Please use this identifier to cite or link to this item: https://doi.org/10.1042/BA20060151
Title: 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
Authors: Boon, C.H. 
Liu, H. 
Ge, Z. 
Cao, T. 
Keywords: Cellular viability
Collagenase
Freeze-thawing
Human embryonic stem cell
Scraping
Trypsin
Issue Date: 2007
Source: Boon, C.H., Liu, H., Ge, Z., Cao, T. (2007). 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. Biotechnology and Applied Biochemistry 47 (1) : 33-37. ScholarBank@NUS Repository. https://doi.org/10.1042/BA20060151
Abstract: 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. © 2007 Portland Press Ltd.
Source Title: Biotechnology and Applied Biochemistry
URI: http://scholarbank.nus.edu.sg/handle/10635/46605
ISSN: 08854513
DOI: 10.1042/BA20060151
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