无血清和有血清培养人脐带间充质干细胞的对比

A comparative study on serum-free and serum culture methods of human umbilical cord mecenchymal stem cells

背景:研究表明有血清培养体系存在若干风险和问题,比如免疫排斥、批次差异、病毒风险等,另外随着外泌体的发现和应用,无血清培养体系变得越来越重要。目的:系统比较人脐带间充质干细胞无血清培养体系与传统有血清培养体系的异同,为人脐带间充质干细胞的临床转化奠定基础和提供实验数据。方法:无菌条件下采集健康剖腹产足月儿脐带,组织块贴壁法培养人脐带间充质干细胞,从第1代开始有血清培养组用体积分数为10%胎牛血清培养,血清替代物组用体积分数为15%血清替代物培养。倒置显微镜观察其形态变化,流式细胞仪检测其表面标记物,CCK-8检测其增殖能力,诱导分化实验检测其多向分化潜能,Western Blot检测干性标志物oct4、nanog、sox2的蛋白水平。结果与结论:(1)倒置显微镜下观察可见血清替代物组人脐带间充质干细胞呈现较均一的漩涡状生长,胎牛血清组人脐带间充质干细胞随着细胞代数的增加逐渐出现细胞分化或者老化现象;(2)两种培养法培养的人脐带间充质干细胞均表达CD73,CD90和CD105,低表达CD34和CD45,二者无明显差异;(3)增殖能力上胎牛血清组优于血清替代物组;(4)两种培养法培养的人脐带间充质干细胞均具有成脂、成骨、成软骨诱导分化能力,二者无明显差异;(5)血清替代物组与胎牛血清组oct4、nanog的表达水平无显著差异,血清替代物组sox2表达水平高于胎牛血清组(P<0.05);(6)血清替代物培养的人脐带间充质干细胞符合间充质干细胞国际标准,血清替代物能够替代胎牛血清成为培养人脐带间充质干细胞的优选方法。

BACKGROUND： Studies have shown increasing risks and problems in the serum culture system, such as immune rejection, batch differences and virus risk. In addition, with the discovery and application of exosomes, the serum-free culture system is becoming an increasing concern. OBJECTIVE： To compare the similarities and differences between the serum-free culture system and the traditional serum culture system, which lays the foundation for the clinical transformation of human umbilical cord mesenchymal stem cells （hUCMSCs） and provides experimental data. METHODS： Umbilical cord was collected from term infants of cesarean section under aseptic condition, and hUCMSCs were isolated and cultured by explant tissue technique. hUCMSCs was cultured with 10% fetal bovine serum （FBS） and 15% serum substitutes （AGS） from the original generation. Then an inverted microscope was used to observe cell morphological changes. Flow cytometry was used to detect cell surface markers. Cell counting kit-8 was used to detect cell proliferation. Induced differentiation experiment was used to detect cell differentiation potential. Western Blot was used to detect the protein levels of oct4, nanog and sox2. RESULTS AND CONCLUSION： Under the inverted microscope, hUCMSCs cultured with AGS showed more uniform vortex-like growth, and those cultured with FBS gradually appeared with cell differentiation or aging with the increase of cell generations. hUCMSCs cultured by both methods expressed CD73,CD90 and CD105 but lowly expressed CD34 and CD45, and there was no significant difference between the two culture methods. FBS method was superior to AGS method in proliferation ability. Results from the induced differentiation experiments showed that hUCMSCs cultured by both methods had adipogenic, osteogenic and chondrogenic abilities, and there was no significant difference between the two culture methods. hUCMSC cultured by both methods expressed oct4 and nanog but showed no significant difference in level, while the expression of sox2 was significantly higher in the hUCMSCs cultured by AGS than by FBS （P 〈 0.05）. To conclude, the hUCMSCs cultured with AGS are in accordance with the international standards of mesenchymal stem cells. The AGS method as an alternative to the FBS method can become a preferred method for hUCMSCs culture.