Technology for high-fidelity podocyte cultivation

高保真足细胞培养技术

• 批准号: 501198-2016
• 负责人: Radisic, Milica
• 金额: $ 2.6万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Collaborative Research and Development Grants
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=662854
• 关键词: Technology; fidelity; podocyte; cultivation

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.******

我们建议建立一种足细胞的3D细胞体外培养系统。足细胞是肾小球中的主要细胞类型，负责血液过滤和排泄物排入尿液。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典型的结构和功能特性。在初步研究中，我们开发了一种“气泡”表面，其中包括模仿肾小球弯曲的球状地形特征，用于足细胞培养。在这个项目中，我们将使用微制造、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，目的是在体外产生分化细胞。该项目的主要假设是，通过地形和生化线索控制细胞微环境将使足细胞能够在体外实现生理表型，这是通过存在裂隙隔膜蛋白(包括neaffin)和采用更具体内足细胞气味的形态来衡量的。具体目标是：1.用地形线索验证培养系统，作为一种手段，通过使用模型毒素来评估neparin的调节；2.利用生物工程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一种明确的表面涂层，以促进足细胞附着在“气泡表面”上。对模型毒素：阿霉素、高糖和嘌呤霉素氨基核苷(PAN)的响应，将探讨neparin表达的调节。在目标2中，我们将通过使用更小直径的球体来优化足细胞培养的“气泡”表面的曲率，以增加neparin的表达。然后，我们将为足细胞和内皮细胞的共培养创造具有可调节渗透性的“气泡”膜的Transwell插入物。新的知识产权将向多伦多大学披露，并按照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18个月的时间表保持一致。HQP将在高度跨学科的环境中进行培训，这是他们成为生物技术、制药和高科技工程行业领导者所必需的。该项目将为足细胞的高保真3D培养提供第一个平台技术。这些结果将被葛兰素史克的密西索加研究中心和世界各地的发现研究所利用。最终，在加拿大，通过在已开发的细胞培养平台中进行复合筛选，将为科学家提供使用该平台的培训的新工作，以及将经历更高效和更快的发展过程的制药行业的增长，将实现经济和社会效益。*