title: An Effective and Reliable Xeno-free Cryopreservation Protocol for Single Human Pluripotent Stem Cells.
abstract: Efficient cryopreservation of human pluripotent stem cells (hPSCs) in chemically defined, xeno-free conditions is highly desirable for medical research and clinical applications such as cell-based therapies. Here we present a simple and effective slow freezing-rapid thawing protocol for the cryopreservation of feeder-free, single hPSCs. This cryopreservation protocol involves the supplementation of 10 % dimethyl sulfoxide (DMSO) and 10 μM Rho-associated kinase inhibitor Y-27632 into two types of xeno-free, defined media supplements (Knockout Serum Replacement and TeSR2). High post-thaw cell recovery (~90 %) and cell expansion (~70 %) can be achieved using this protocol. The cryopreserved single cells retain the morphological characteristics of hPSCs and differentiation capabilities of pluripotent stem cells.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB, Canada.
authors.forename: Guoliang
authors.initials: G
authors.lastname: Meng
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB, Canada.
authors.forename: Anna
authors.initials: A
authors.lastname: Poon
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB, Canada.
authors.forename: Shiying
authors.initials: S
authors.lastname: Liu
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB, Canada. rancourt@ucalgary.ca.
authors.forename: Derrick E
authors.initials: DE
authors.lastname: Rancourt
mesh_terms: D000577:Amides / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects*; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology; D016895:Culture Media, Serum-Free / Q000737:chemistry; D005615:Freezing; D006801:Humans; D039904:Pluripotent Stem Cells / Q000187:drug effects*; D011725:Pyridines / Q000494:pharmacology; D059010:Single-Cell Analysis / Q000379:methods*
chemical_list: D000577:Amides; D003451:Cryoprotective Agents; D016895:Culture Media, Serum-Free; D011725:Pyridines; C108830:Y 27632
keywords: Chemically defined; Cryopreservation; Dimethyl sulfoxide; Human pluripotent stem cells; Rapid thawing; Rho-associated kinase inhibitor; Slow freezing; Xeno-free