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基金开发和验证基于微载体的细胞培养系统，用于选择、扩大规模和无饲养层批量生产iPS细胞，该系统基于Primorigen专有的多能干细胞附着因子（StemCadhere”），用于直接物理选择和繁殖iPSC。I期研究将定义优化E-钙粘蛋白介导的细胞粘附至StemCadhere”-包被的微载体的偶联化学，分析iPSC在StemCadhere”-包被的微载体上的粘附效率和增殖，分析StemCadhere-包被的微载体用于直接物理选择新诱导的iPS细胞的效用，并比较冷冻保存的iPSC在StemCadhere”-包被的微载体与Matrigel-包被的微载体上的性能。这项工作将为II期研究提供基础，该研究将研究直接在微载体上生产分化细胞。将使用类似的方法来测试和选择对每个胚层（外胚层、中胚层和内胚层）和有限数量的终末分化谱系（如肝细胞、心肌细胞和造血细胞）特异性的细胞粘附分子。最初的商业化产品将是a）附着在微载体上的即用型（冷冻保存的）iPS细胞，准备加入分化因子和下游方案;和B）与微载体偶联的附着因子的新应用，Primorigen可以向从事iPS分化细胞生产的制药和生物技术公司广泛销售。 公共卫生相关性：Primorigen Biosciences LLC SBIR项目叙述目前用于生产iPS细胞的方法需要劳动密集型人工培养步骤、培养物的频繁补料以及包含药物选择性抗性标记以实现从非诱导的亲本细胞（例如包皮成纤维细胞）纯化iPS。为了实现iPS细胞作为细胞疗法来源和用于药物开发的分化细胞的承诺，必须避免使用慢病毒载体和其他能够基因组整合的试剂，这排除了使用整合的耐药标记物进行iPS选择。此外，为了降低生产批次的可变性，期望使尽可能多的培养自动化，同时保持iPS细胞的增殖和多能性。Primorigen的SBIR提案将通过利用Primorigen专有的干细胞附着因子在微载体悬浮培养中进行iPS细胞的无饲养层繁殖，开发一种用于选择和大规模生产iPS细胞的全面综合解决方案来满足这一需求。

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.