title: Optimization of slow cooling cryopreservation for human pluripotent stem cells.
abstract: Human pluripotent stem cells (hPSCs) have the potential for unlimited expansion and differentiation into cell types of all three germ layers. Cryopreservation is a key process for successful application of hPSCs. However, the current conventional method leads to poor recovery of hPSCs after thawing. Here, we demonstrate a highly efficient recovery method for hPSC cryopreservation by slow freezing and single-cell dissociation. After confirming hPSC survivability after freeze-thawing, we found that hPSCs that were freeze-thawed as colonies showed markedly decreased survival, whereas freeze-thawed single hPSCs retained the majority of their viability. These observations indicated that hPSCs should be cryopreserved as single cells. Freeze-thawed single hPSCs efficiently adhered and survived in the absence of a ROCK inhibitor by optimization of the seeding density. The high recovery rate enabled conventional colony passaging for subculture within 3 days post-thawing. The improved method was also adapted to a xeno-free culture system. Moreover, the cell recovery postcryopreservation was highly supported by coating culture surfaces with human laminin-521 that promotes adhesion of dissociated single hPSCs. This simplified but highly efficient cryopreservation method allows easy handling of cells and bulk storage of high-quality hPSCs.
authors.affiliation: Department of Embryonic Stem Cell Research, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan.
authors.forename: Takamichi
authors.initials: T
authors.lastname: Miyazaki
authors.forename: Norio
authors.initials: N
authors.lastname: Nakatsuji
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Suemori
mesh_terms: D002448:Cell Adhesion; D002470:Cell Survival; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents; D003470:Culture Media; D005615:Freezing; D006801:Humans; D059785:Karyotype; D007797:Laminin / Q000502:physiology*; D039904:Pluripotent Stem Cells / Q000166:cytology*; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors*
chemical_list: D003451:Cryoprotective Agents; D003470:Culture Media; D007797:Laminin; D054460:rho-Associated Kinases
keywords: early development; ectoderm; process; single cell dissociation; slow freezing; tissue endoderm; tissue germ layer; tissue mesoderm; tissue other