RRC ID |
44916
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著者 |
Imaizumi K, Nishishita N, Muramatsu M, Yamamoto T, Takenaka C, Kawamata S, Kobayashi K, Nishikawa S, Akuta T.
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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.
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ジャーナル |
PLoS One
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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.
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巻・号 |
9(2)
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ページ |
e88696
|
公開日 |
2014-1-1
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DOI |
10.1371/journal.pone.0088696
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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
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WOS 分野
|
HEMATOLOGY
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リソース情報 |
ヒト・動物細胞 |
|