组织块法与酶消化法培养人脐带间充质干细胞的比较

Tissue explant method versus enzymatic digestion method for culture of human umbilical cord mesenchymal cells

目的比较组织块法与酶消化法对分离培养人脐带间充质干细胞(hUC-MSCs)的影响。方法选取5根足月妊娠自然分娩的健康婴儿的废弃脐带,采用组织块法和酶消化法进行原代分离培养,比较两种培养方法的细胞增殖情况,并测定其细胞表面免疫标志物。结果hUC-MSCs的融合状况:组织块法培养的第5天,细胞从组织块内爬出,培养2周左右细胞融合率可达80%以上;酶消化法培养5 d后,可见少量形态不规则的细胞贴壁,培养2周后细胞融合可达40%~50%。hUC-MSCs的增殖情况:组织块法原代及传代后的hUC-MSCs的增殖速度远高于酶消化法,差异有统计学意义(P<0.05)。hUC-MSCs的标志物:组织块法分离培养的P3细胞CD34、CD45、HLA-DR表达<1%,CD44、CD73、CD90和CD105表达>95%;酶消化法分离培养的P3细胞表面CD34、CD45、HLA-DR、CD105表达<1%,CD73、CD90表达>95%。结论组织块法与酶消化法相比,能获得较纯的hUC-MSCs,且方法简单、成本低,是获得hUC-MSCs的理想方法。

Objective:To compare the effects of tissue explant method and enzymatic digestion method on the isolated and cultured human umbilical cord mesenchymal stem cells(hUC-MSCs).Methods:The umbilical cords of 5 healthy full-term infants were selected to obtain human umbilical cord mesenchymal stem cells.The primary hUC-MSCs were isolated and cultured by using tissue explant method or enzymatic digestion method.The cell proliferation was compared between the two methods.The cell surface markers were measured by flow cytometry.Results:Fusion status of hUC-MSCs:the spindled-shaped cells began to migrate from the umbilical cord tissue after 5 days culture in tissue explant method.The cell fusion rate was over 80%after 2 weeks of culture.After 5 days of culture with enzyme digestion method,a small number of irregular cells adhered to the wall were observed.After 2 weeks of culture,the fusion rate was 40%-50%.Proliferation status of hUC-MSCs:the proliferation rate of hMSCs by tissue explant method was significantly higher than that by enzyme digestion method(P<0.05).Markers of hUC-MSCs were as follows:the expression of CD34,CD45 and HLA-DR on the third passage(P3)of cells was less than 1%,and the expression of CD44,CD73,CD90 and CD105 was more than 95%by the tissue explant method.The expression of CD34,CD45,HLA-DR and CD105 on the P3 cells was less than 1%,and the expression of CD73 and CD90 was more than 95%by enzyme digestion method.Conclusions:Compared with enzymatic digestion method,the tissue explant method can obtain the purer hUC-MSCs,which is an ideal method for obtaining hUC-MSCs because of its simple and low cost.