title: Passaging and colony expansion of human pluripotent stem cells by enzyme-free dissociation in chemically defined culture conditions.
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.
authors.affiliation: Center for Molecular Medicine, National Heart, Lung, and Blood Institute (NHLBI), Bethesda, Maryland, USA.
authors.forename: Jeanette
authors.initials: J
authors.lastname: Beers
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Gulbranson
authors.forename: Nicole
authors.initials: N
authors.lastname: George
authors.forename: Lauren I
authors.initials: LI
authors.lastname: Siniscalchi
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Jones
authors.forename: James A
authors.initials: JA
authors.lastname: Thomson
authors.forename: Guokai
authors.initials: G
authors.lastname: Chen
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D003094:Collagen; D003470:Culture Media / Q000737:chemistry; D004338:Drug Combinations; D004492:Edetic Acid / Q000737:chemistry; D005260:Female; D006801:Humans; D007797:Laminin; D051379:Mice; D016513:Mice, SCID; D039904:Pluripotent Stem Cells / Q000166:cytology*; D011509:Proteoglycans
chemical_list: D003470:Culture Media; D004338:Drug Combinations; D007797:Laminin; D011509:Proteoglycans; C059485:matrigel; D003094:Collagen; D004492:Edetic Acid