Passaging and colony expansion of human pluripotent stem cells by enzyme-free dissociation in chemically defined culture conditions.
Passaging and colony expansion of human pluripotent stem cells by enzyme-free dissociation in chemically defined culture conditions.
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DOI：
10.1038/nprot.2012.130
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发表时间：
2012-11
期刊：
影响因子：
14.8
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中科院分区：
文献类型：
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本协议描述了一种基于edta的传代程序，用于化学定义的E8培养基，作为人类多能干细胞（iPSCs）基础和转化研究的工具。在这个方案中，传代一个6孔或10厘米的细胞板大约需要6-7分钟。这种无酶方案实现最大的细胞存活，无需酶中和，离心，或药物治疗。它还允许更高的吞吐量，需要最少的材料和限制污染。在这里，我们描述了如何生产一致的E8培养基用于日常维护和重编程，以及如何将基于edta的传代程序纳入人诱导的PSC （iPSC）衍生，菌落扩增，冷冻保存和畸胎瘤形成。该方法在常规细胞扩增中是成功的，对于大容量培养或具有psc优先解离的大量细胞是有效的。该方法对所有培养阶段都有效，为在E8培养基中传代人类多能干细胞提供了一致和通用的方法。
This protocol describes an EDTA-based passaging procedure to be used with chemically defined E8 medium that serves as a tool for basic and translational research into human pluripotent stem cells (iPSCs). In this protocol, passaging one six-well or 10 cm plate of cells takes about 6–7 min. This enzyme-free protocol achieves maximum cell survival without enzyme neutralization, centrifugation, or drug treatment. It also allows for higher throughput, requires minimal material and limits contamination. Here we describe how to produce a consistent E8 medium for routine maintenance and reprogramming and how to incorporate the EDTA-based passaging procedure into human induced PSC (iPSC) derivation, colony expansion, cryopreservation and teratoma formation. This protocol has been successful in routine cell expansion, and efficient for expanding large-volume cultures or a large number of cells with preferential dissociation of PSCs. Effective for all culture stages, this procedure provides a consistent and universal approach to passaging human pluripotent stem cells in E8 medium.