Efficient and scalable culture of single dissociated human pluripotent stem cells using recombinant E8 fragments of human laminin isoforms.
2015
This unit describes a protocol for efficient expansion of human pluripotent stem cells (hPSCs). A key feature of this method is subculture of hPSCs by single-cell dissociation passaging on substrates coated with recombinant E8 fragments of human laminin isoforms (LM-E8s). LM-E8s, provide superior adhesion over intact laminin isoforms and Matrigel. Single hPSCs seeded on LM-E8s show accelerated migration and rapid reconstruction of clusters, resulting in robust survival and proliferation. This protocol yields 200-fold more hPSCs than conventional subculture methods in 1 month of culture. Furthermore, this protocol can be easily adapted to most hPSC lines in combination with the use of various xeno-free, defined culture media, and large-scale expansion of hPSCs is easily achievable to facilitate the practical applications of hPSCs. © 2015 by John Wiley & Sons, Inc.
Keywords:
human pluripotent stem cells (hPSCs);
single-cell dissociation culture;
laminin isoform;
laminin fragment;
culture substrate
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