔比星、高糖和嘌呤霉素氨基核苷（PAN）模型毒素反应中的调节。在Aim 2中，我们将通过使用较小直径的球体来优化足细胞培养的“气泡”表面曲率，以增加肾素的表达。然后，我们将创建带有可调节渗透性的“气泡”膜的transwell插入物，用于足细胞和内皮细胞的共培养。新的知识产权将向多伦多大学披露，并按现行知识产权政策处理。拟培养博士后1人、博士生1人、本科生1人、副研究员1人，培养时间为18个月。HQP将在一个高度跨学科的环境中培养他们成为生物技术、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培养提供有史以来第一个平台技术。这些结果将在GSK的密西沙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，通过在已开发的细胞培养平台上进行化合物筛选，将实现经济和社会效益，为科学家提供新的就业机会，这些科学家将接受使用该平台的培训，制药行业的增长将经历更有效和更快的开发过程。******

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.******