小鼠骨髓间充质干细胞的分离与培养

Isolation and Cultivation of Mouse Bone Marrow Mesenchymal Stem Cells

探索在体外分离培养小鼠骨髓间充质干细胞 (Bone m arrow m esenchym al stem cells,BMSCs)的最适条件。以密度梯度离心技术及贴壁筛选法相结合 ,在体外分离 BAL B/C小鼠的 BMSCs,通过 MTT法测定了不同离心力、不同换液时间、不同血清浓度、不同种植密度等因素对 BMSCs分离和培养的影响。在 5 0 0 g× 30 min的离心力下分离细胞 ,加入 10 %的胎牛血清 ,原代按 (12～ 2 0 )× 10 5/m l的细胞密度接种 ,接种 2 4 h后换液 ,继代种植密度为 6 .4～ 2 5 .6× 10 4 /ml时最适宜细胞生长 ;在此条件下 ,细胞快速增殖 ,传代后 8h贴壁达 90 %以上 ,2 4 h便进入对数生长期 ,直至第 5 d,细胞约增殖 3倍。本研究建立了在体外分离培养骨髓间充质干细胞的细胞生物学方法和技术参数。

To establish the best condition of isolation and cultivation of mouse bone marrow mesenchymal stem cells (BMSCs) in vitro, we isolated BMSCs from BALB/C mouse using density gradient centrifugation and adherent selecting. The effects of different centrifuge power, adherent time, serum concentration and cell density on the isolation and cultivation of BMSCs were investigated. The best isolating condition is:500g×30min,24 hours adhering. The best cell density is 12-20×105/ml of primary cells and 6.4-25.6×104/ml of secondary cells. The best serum concentration is 10%. More than 90% of subcultured cells were adhesive in 8 hours. Thus we have established a cell biological method of isolation and cultivation of BMSCs.