Int. J. Dev. Biol. 55: 305 - 311 (2011)
doi: 10.1387/ijdb.103145tn
© UPV/EHU Press

Highly efficient cryopreservation of human induced pluripotent stem cells using a dimethyl sulfoxide-free solution

Tatsuya Nishigaki1, Yuji Teramura2, Akira Nasu3, Kei Takada4, Junya Toguchida3 and Hiroo Iwata*,1

1Department of Reparative Materials, Institute for Frontier Medical Sciences, 2Radioisotope Research Center, 3Department of Tissue Regeneration, Institute for Frontier Medical Sciences, 4Laboratory of Cell Processing, Stem Cell Research Center, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan

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.

Keywords:

cryopreservation, human induced pluripotent stem (iPS) cell, vitrification, Me2SO-free, ethylene glycol

*Corresponding author e-mail: iwata@frontier.kyoto-u.ac.jp