Defined Essential 8™ Medium and Vitronectin Efficiently Support Scalable Xeno-Free Expansion of Human Induced Pluripotent Stem Cells in Stirred Microcarrier Culture Systems.
Defined Essential 8™ Medium and Vitronectin Efficiently Support Scalable Xeno-Free Expansion of Human Induced Pluripotent Stem Cells in Stirred Microcarrier Culture Systems.
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DOI：
10.1371/journal.pone.0151264
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Cabral JM
中科院分区：
文献类型：
--
作者：
Badenes SM;Fernandes TG;Cordeiro CS;Boucher S;Kuninger D;Vemuri MC;Diogo MM;Cabral JM
使用 Essential 8™ 无异源培养基和确定的无异源基质玻连蛋白在贴壁条件下成功进行了人诱导多能干 (hiPS) 细胞培养。该基质能够支持 hiPS 细胞在包被的板或聚苯乙烯包被的微载体上扩增，同时保持 hiPS 细胞的功能和多能性。重要的是，基于微载体的系统的放大是使用 50 mL 旋转烧瓶在动态条件下完成的。进行三水平因子设计实验，以确定以下方面的最佳条件：a）初始细胞密度，b）搅拌速度，c）最大化转瓶培养物中的细胞产量。通过以 55,000 个细胞/cm2 的微载体表面积接种并使用 44 rpm，可实现 3.5 的最大细胞产量，培养 10 天后产生的细胞密度为 1.4x106 个细胞/mL。动态培养后，hiPS细胞在重新铺板时保持其典型形态，表现出多能性相关标记物表达以及三谱系分化能力，这通过胚状体形成诱导其自发分化得到验证，随后成功完成向神经和心脏命运等特定谱系的下游分化。总之，成功开发并实施了一种可扩展、稳健且具有成本效益的无异源培养系统，用于 hiPS 细胞的规模化生产。
Human induced pluripotent stem (hiPS) cell culture using Essential 8™ xeno-free medium and the defined xeno-free matrix vitronectin was successfully implemented under adherent conditions. This matrix was able to support hiPS cell expansion either in coated plates or on polystyrene-coated microcarriers, while maintaining hiPS cell functionality and pluripotency. Importantly, scale-up of the microcarrier-based system was accomplished using a 50 mL spinner flask, under dynamic conditions. A three-level factorial design experiment was performed to identify optimal conditions in terms of a) initial cell density b) agitation speed, and c) to maximize cell yield in spinner flask cultures. A maximum cell yield of 3.5 is achieved by inoculating 55,000 cells/cm2 of microcarrier surface area and using 44 rpm, which generates a cell density of 1.4x106 cells/mL after 10 days of culture. After dynamic culture, hiPS cells maintained their typical morphology upon re-plating, exhibited pluripotency-associated marker expression as well as tri-lineage differentiation capability, which was verified by inducing their spontaneous differentiation through embryoid body formation, and subsequent downstream differentiation to specific lineages such as neural and cardiac fates was successfully accomplished. In conclusion, a scalable, robust and cost-effective xeno-free culture system was successfully developed and implemented for the scale-up production of hiPS cells.