title: A Simple and Efficient Method of Slow Freezing for Human Embryonic Stem Cells and Induced Pluripotent Stem Cells.
abstract: Protocols available for the cryopreservation of human embryonic stem (ES) and induced pluripotent stem (iPS) cells are very inefficient and laborious compared to those for the cryopreservation of murine ES/iPS cells or other general cell lines. While the vitrification method may be adequate when working with small numbers of human ES/iPS cells, it requires special skills and is unsuitable when working with large cell numbers. Here, we describe a simple and efficient method for the cryopreservation of hES/hiPS cells that is based on a conventional slow freezing method that uses a combination of Pronase/EDTA for Stem™ and CP-5E™ [final concentrations: 6 % hydroxyethyl starch, 5 % DMSO, and 5 % ethylene glycol in saline]. CP-5E™ is highly effective for the cryopreservation of small cell clumps produced by hES/hiPS colony detachment in the presence of Pronase and EDTA (Pronase/EDTA for Stem™, a formulation containing multiple digestive enzymes from Streptomyces griseus). This novel method would be quite useful for large-scale hES/iPS cell banking for use in clinical applications.
authors.affiliation: Laboratory for Stem Cell Biology, RIKEN Center for Developmental Biology, 2-2-3, Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo, 650-0047, Japan.|Kyokuto Pharmaceutical Industrial Co. Ltd., 7-8, Nihonbashi Kobunecho, Chuo-ku, Tokyo, 103-0024, Japan.
authors.forename: Keitaro
authors.initials: K
authors.lastname: Imaizumi
authors.affiliation: Kyokuto Pharmaceutical Industrial Co. Ltd., 7-8, Nihonbashi Kobunecho, Chuo-ku, Tokyo, 103-0024, Japan.
authors.forename: Momoe
authors.initials: M
authors.lastname: Iha
authors.affiliation: Division of Cell Therapy, Foundation for Biomedical Research and Innovation, TRI#308, 1-5-4 Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo, 650-0043, Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Nishishita
authors.affiliation: Division of Cell Therapy, Foundation for Biomedical Research and Innovation, TRI#308, 1-5-4 Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo, 650-0043, Japan. kawamata@fbri.org.
authors.forename: Shin
authors.initials: S
authors.lastname: Kawamata
authors.affiliation: Laboratory for Stem Cell Biology, RIKEN Center for Developmental Biology, 2-2-3, Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo, 650-0047, Japan.
authors.forename: Shinichi
authors.initials: S
authors.lastname: Nishikawa
authors.affiliation: Laboratory for Stem Cell Biology, RIKEN Center for Developmental Biology, 2-2-3, Minatojima-Minamimachi, Chuo-ku, Kobe, Hyogo, 650-0047, Japan. t.akuta@kyokutoseiyaku.co.jp.|Kyokuto Pharmaceutical Industrial Co. Ltd., 7-8, Nihonbashi Kobunecho, Chuo-ku, Tokyo, 103-0024, Japan. t.akuta@kyokutoseiyaku.co.jp.
authors.forename: Teruo
authors.initials: T
authors.lastname: Akuta
mesh_terms: D018929:Cell Culture Techniques / Q000379:methods; D049109:Cell Proliferation; D015925:Cryopreservation / Q000191:economics / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D019855:Ethylene Glycol / Q000494:pharmacology; D005615:Freezing; D000066449:Human Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects; D006801:Humans; D006895:Hydroxyethyl Starch Derivatives / Q000494:pharmacology; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000187:drug effects; D011402:Pronase / Q000494:pharmacology; D013194:Staining and Labeling / Q000379:methods; D013305:Streptomyces griseus / Q000201:enzymology
chemical_list: D003451:Cryoprotective Agents; D006895:Hydroxyethyl Starch Derivatives; D011402:Pronase; D019855:Ethylene Glycol; D004121:Dimethyl Sulfoxide
keywords: Cryopreservation; Dimethyl sulfoxide; Ethylene glycol; Human embryonic stem cells; Human induced pluripotent stem cells; Hydroxyethyl starch; Pronase; Slow freezing