title: Highly efficient cryopreservation of human induced pluripotent stem cells using a dimethyl sulfoxide-free solution.
abstract: Human induced pluripotent stem (hiPS) cells have great potential for regenerative medicine and drug discovery. It is essential to establish highly efficient and reliable methods for hiPS cell cryopreservation. We examined cryopreservation of hiPS cells by the vitrification method using a dimethyl sulfoxide Me2SO-free and serum-free medium, VS2E, that uses Euro-Collins solution as a base with 40% (v/v) ethylene glycol and 10% (w/v) polyethylene glycol as cryoprotectants. This combination of vitrification and cryoprotectants resulted in a higher recovery rate of hiPS cells than with a commercially-available vitrification solution, DAP213, which contained Me2SO and serum components. After vitrification and warming, hiPS cells were cultured easily. Even after several subculturing steps, cells expressed undifferentiated cell markers, such as Oct-3/4 and SSEA-4, and also exhibited alkaline phosphatase activity. The pluripotency of hiPS cells was maintained, as demonstrated by teratoma formation upon hiPS cell transplantation into severe combined immunodeficient mice. Thus, we successfully preserved hiPS cells under liquid nitrogen with high efficiency using Me2SO-free vitrification solution and rapid cooling.
authors.affiliation: Department of Reparative Materials, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan.
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Nishigaki
authors.forename: Yuji
authors.initials: Y
authors.lastname: Teramura
authors.forename: Akira
authors.initials: A
authors.lastname: Nasu
authors.forename: Kei
authors.initials: K
authors.lastname: Takada
authors.forename: Junya
authors.initials: J
authors.lastname: Toguchida
authors.forename: Hiroo
authors.initials: H
authors.lastname: Iwata
mesh_terms: D000081:Acetamides; D000469:Alkaline Phosphatase / Q000096:biosynthesis / Q000378:metabolism; D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D019855:Ethylene Glycol / Q000494:pharmacology; D006801:Humans; D006982:Hypertonic Solutions / Q000494:pharmacology; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D016513:Mice, SCID; D050814:Octamer Transcription Factor-3 / Q000096:biosynthesis; D011092:Polyethylene Glycols / Q000494:pharmacology; D011409:Propylene Glycols; D055746:Stage-Specific Embryonic Antigens / Q000096:biosynthesis
chemical_list: D000081:Acetamides; D003451:Cryoprotective Agents; C093932:DAP213 solution; C037096:Euro-Collins' solution; D006982:Hypertonic Solutions; D050814:Octamer Transcription Factor-3; D011409:Propylene Glycols; D055746:Stage-Specific Embryonic Antigens; C066584:stage-specific embryonic antigen-4; D011092:Polyethylene Glycols; D000469:Alkaline Phosphatase; D019855:Ethylene Glycol; D004121:Dimethyl Sulfoxide