Document Detail


Cryopreservation by slow cooling with DMSO diminished production of Oct-4 pluripotency marker in human embryonic stem cells.
MedLine Citation:
PMID:  16839540     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
We tested a "standard" cryopreservation protocol (slow cooling with 10% DMSO) on the human embryonic stem cell (hESC) line H9 containing an Oct-4 (POU5F1) promoter-driven, enhanced green fluorescent protein (EGFP) reporter to monitor maintenance of pluripotency. Cells were cooled to -80 degrees C in cryovials and then transferred to a -80 degrees C freezer. Cells were held at -80 degrees C for 3 days ("short-term storage") or 3 months ("long-term storage"). Vials were thawed in a +36 degrees C water bath and cells were cultured for 3, 7, or 14 days. Propidium iodide (PI) was used to assess cell viability by flow cytometry. Control cells were passaged on the same day that the frozen cells were thawed. The majority of cells in control hESC cultures were Oct-4 positive and almost 99% of EGFP+ cells were alive as determined by exclusion of PI. In contrast, the frozen cells, even after 3 days of culture, contained only 50% live cells, and only 10% were EGFP-positive. After 7 days in culture, the proportion of dead cells decreased and there was an increase in the Oct-4-positive population but microscopic examination revealed large patches of EGFP-negative cells within clusters of colonies even after 14 days of culturing. After 3 months of storage at -80 degrees C the deleterious effect of freezing was even more pronounced: the samples regained a quantifiable number of EGFP-positive cells only after 7 days of culturing following thawing. It is concluded that new protocols and media are required for freezing hESC and safe storage at -80 degrees C as well as studies of the mechanisms of stress-related events associated with cell cryopreservation.
Authors:
Igor I Katkov; Min S Kim; Ruchi Bajpai; Yoav S Altman; Marc Mercola; Jeanne F Loring; Alexey V Terskikh; Evan Y Snyder; Fred Levine
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2006-07-12
Journal Detail:
Title:  Cryobiology     Volume:  53     ISSN:  0011-2240     ISO Abbreviation:  Cryobiology     Publication Date:  2006 Oct 
Date Detail:
Created Date:  2006-09-18     Completed Date:  2007-01-16     Revised Date:  2014-09-22    
Medline Journal Info:
Nlm Unique ID:  0006252     Medline TA:  Cryobiology     Country:  United States    
Other Details:
Languages:  eng     Pagination:  194-205     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Animals
Cell Differentiation
Cell Survival
Cryopreservation / instrumentation,  methods*
Cryoprotective Agents / pharmacology*
Dimethyl Sulfoxide / pharmacology*
Embryo, Mammalian / cytology*
Free Radical Scavengers / pharmacology
Green Fluorescent Proteins / metabolism
Humans
Kinetics
Mice
Microscopy, Fluorescence
Octamer Transcription Factor-3 / metabolism*
Stem Cells / cytology*
Grant Support
ID/Acronym/Agency:
R33 HL088266/HL/NHLBI NIH HHS; R33 HL088266-03/HL/NHLBI NIH HHS; R37 HL059502/HL/NHLBI NIH HHS; R37 HL059502-11S1/HL/NHLBI NIH HHS
Chemical
Reg. No./Substance:
0/Cryoprotective Agents; 0/Free Radical Scavengers; 0/Octamer Transcription Factor-3; 147336-22-9/Green Fluorescent Proteins; YOW8V9698H/Dimethyl Sulfoxide

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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