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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。
Objective To culture high purity bone marrow mesenchymal stem cells. Methods SPF S-D rats aged 6 to 8 weeks old were cultured with modified adherent method to culture bone marrow-derived mesenchymal stem cells (BM-MSCs). When the cell fusion degree reached 70% -80%, passage was performed. Morphological and adhesive properties of the cells were observed. Cell phenotypes were detected by flow cytometry at P2 generation. Results There were no round cells in P1 and the shape of cells tended to be completely consistent. Cells showed typical adhesion characteristics; for the first time to reach 80% confluency for 9 ~ 11d, P1 to P2 for 7 ~ 8d, P2 to P3 for 5 ~ 6d. After P3, about 3 ~ 5 × 106 cells of MSCs were obtained from each rat. Cell phenotype CD29 positive rate was 99.6%, CD90 positive rate was 94.9%, CD44 positive rate was 68.1%; CD34 negative rate was 98.6%, CD45 negative rate was 99.9%. Conclusion The improved method of adherent method is simple, easy to operate, does not require centrifugation and other operations, basically excluding non-target cell contamination, reduce damage to cells, complete biological characteristics, access to high purity MSCs.