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背景:脐血中存在间充质干细胞,目前国内外尚未见到对脐血间充质干细胞体外分离、培养及扩增较统一且有效的方法。目的:探讨影响人脐血间充质干细胞成功分离培养的相关因素。方法:分别从不同胎龄(≥40 周,37 周和≤32 周),脐血中单个核细胞数量(≥2.5×109L-1,<2.5×109 L-1), 不同细胞接种浓度(1×107,1×109,1×1011 L-1),不同体积分数胎牛血清(5%,10%,15%,20%)以及培养瓶是否被胎牛血清包被等方面对人脐血间充质干细胞分离培养成功率进行比较。结果与结论:脐血间充质干细胞的分离培养成功率为 58.3%,且随胎龄的增高而培养成功率降低(P<0.01);脐血中单个核细胞浓度≥2.5×109 L-1组培养成功率高于<2.5×109 L-1组(P<0.01);相同容量脐血中单个核细胞数量与胎龄呈负相关(r=-0.95,P<0.01);1×1011 L-1 组原代及传代培养的间充质干细胞生长及扩增情况高于 1×107,1×109 L-1;体积分数 5%FBS 组间充质干细胞贴壁速度较其他 3 组略慢,但细胞纯度较高,且细胞传代速度与其他 3 组无明显差别;胎牛血清包被组脐血间充质干细胞原代和传代后的纯度及扩增能力均高于未包被组。提示脐血间充质干细胞分离培养成功率受多种因素的影响:通过选择较低胎龄的胎儿,采集足够量的脐血,以较高的细胞密度接种,培养基中添加较低浓度的胎牛血清,并将培养瓶预先用胎牛血清进行包被,能在体外建立稳定的脐血间充质干细胞培养体系。
BACKGROUND: Mesenchymal stem cells are present in umbilical cord blood. At present, no uniform and effective method of isolation, culture and expansion of umbilical cord blood mesenchymal stem cells has been found at home and abroad. Objective: To explore the factors affecting the successful isolation and culture of human umbilical cord blood mesenchymal stem cells. Methods: The number of mononuclear cells (≥2.5 × 109L-1, <2.5 × 109 L-1) and the concentration of mononuclear cells in different cell seeding concentrations (≥40 weeks, 37 weeks and ≤32 weeks) × 107,1 × 109, 1 × 1011 L-1), different volume fraction of fetal bovine serum (5%, 10%, 15%, 20%) and whether the culture flask was coated with fetal bovine serum, Mesenchymal stem cell isolation and culture success rates were compared. RESULTS AND CONCLUSION: The successful rate of isolation and culture of umbilical cord blood mesenchymal stem cells was 58.3%, and the success rate of culture was decreased with the increase of gestational age (P <0.01). The concentration of mononuclear cells in cord blood was ≥2.5 × 109 L-1 The success rate of group culture was higher than that of <2.5 × 109 L-1 group (P <0.01). The number of mononuclear cells in cord blood of the same volume was negatively correlated with gestational age (r = -0.95, P <0.01) The growth and proliferation of mesenchymal stem cells in primary and subcultured -1 group were higher than that of 1 × 107 and 1 × 109 L-1 groups, and the adherent rate of mesenchymal stem cells in 5% FBS group was slightly slower than that of the other three groups , But the cell purity was higher, and the cell passage speed was not significantly different from the other three groups. The purity and the ability of expansion of umbilical cord blood mesenchymal stem cells of fetal bovine serum-coated group were higher than those of the non-coated group. Tip The success rate of cord blood MSCs isolation and culture is influenced by many factors: by selecting fetuses of lower gestational age, collecting a sufficient amount of cord blood and inoculating at a higher cell density with a lower concentration of Fetal bovine serum, and the culture flask pre-coated with fetal bovine serum, in vitro to establish a stable culture system of umbilical cord blood mesenchymal stem cells.