密度梯度离心法结合贴壁法体外分离培养大鼠骨髓间充质干细胞

目的 研究密度梯度离心法结合贴壁法分离大鼠骨髓间充质干细胞(BMSCs)及不同首次换液时间对细胞增殖的影响,探索一种简单易行、扩增效率高的方法。方法 3月龄雄性SD大鼠提取原代BMSCs,密度梯度离心法结合贴壁法分离培养,并应用3组不同首次半量换液时间(24h,48h,72h)进行培养传代,测定生长曲线和细胞的存活率;免疫细胞化学染色分析细胞表面抗原CD44和CD166的表达。结果 分离的BMSCs48h后细胞完全贴壁生长,呈形态均一的纺锤形单核细胞状,第3代细胞培养1w后呈长梭型,呈平行排列生长。48h半量换液组细胞增殖活性及生存率最高,其次为24h和72h组;表面标志物CD44和CD166均呈阳性。结论 联合应用密度梯度离心法及贴壁筛选法可成功分离BMSCs,且效率高、纯度高,48h首次半量换液更适合BMSCs的生长繁殖。

In vitro isolation and culture of rat bone marrow mesenchymal stem cells using density gradient centrifugation combined with adherence method

Objective The rat BMSCs were isolated and cultured using density gradient centrifugation combined with adherence method, and different first replacing culture medium time was performed to study the different cell proliferation and approach simple and efficient amplification methods of BMSCs. Methods Primary BMSCs were extracted sterilely from 3 month male SD rat and cultured using density gradient centrifugation combined with adherence method, with three different first replacing semis culture medium time(24h, 48h, 72h). MTT method was used to study the growth curve and cell survival rate, the expression of CD44 and CD166 was analyzed by immunocytochemical staining. Results The isolated BMSCs were well grown and adhered to wall 48h after culture with fusiform histomonocyte morphology. The 3rd generation cells 1 week after culture presented fusiform and well-distributed regularlly, the cell proliferation activity and survival rate were higher while first replacing semis culture medium time was 48 h, the expression of cell surface antigen CD44 and CD166 was positive. Conclusion BMSCs were isolated successfully using density gradient centrifugation combined with adherence method, 48h first replacing semis culture medium is suitable to the growth and amplification of BMSCs。