人骨髓间充质干细胞在不同输注液中的生存和生长

Survival and growth of human bone marrow mesenchymal stem cells in different infusion solutions

背景:间充质干细胞移植技术快速发展,需要异地移植病例不断增加,但是关于细胞保存运输条件的研究甚少。目的:比较几种临床常用输注液及实验室配制的输注液对间充质干细胞生存和生长的影响。方法:抽取健康志愿者髂骨骨髓,采用密度梯度离心和贴壁筛选相结合的方法,分离培养骨髓间充质干细胞。对骨髓间充质干细胞进行细胞形态、细胞表面标记和多向分化能力的鉴定。25℃条件下,用10%FBS-DMEM、细胞输注液和5种临床常用细胞输注液(9g/L氯化钠注射液、50g/L葡萄糖注射液、50g/L葡萄糖氯化钠注射液、2%和5%的人血白蛋白溶液)保存第3代骨髓间充质干细胞,检测1,2,4和6h细胞的存活率。然后4℃条件下,以细胞输注液保存骨髓间充质干细胞,检测2,4,8,12,24h细胞的存活率和存活细胞的生长曲线。结果与结论:第3代骨髓间充质干细胞呈均匀一致的长梭形,平行或旋涡状紧密排列。细胞表面标记CD44、CD105表达阳性,CD45表达阴性。成骨诱导3周后,长梭形骨髓间充质干细胞变成多角形,vonKossa染色可见典型的黑色钙化小结;成脂诱导2周后,细胞变成短梭形或椭圆形,油红O染色可见胞浆内密集的被染成红色的脂滴。在25℃条件下,细胞输注液中保存的骨髓间充质干细胞,其存活率远高于在上述5种临床常用输注液中所保存细胞;4℃条件下,细胞输注液和完全培养基中保存的骨髓间充质干细胞存活率和再生长能力基本相同。提示细胞完全培养基成分配制的输注液是维持间充质干细胞脱离培养环境后保持细胞活性,且延长细胞存活时间最适宜的溶液。

BACKGROUND：Mesenchymal stem cells（MSCs） transplantation technology has developed rapidly and the number of patients requiring remote transplantation is increasing gradually.However,the study on cell preservation and transportation is currently inadequate.OBJECTIVE：To compare the effects of common infusion solutions and the infusion solution prepared by our lab on the survival and growth of MSCs.METHODS：Bone marrow of healthy volunteer was extracted to isolate and culture human bone marrow mesenchymal stem cells（hBMSCs） by density gradient centrifugation and adherence screening methods.Cell morphology,cell surface markers and multi-directional differentiation potential were observed and identified.The third-generation hBMSCs were preserved using 10% fetal bovine serum-Dulbecco＇s modified Eagle＇s medium（FBS-DMEM）,the infusion solution prepared by our lab and 5 kinds of common infusion solutions（9 g/L sodium chloride injection,50 g/L glucose injection,50 g/L glucose and sodium chloride injection,9 g/L sodium chloride injection containing 2% human albumin,9 g/L sodium chloride injection containing 5% human albumin）at 25 ℃ for 1,2,4 and 6 hours,cell survival rate were determined.Then,the third-generation hBMSCs were preserved using the infusion solution prepared by our lab at 4 ℃ for 2,4,8,12 and 24 hours.Cell survival rate and cell growth curve were determined.RESULTS AND CONCLUSION：The third-generation hBMSCs were uniform long spindle,closely arranged parallel or swirling.hBMSCs highly expressed CD44 and CD105,and were negative for CD45.After 3 weeks of osteogenic induction,long-spindle BMSCs became polygonal,and von Kossa staining showed the typical black calcified nodules.After 2 weeks of adipogenic induction,cells became short spindle or oval,and oil red O staining showed dense red-stained lipid droplets.At 25 ℃,the survival rate of hBMSCs stored in the infusion solution prepared by our lab was much higher than hBMSCs stored in 5 kinds of common infusion solutions mentioned above.At 4 ℃,the survival and growth ability of hBMSCs stored in the infusion solution prepared by our lab and 10% FBS-DMEM were identical essentially.Results suggested that the infusion solution prepared by our lab according to cell culture medium is a more appropriate solution for maintaining cell activity and extending the extension of cell survival.