Passaging and colony expansion of human pluripotent stem cells by enzyme-free dissociation in chemically defined culture conditions View Full Text


Ontology type: schema:ScholarlyArticle      Open Access: True


Article Info

DATE

2012-11

AUTHORS

Jeanette Beers, Daniel R Gulbranson, Nicole George, Lauren I Siniscalchi, Jeffrey Jones, James A Thomson, Guokai Chen

ABSTRACT

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 (PSCs). 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 PSCs in E8 medium. More... »

PAGES

2029

Identifiers

URI

http://scigraph.springernature.com/pub.10.1038/nprot.2012.130

DOI

http://dx.doi.org/10.1038/nprot.2012.130

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1021200313

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/23099485


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