贴壁法体外分离培养大鼠骨髓间充质干细胞的效果验证

背景:骨髓中的间充质干细胞含量不高,且随着年龄增加或体质衰弱,骨髓间充质干细胞的数量会逐渐减少.目的:验证贴壁法分离培养大鼠骨髓间充质干细胞的效果.方法:大鼠麻醉后取双侧股骨和胫骨,剪去骨骺端,暴露骨髓腔,用含小牛血清的DMEM培养基冲洗骨髓腔,收集骨髓细胞,反复吹打制成单细胞悬液,接种后置于37 ℃、体积分数为5%的CO_2培养箱内孵育,24 h后全量换液,以后每周全量换液1次,筛选易贴壁但贴壁不牢的细胞进行传代培养.观察细胞形态,绘制细胞生长曲线,流式细胞仪及免疫细胞化学染色鉴定骨髓间充质干细胞表面标志的表达.结果与结论:培养24 h后细胞能够贴壁生长,呈梭形或三角形;第二三天贴壁细胞迅速增殖;培养15 d左右出现致密的贴壁细胞层,呈漩涡状生长或成簇生长.细胞在接种后2 d进入对数生长期,12 d左右进入平台期,约15 d细胞可铺满瓶底.分离培养的大鼠骨髓间充质干细胞CD90和CD54均呈阳性表达.结果验证了采用贴壁法可在体外成功分离培养大鼠骨髓间充质干细胞,操作简单,造成污染的环节和机会较少,不需离心,可以更好的保持细胞活性.

Effect validation of bone marrow-derived mesenchymal stem cells cultured by adherent method in vitro

BACKGROUND: A small number of mesenchymal stem cells (MSCs) present in bone marrow, which would gradually drop with age. OBJECTIVE: To verify the effect of adherent method on culture of bone marrow-derived MSCs. METHODS: Under anaesthesia, bone marrow cells were obtained from femur and tibia of rats, cultured by DMEM containing calf serum, placed in an incubator containing 5% CO_2 at 37 ℃. The culture medium was renewed after 24 hours, and remained periodical medium change with once per week. The weakly adherent cells were passaged. The cell morphology, growth curve, and the expression of cell-surface markers were identified by flow cytometry and immunocytochemical staining. RESULTS AND CONCLUSION: After 24 hours of culture, the cells could adhere to the walls with fusiform or triangle shapes, proliferated faster after 2-3 days, and presented whirlpool-like or clustering. The cells reached a logarithmic growth phase after 2 days, and into the late stationary phase after 12 days, which covered the bottle after 15 days. The cultured cells were positive to CD90 and CD54. The results verified that bone marrow-derived MSCs can be isolated by adherent method. This method is easy operation, and can maintain cell activity preferably.