大鼠骨髓间充质干细胞最适培养基的筛选

王景阁; 俞小琴; 文继锐; 朱光光; 包明月; 何学令; 李良

大鼠骨髓间充质干细胞最适培养基的筛选

Screening of the Optimum Medium for Rat Mesenchymal Stem Cells

摘要

摘要:
目的探讨不同培养基对大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells，BMSCs)生长的影响，筛选对大鼠BMSCs生长较适宜的培养基。
方法采用全骨髓差速贴壁分离法从SD大鼠股骨和胫骨分离BMSCs，分别选用DMEM-LG、α-MEM、DMEM/F12三种培养基分离培养大鼠BMSCs。倒置相差显微镜下观察不同培养基对大鼠BMSCs形态均一化程度、克隆形成时间与第14天克隆形成数量、第1次传代时间、细胞增殖率以及细胞贴壁率等的影响；流式细胞术鉴定并观察不同培养基对大鼠BMSCs表面抗原表达的影响。
结果与其他两组相比，体外DMEM-LG培养基培养的大鼠BMSCs形态均一、克隆形成时间和第1次传代时间短、克隆形成数量多达(14±2)个、细胞贴壁率高达(47.0±2.8)%；同时，BMSCs进入对数生长期仅需4 d，且平均增殖率最高，单位时间内平均扩增数量最多，3 d总扩增数量达(2.2~2.7)×10⁵ mL^-1；采用DMEM-LG、α-MEM、DMEM/F12三种培养基分离培养的细胞均为大鼠BMSCs，且不同培养基对大鼠BMSCs表面抗原表达无明显影响。
结论 DMEM-LG培养基较适合大鼠BMSCs的生长。

Abstract:
Objective To investigate the effect of three different cell culture mediums, DMEM-LG, α-MEM and DMEM/F12, on the growth of rat bone marrow mesenchymal stem cells (BMSCs) in vitro, and so that to screen out the most suitable medium for in vitro culturing the rat BMSCs.
Methods BMSCS were isolated from the femur and tibia of SD rats by whole bone marrow differential adherence method. The isolated cells were then cultured with three culture mediums, DMEM-LG, α-MEM and DMEM/F12. The rat BMSCs morphology, adhesion, proliferation, the time of passage and the number the colony at day 14 in three mediums respectively were observed with inverted phase contrast microscopy and compared. Flow cytometry was used to identify and observe the effects of different mediums on the surface antigen expression of rats BMSCs.
Results Compared with the other two groups of media, BMSCs cultured in DMEM-LG had shorter colony formation time, shorter first passage time, more clone formation (14±2) and showed uniform morphology and the highest attachment efficiency (47.0±2.8)%. Meanwhile, BMSCs cultured with DMEM-LG entered logarithmic growth phase after only 4 days of culturing and showed the highest average specific growth rate and the largest average number of propagations per unit time. The total number of cells reached about (2.2-2.7)×10⁵ mL^-1 within three days. The cells cultured with 3 mediums were all identified as rat BMSCs, and the expression of surface antigen in BMSCs was not significantly affected by different media.
Conclusion DMEM-LG is more suitable for proliferation of rat BMSCs in vitro.

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