A Novel System for Mass Production of iPS Cells

描述（由申请人提供）：Primorigen Biosciences LLC SBIR项目摘要Primorigen将使用SBIR资金开发和验证基于微载体的细胞培养系统，用于iPS细胞的选择、放大和无投料批量生产，该系统基于Primorigen专有的多能干细胞附着因子（StemCadhere”），用于iPSC的直接物理选择和繁殖。I期研究将定义偶联化学，优化E-cadherin介导的细胞与stemcadherin包被微载体的粘附，分析iPSC在stemcadherin包被微载体上的附着效率和增殖，分析stemcadherin包被微载体对新诱导的iPS细胞的直接物理选择的效用，并比较stemcadherin包被微载体和matrigel包被微载体上冷冻保存的iPSC的性能。这项工作将为II期研究提供基础，II期研究将研究直接在微载体上产生分化细胞。类似的方法将用于测试和选择每个胚层（外胚层、中胚层和内胚层）以及有限数量的终末分化谱系（如肝细胞、心肌细胞和造血细胞）的细胞粘附分子。最初的商业化产品将是a)附着在微载体上的即用（冷冻保存）iPS细胞，准备添加分化因子和下游协议；b)附着因子与微载体耦合的新应用，Primorigen可以向从事iPS分化细胞生产的制药和生物技术公司大规模推销。

DESCRIPTION (provided by applicant): Primorigen Biosciences LLC SBIR Project Abstract Primorigen will use SBIR funds to develop and validate a microcarrier-based cell culture system for selection, scale-up and feeder-free mass production of iPS cells, based on Primorigen's proprietary pluripotent stem cell attachment factor (StemCadhere") for direct physical selection and propagation of iPSC. Phase I studies will define coupling chemistry that optimizes E-cadherin mediated cell adhesion to StemCadhere"-coated microcarriers, analyze iPSC attachment efficiency and propagation on the StemCadhere"-coated microcarriers, analyze the utility of StemCadhere-coated microcarriers for direct physical selection of newly induced iPS cells, and compare performance of cryopreserved iPSC on StemCadhere"-coated microcarriers with Matrigel-coated microcarriers. This work will provide the foundation for Phase II studies, which will investigate production of differentiated cells directly on the microcarriers. Similar methods will be used to test and select cellular adhesion molecules specific for each germ layer (ectoderm, mesoderm and endoderm), and for a limited number of terminally differentiated lineages such as hepatocytes, cardiomyocytes, and hematopoietic cells. The initial commercialized products will be a) ready-to-use (cryopreserved) iPS cells attached to the microcarriers, ready for addition of differentiation factors and downstream protocols; and b) a new application for attachment factors coupled to microcarriers that Primorigen can market at large to pharmaceutical and biotechnology firms engaged in differentiated cell production from iPS. PUBLIC HEALTH RELEVANCE: Primorigen Biosciences LLC SBIR Project Narrative Current methods for production of iPS cells require labor intensive manual culturing steps, frequent feeding of cultures, and inclusion of drug-selectable resistance markers to achieve purification of iPS from non-induced parental cells (such as foreskin fibroblasts). To fulfill the promise of iPS cells as a source for cellular therapies and differentiated cells for pharmaceutical development, it is mandatory to avoid the use of lentiviral vectors and other agents capable of genomic integration, which precludes the use of integrated drug-resistance markers for iPS selection. In addition, to decrease variability in production lots, it is desirable to automate as much of the culture as possible, while maintaining the proliferation and pluripotency of the iPS cells. Primorigen's SBIR proposal will address this need by developing a comprehensive, integrated solution for selection and mass production of iPS cells by taking advantage of Primorigen's proprietary stem cell attachment factor for feeder-free propagation of iPS cells in microcarrier suspension culture.