胰酶和Ⅳ型胶原酶在人胚胎干细胞传代中的作用特点

目的对比人胚胎干细胞(hESC)培养过程中不同的酶消化细胞的特点,探讨适合hESC长期体外培养的消化传代方法。方法将正常生长在小鼠胚胎成纤维细胞(MEF)滋养层上的hESC分别用0.05%胰酶-EDTA和Ⅳ型胶原酶消化,重新接种,了解两种酶消化后的细胞存活率、克隆形成数量、细胞产量、冻存-复苏后细胞的存活率以及长期传代后细胞核型情况。结果用0.05%胰酶-EDTA消化传代后形成的克隆大小均一,每代第3d可获得碱性磷酸酶(AP)阳性克隆数(21.2±3.5)个/10倍视野,每次传代时hES细胞产量可达(4.56±1.29)×105/cm2,传代时细胞存活率高达95.60%,经冻存-复苏后也有81.20%细胞存活率。用Ⅳ型胶原酶消化组每代仅可获得低浓度胰酶消化组AP阳性克隆数的一半,克隆大小不均,细胞产量也比低浓度胰酶消化组少,传代时细胞存活率和复苏后细胞存活率和低浓度胰酶组相似。分别用两种酶处理传代十余代,核型均无异常。结论两种酶消化均适用于hESC培养,二者作用特点不完全相同,分别适用于不同的实验需要。

Comparison between Trypsinization and Collagenase Treatment in Passaging of Human Embryonic Stem Cells

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.