Document Detail


Mechanical dissociation of human embryonic stem cell colonies by manual scraping after collagenase treatment is much more detrimental to cellular viability than is trypsinization with gentle pipetting.
MedLine Citation:
PMID:  17115976     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Because hESC (human embryonic stem cells) are 'social cells' that require co-operative interactions and intimate physical contact with each other, it is absolutely essential to dissociate hESC colonies into cellular clumps rather than into a single-cell suspension during serial passage. The present study compared two commonly used protocols for dissociating hESC colonies. The first protocol involved mild enzymatic treatment with collagenase type IV (1 mg/ml) for approx. 5-10 min, prior to mechanical dissociation into cellular clumps through manual scraping with a plastic pipette tip. The second protocol involved a short duration of exposure (2-3 min) to low concentrations of trypsin (0.05%), followed by gentle pipetting. The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay was used to compare the recovery of viable cells after dissociating hESC colonies with these two protocols, before and after conventional freeze-thawing with 10% (v/v) DMSO. Besides undifferentiated hESC, the randomly differentiated fibroblastic progenies of hESC at various passages (P0-P4), together with an immortalized cell line (CRL-1486), were also utilized to compare the two protocols. The results demonstrated that the second protocol (trypsinization with gentle pipetting) is much less detrimental to cellular viability than is the first protocol (collagenase treatment with scratching). This in turn translated to higher freeze-thaw survival rates. It is hypothesized that scratching after collagenase treatment (first protocol) somehow induces physical damage to the cells, thereby leading to a lower recovery of viable cells, both before and after freeze-thawing.
Authors:
Boon Chin Heng; Hua Liu; Zigang Ge; Tong Cao
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Publication Detail:
Type:  Comparative Study; Journal Article    
Journal Detail:
Title:  Biotechnology and applied biochemistry     Volume:  47     ISSN:  1470-8744     ISO Abbreviation:  Biotechnol. Appl. Biochem.     Publication Date:  2007 May 
Date Detail:
Created Date:  2007-04-16     Completed Date:  2007-05-01     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  8609465     Medline TA:  Biotechnol Appl Biochem     Country:  England    
Other Details:
Languages:  eng     Pagination:  33-7     Citation Subset:  IM    
Affiliation:
Stem Cell Laboratory, Faculty of Dentistry, National University of Singapore, 5 Lower Kent Ridge Road, 119074 Singapore.
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MeSH Terms
Descriptor/Qualifier:
Cell Culture Techniques
Cell Line
Cell Survival / drug effects
Collagenases / pharmacology*
Cryopreservation
Embryonic Stem Cells / cytology,  drug effects*
Humans
Trypsin / pharmacology*
Chemical
Reg. No./Substance:
EC 3.4.21.4/Trypsin; EC 3.4.24.-/Collagenases

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


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