改良贴壁法培养大鼠骨髓间充质干细胞

目的 培养高纯度骨髓间充质干细胞. 方法 取雄性6～8周龄SPF级S-D大鼠,采用改良贴壁法培养骨髓间充质干细胞(BM-MSCs),细胞整体融合度达70％～80％时传代.观察细胞形态、贴壁性能,在P2代时以流式细胞仪鉴定检测细胞表型. 结果 P1后镜下无圆形细胞,细胞形态逐渐趋于完全一致.细胞均表现典型之贴壁特性；首次达到80％融合度时间为9～11d,P1至P2为7～8d,P2至P3为5～6d.至P3时后,每只大鼠约可得MSCs 3～5×106个细胞.细胞表型CD29阳性率为99.6％,CD90阳性率为94.9％,CD44阳性率为68.1％; CD34阴性率为98.6％,CD45阴性率为99.9％. 结论 应用改良贴壁法较为简单、易行,不需要离心等操作,基本排除了非目标细胞的污染,降低对细胞的损害,生物学特性完整,获得纯度较高的MSCs.

Isolation and culture of Bone marrow-derived mesenchymal stem cells using a modified plastic-adherent method

Objective To isolate and culture high-grade mesenchymal stem ceils from rats bone marrow and determine its property of plastic-adherent and specific cell surface markers. Methods Mesenchymal stem ceils were isolated from male S-D rats bone marrow weighed 190-220g and cultured by using modified plastic-adherent method . After the first passage, the second passage would be conducted as the overall alignment of the ceils reached 70-80%. Then the morphdogy and performance of the ceils were observed with phase-contrast microscopy and the specific cell surface markers was also determined by flow cytometry. Results After the frrst passage there were round cells and the morphology of the cells were uniform showing typically plastic-adherent property. The time for the first adherence of 70-80% was 7-gdays,and first passage to the second one and the second to the third one were 7-8days and 5--6 days,then 3-5 ×10^6 MSCs could be obtained from each rat.The positive rats of CD29 ,CD60 and CD44were 99.6% ,94.9%.and 68.1%. While the negative rates of CD34 and Cd45 were 98.6% and 99.9%. Conclusions Reforming plastic-adherent method is a convenient and effective procedure without the need of centrifuge and other manipulation and generally excluded the non-cell pollution that reduced the damage to the cells and maintained the integrity of the cells, finally to ensured the procurement of relatively high -purity MSCs from rats bone marrow.