title: A simple and highly effective method for slow-freezing human pluripotent stem cells using dimethyl sulfoxide, hydroxyethyl starch and ethylene glycol.
abstract: Vitrification and slow-freezing methods have been used for the cryopreservation of human pluripotent stem cells (hPSCs). Vitrification requires considerable skill and post-thaw recovery is low. Furthermore, it is not suitable for cryopreservation of large numbers of hPSCs. While slow-freezing methods for hPSCs are easy to perform, they are usually preceded by a complicated cell dissociation process that yields poor post-thaw survival. To develop a robust and easy slow-freezing method for hPSCs, several different cryopreservation cocktails were prepared by modifying a commercially available freezing medium (CP-1™) containing hydroxyethyl starch (HES), and dimethyl sulfoxide (DMSO) in saline. The new freezing media were examined for their cryopreservation efficacy in combination with several different cell detachment methods. hPSCs in cryopreservation medium were slowly cooled in a conventional -80°C freezer and thawed rapidly. hPSC colonies were dissociated with several proteases. Ten percent of the colonies were passaged without cryopreservation and another 10% were cryopreserved, and then the recovery ratio was determined by comparing the number of Alkaline Phosphatase-positive colonies after thawing at day 5 with those passaged without cryopreservation at day 5. We found that cell detachment with Pronase/EDTA followed by cryopreservation using 6% HES, 5% DMSO, and 5% ethylene glycol (EG) in saline (termed CP-5E) achieved post-thaw recoveries over 80%. In summary, we have developed a new cryopreservation medium free of animal products for slow-freezing. This easy and robust cryopreservation method could be used widely for basic research and for clinical application.
authors.affiliation: Laboratory for Stem Cell Biology, RIKEN Center for Developmental Biology, Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo, Japan ; Kobe office, RIKEN Cell Tech Co. Ltd., Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo, Japan.
authors.forename: Keitaro
authors.initials: K
authors.lastname: Imaizumi
authors.affiliation: Laboratory for Stem Cell Biology, RIKEN Center for Developmental Biology, Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo, Japan ; Division of Cell Therapy, Foundation for Biomedical Research and Innovation, Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo, Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Nishishita
authors.affiliation: Division of Cell Therapy, Foundation for Biomedical Research and Innovation, Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo, Japan.
authors.forename: Marie
authors.initials: M
authors.lastname: Muramatsu
authors.affiliation: Division of Cell Therapy, Foundation for Biomedical Research and Innovation, Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo, Japan.
authors.forename: Takako
authors.initials: T
authors.lastname: Yamamoto
authors.affiliation: Laboratory for Stem Cell Biology, RIKEN Center for Developmental Biology, Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo, Japan ; Division of Cell Therapy, Foundation for Biomedical Research and Innovation, Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo, Japan.
authors.forename: Chiemi
authors.initials: C
authors.lastname: Takenaka
authors.affiliation: Laboratory for Stem Cell Biology, RIKEN Center for Developmental Biology, Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo, Japan ; Division of Cell Therapy, Foundation for Biomedical Research and Innovation, Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo, Japan.
authors.forename: Shin
authors.initials: S
authors.lastname: Kawamata
authors.affiliation: Laboratory for Stem Cell Biology, RIKEN Center for Developmental Biology, Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo, Japan ; Department of Pediatric Hematology and Oncology Research, National Research Institute for Child Health and Development, Okura, Setagaya-ku, Tokyo, Japan.
authors.forename: Kenichiro
authors.initials: K
authors.lastname: Kobayashi
authors.affiliation: Laboratory for Stem Cell Biology, RIKEN Center for Developmental Biology, Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo, Japan.
authors.forename: Shin-Ichi
authors.initials: S
authors.lastname: Nishikawa
authors.affiliation: Laboratory for Stem Cell Biology, RIKEN Center for Developmental Biology, Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo, Japan ; Kobe office, RIKEN Cell Tech Co. Ltd., Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo, Japan.
authors.forename: Teruo
authors.initials: T
authors.lastname: Akuta
mesh_terms: D000469:Alkaline Phosphatase / Q000737:chemistry; D002454:Cell Differentiation; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000737:chemistry*; D004121:Dimethyl Sulfoxide / Q000737:chemistry*; D004492:Edetic Acid / Q000737:chemistry; D019855:Ethylene Glycol / Q000737:chemistry*; D005434:Flow Cytometry; D005615:Freezing; D006801:Humans; D006895:Hydroxyethyl Starch Derivatives / Q000737:chemistry*; D007621:Karyotyping; D039904:Pluripotent Stem Cells / Q000166:cytology*; D013696:Temperature; D058989:Vitrification
chemical_list: D003451:Cryoprotective Agents; D006895:Hydroxyethyl Starch Derivatives; D004492:Edetic Acid; D000469:Alkaline Phosphatase; D019855:Ethylene Glycol; D004121:Dimethyl Sulfoxide