RRC ID 44916
著者 Imaizumi K, Nishishita N, Muramatsu M, Yamamoto T, Takenaka C, Kawamata S, Kobayashi K, Nishikawa S, Akuta T.
タイトル A simple and highly effective method for slow-freezing human pluripotent stem cells using dimethyl sulfoxide, hydroxyethyl starch and ethylene glycol.
ジャーナル PLoS One
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.
巻・号 9(2)
ページ e88696
公開日 2014-1-1
DOI 10.1371/journal.pone.0088696
PII PONE-D-13-36920
PMID 24533137
PMC PMC3922972
MeSH Alkaline Phosphatase / chemistry Cell Differentiation Cryopreservation / methods* Cryoprotective Agents / chemistry* Dimethyl Sulfoxide / chemistry* Edetic Acid / chemistry Ethylene Glycol / chemistry* Flow Cytometry Freezing Humans Hydroxyethyl Starch Derivatives / chemistry* Karyotyping Pluripotent Stem Cells / cytology* Temperature Vitrification
IF 2.74
引用数 17
WOS 分野 HEMATOLOGY
リソース情報
ヒト・動物細胞