Document Detail

A controlled-cooling protocol for cryopreservation of human and non-human primate embryonic stem cells.
MedLine Citation:
PMID:  18453247     Owner:  NLM     Status:  MEDLINE    
Freeze storage of human embryonic stem (hES) cells has not proven effective using the methods employed for mouse ES (mES) cells, while rhesus ES (rhES) cells are only modestly effectively frozen using common mES cell methods. Because human and rhES cells are passaged and frozen in clusters that approximate the size of embryos, we employed a mammalian embryo freezing method to cryopreserve primate ES cells. This protocol involves freezing in a dimethylsulfoxide cryoprotectant using straws. An ice crystal seed is induced at -10 degrees C followed by controlled cooling at -1 degrees C per minute down to -33 degrees C with a plunge from there directly into liquid nitrogen (LN2) at -196 degrees C. Thaw is effected rapidly by moving the frozen cells directly from LN2 into a water bath and placing directly into culture medium without step-wise cryoprotectant removal. Using this protocol, we have increased the survival of human ES cells from < or = 1 to approximately 80% and rhES cells from approximately 30 to > or = 90%. Thus, this protocol describes a technically simple but effective means of long-term storage of primate ES cells.
Carol B Ware; Szczepan W Baran
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Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Methods in molecular biology (Clifton, N.J.)     Volume:  407     ISSN:  1064-3745     ISO Abbreviation:  Methods Mol. Biol.     Publication Date:  2007  
Date Detail:
Created Date:  2008-05-05     Completed Date:  2008-06-05     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  9214969     Medline TA:  Methods Mol Biol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  43-9     Citation Subset:  IM    
Department of Comparative Medicine, University of Washington, Seattle, USA.
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MeSH Terms
Cell Culture Techniques / methods*
Cells, Cultured
Cryopreservation / methods*
Dimethyl Sulfoxide
Embryonic Stem Cells / cytology*
Reg. No./Substance:
67-68-5/Dimethyl Sulfoxide

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