Abstract: Objective Human embryonic stem cells(hESC) are typically passaged by two different enzymes,0.05% Trypsin-EDTA and Collagenase,type Ⅳ.The effect of the two enzymes on hESC cell viability,growth and genotype stability is to be analyzed.Methods We cultured hESC on mouse embryonic fibroblasts(MEF),digested and passaged the cells by the two enzymes,and then counted the cell survival rate,colony formation rate and total cell number.Furthermore,we identified the survival rate of human ES cells after passaging and cryopreservation,and the karyotype profile after more than 10 passages.Results We found that,passaged by trypsin,the cell colony was uniform,the colony formation rate was higher and total cell number increased faster than that of collagenase treatment.The two enzyme treatments were yield nearly the same survival rate after digestion or cryopreservation,and are able to maintain normal karyotype after more than 10 passages.Conclusion 0.05% Trypsin-EDTA and Collagenase type Ⅳ are both good choice in hES cells passaging,but suitable for different experiments.

Key words: human embryonic stem cell, cell culture, trypsin, collagenase Ⅳ