改良原代培养体系提高人脐带间充质干细胞的产量  

Improved primary culture system increases the yield of human umbilical cord mesenchymal stem cells

作　　者：高彦琳 张宁坤 陈厚良 高连如 朱智明 

机构地区：[1]安徽医科大学海军临床学院,北京市100048 [2]解放军海军总医院,北京市100048

出　　处：《中国组织工程研究》2015年第10期1477-1481,共5页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金面上项目(81170094)~~

摘　　要：背景:目前关于脐带间充质干细胞培养方法的研究很多,但尚无关于初次培养后废弃物的相关研究。目的:探讨优化人脐带来源间充质干细胞体外培养的最佳方法。方法:采用组织块贴壁法分离培养人脐带间充质干细胞,记为初次培养组。将原代培养瓶中的培养液及组织离心,重新分成组织组、混合组和纯液组进行再次培养。观察4组原代细胞的细胞形态、获得时间和细胞得率;MTT法绘制细胞生长曲线,流式细胞仪检测细胞周期及免疫表型。结果与结论:初次培养组、再次培养组织组、再次培养混合组、再次培养纯液组获取细胞的平均时间分别为(15.00±0.45)d,(7.0±0.3)d,(8.00±0.25)d,(8.00±0.25)d。每个T75培养瓶可获取的第1代细胞数分别为(4.0±0.5)×105、(9.0±0.55)×105、(15.0±0.2)×105、(7.0±0.33)×105个。倒置显微镜下观察4组细胞为形态相对均一的梭形贴壁细胞,呈平行排列生长或漩涡状生长。4组细胞的生长曲线、增殖活性、表面标记物检测均无明显差异。结果表明对脐带间充质干细胞的原代培养体系进行再次培养,可在短时间内扩增出大量原代细胞。BACKGROUND：At present, a lot of research about culture methods for umbilical cord mesenchymal stem cels, but not for the waste of primary system. OBJECTIVE：To explore the best culture method of human umbilical cord mesenchymal stem celsin vitro. METHODS：Human umbilical cord mesenchymal stem cels were prepared by tissue explants method, recorded as initial culture group. The centrifugal fluid and tissue of the primary culture flask were centrifuged and divided into three groups for secondary culture： tissue group, mixed group and pure liquid group. Cel morphology, time for cel acquisition, and yield of primary cels in the four groups were observed; the cel growth curve was analyzed by MTT assay; and cel cycle and phenotype were detected by flow cytometry. RESULTS AND CONCLUSION： The average time for cel acquisition in the initial culture group, tissue group, mixed group and pure liquid group were （15.00±0.45）, （7.0±0.3）, （8.00±0.25） and （8.00±0.25） days, respectively. The number of cels at first generation was （4.0±0.5）×10^5, （9.0±0.55）×10^5, （15.0±0.2）×10^5 and （7.0±0.33）×10^5 markers of the four groups had no significant differences. The human umbilical cord mesenchymal stem cels can be obtained rapidly and largely through the secondary culture to the primary culture system. T75 culture bottle, respectively. Under the inverted microscope, cels in the four groups were fusiform-like adherent cels, which were in paralel or circinate arrangement. Growth curve, proliferative activity, surface markers of the four groups had no significant differences. The human umbilical cord mesenchymal stem cells can be obtained rapidly and largely through the secondary culture to the primary culture system.

关 键 词：脐带 间质干细胞 细胞 培养的 干细胞 脐带脐血干细胞 脐带间充质干细胞 细胞培养 华通胶 国家自然科学基金 

分 类 号：R318[医药卫生—生物医学工程]