一种改良的人脐带间充质干细胞培养方法  

A modified culture method of human umbilical cord mesenchymal stem cells

作　　者：张俊河[1] 高建辉[1] 马静 任文杰 Zhang Junhe;Gao Jianhui;Ma Jing;Ren Wenjie(Institutes of Health Central Plains,Xinxiang Medical University,Xinxiang 453003,China)

机构地区：[1]新乡医学院健康中原研究院,新乡453003

出　　处：《中华实验外科杂志》2023年第4期660-662,共3页Chinese Journal of Experimental Surgery

基　　金：国家重点研发计划课题(2018YFC1105804);河南省自然科学基金面上项目(232300421166);新乡市科技攻关计划项目(GG2021008)。

摘　　要：目的建立一种改良的人脐带间充质干细胞(MSCs)的培养方法,并分析其生物学特性。方法采用组织块贴壁法原代分离人脐带MSCs,首先在杜尔伯科改良伊格尔(DMEM)高糖培养基培养,接着改用含有间充质干细胞生长添加物的DMEM培养基传代培养;传代两次后,重新换回DMEM培养基进行培养(三联法培养组)。同时以DMEM高糖培养基作为对照组。通过绘制生长曲线,细胞表面标志物检测,成脂分化检测等,对培养获得的人脐带MSCs进行鉴定。两组间比较采用t检验。结果三联法培养组获得人脐带MSCs成功率高,组织块加放无菌盖玻片的细胞爬出时间、原代培养时间和细胞密度明显优于未加无菌盖玻片[(96.16±4.61)h比(139.50±6.26)h,(15.17±2.36)d比(21.47±2.83)d,30.57%比14.27%,t=9.650、2.960、9.669,P<0.05],差异均有统计学意义;人脐带MSCs细胞表面高表达CD44(99.05%)和CD29(98.51%),不表达造血干细胞表型CD45(0.05%),且具有多向分化潜能。结论本方法分离培养的人脐带MSCs,步骤简单、成功率高、成本较低,适用于人脐带MSCs的大规模培养。Objective To establish a modified culture method of human umbilical cord mesenchymal stem cells(MSCs),and analyze its biological characteristics.Methods Human umbilical cord MSCs were isolated using the tissue explants adherent method.Firstly,MSCs were cultured in ordinary dulbecco’s modified eagle’s medium(DMEM)with high glucose,then DMEM supplemented with mesenchymal stem cell growth supplement was used for subculture.After two rounds of passaging,ordinary DMEM was used for culture(triple method culture group).At the same time,DMEM with high glucose was used as control group.By drawing the growth curve,cell surface markers were detected,the adipogenic differentiation was examined,and the cultured human umbilical cord MSCs were identified.T-test was used for comparison of two groups.Results The triple method culture group had a high success rate in obtaining human umbilical cord MSCs.The cell extension time,primary culture time and cell density of tissue explants with sterile coverslips were significantly better than those without sterile coverslips[(96.2±4.6)h vs.(139.5±6.2)h,(15.2±2.3)d vs.(21.5±2.8)d,30.6%vs.14.3%]with the difference being statistically significant(t=9.650,2.960,9.669,all P<0.05).Human umbilical cord MSCs cells stably expressed the stem cell markers CD44(99.05%)and CD29(98.51%),while no expression of the hematopoietic marker CD45(0.05%)was detected,which had the potential for multi-directional differentiation.Conclusion The human umbilical cord MSCs isolated and cultured by this method have simple steps,high success rate and low cost,which are suitable for large-scale culture of human umbilical cord MSCs.

关 键 词：脐带 间充质干细胞 组织块贴壁法 

分 类 号：Q813.11[生物学—生物工程] R318[医药卫生—生物医学工程]