title: Adaptation of human pluripotent stem cells to feeder-free conditions in chemically defined medium with enzymatic single-cell passaging.
abstract: This protocol describes the culture of human pluripotent stem cells (PSCs) under feeder-free conditions in a commercially available, chemically defined, growth medium, using Matrigel as a substrate and the enzyme solution Accutase for single-cell passaging. This system is strikingly different from traditional PSC culture, where the cells are co-cultured with feeder cells and in medium containing serum replacement. PSCs cultured in this new system have a different morphology than those cultured on feeder cells but retain their characteristic pluripotency. This feeder-free PSC culture system is conceptually similar to feeder-free systems that use mouse embryonic fibroblast (MEF)-conditioned medium (MEF-CM) and Matrigel substratum. Instead of MEF-CM, a very complex and undefined medium, this new system uses StemPro SFM, a chemically defined medium that permits enzymatic passaging with Accutase to disaggregate the colonies into single cells. Accutase passaging has been used in conjunction with Stempro in our hands for 20+ passages without detectable karyotypic abnormalities. We will also review techniques for adapting cultures previously grown on MEFs, routine passaging of the cells, and cryopreservation.
authors.affiliation: Neuroscience Laboratories, Centers for Neuroscience and Translational Research, CHOC Research Institute, Children's Hospital of Orange County Research Institute, Orange, CA, USA.
authors.forename: Alexander E
authors.initials: AE
authors.lastname: Stover
authors.forename: Philip H
authors.initials: PH
authors.lastname: Schwartz
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D049109:Cell Proliferation / Q000187:drug effects; D048430:Cell Shape / Q000187:drug effects; D018920:Coculture Techniques; D003094:Collagen / Q000494:pharmacology; D015925:Cryopreservation; D003470:Culture Media / Q000494:pharmacology*; D004338:Drug Combinations; D053595:Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects; D004798:Enzymes / Q000378:metabolism*; D006801:Humans; D007797:Laminin / Q000494:pharmacology; D051379:Mice; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000187:drug effects; D011509:Proteoglycans / Q000494:pharmacology
chemical_list: D003470:Culture Media; D004338:Drug Combinations; D004798:Enzymes; D007797:Laminin; D011509:Proteoglycans; C059485:matrigel; D003094:Collagen