影响脐血间充质干细胞分离培养的关键环节

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× 10⁹ L^-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。

Factors influencing yields of mesenchymal stem cells derived from human umbilical cord blood

BACKGROUND: Mesenchymal stem cells (MSCs) exist in umbilical cord blood (UCB), currently, there is not a method to in vitro separate, culture and amplificate human UCB-MSCs effectively. OBJECTIVE:To explore factors that influence yields of UCB-MSCs. METHODS:The relationship between the success rate of yielding UCB-MSCs and several factors, such as gestational ages (≥40 weeks, 37 weeks and ≤32 weeks), the number of mononuclear cells (MNCs) in UCB (≥2.5×10⁹/L, <2.5×10⁹/L), the inoculum density of MNCs (1×10⁷, 1×10⁹, 1×10¹¹/L), the concentration of fetal bovine serum (FBS, 5%, 10%, 15%, 20%) in culture medium, and whether the culture flask being coated with FBS or not beforehand, as well as relationships among these factors were investigated. RESULTS AND CONCLUSION:The success rate of yielding UCB-MSCs was up to 58.3%. The success rate decreased as the gestational ages increasing (P < 0.01). The success rate could be enhanced to 76.9% when the MNCs count was more than 2.5×10⁹/L, and there was significant difference when comparing to that of the group (36.4%) with MNCs count less than 2.5×109/L (x²=8.07, P=0.005). There was a negative correlation between the MNCs count and the gestational ages in the specimens with the same volume of UCB (r=-0.95, P < 0.01). In the group with the cell inoculum density of 1×10¹¹/L, the growth and proliferation of primary and subculturing MSCs were better than that of the groups with the cell inoculum density lower than 1×10¹¹/L. The adherence of MSCs in the group with the culture medium containing 5% FBS happened much later than other 3 groups, while the purity of MSCs in this group was much higher. When comparing the passage rate, there was no significant difference among the 4 groups with different concentration of FBS. In the group of culture flask being coated with FBS beforehand, the purity and proliferation ability of MSCs was higher than that in the groups with culture flask not being coated. It is suggested that culture of UCB-MSCs was influenced by several factors. The success rate could be increased by choosing the fetus with relative lower gestational ages, collecting enough volume of UCB, inoculating cells with a higher density, choosing the medium with lower concentration of FBS, and coating the culture flask with FBS beforehand.