临床治疗用人骨髓间充质干细胞保存条件的研究

Influence of preservation conditions on human mesenchymal stem cells

目的探讨不同输注液、保存时间及温度对临床治疗用人骨髓间充质干细胞(hBMSCs)存活率、表型及增殖能力等状况的影响。方法采用密度梯度离心法从人骨髓分离培养hBMSCs,流式细胞术检测细胞表型,并进行成骨、成脂等多向分化能力鉴定。在4℃和25℃条件下,将培养的第3代hBMSCs保存在3种临床常用输注液(9g/L氯化钠注射液、5%人血清清蛋白氯化钠注射液、50g/L葡萄糖氯化钠注射液)中,检测hBMSCs在不同时间点(0.5、1.0、2.0、4.0、6.0h)的细胞存活率、细胞表型和存活细胞的增殖能力。结果 hBMSCs间充质干细胞形态为均一的长梭形,表达CD29、CD105,不表达CD34。成脂诱导2周,油红O染色,可见脂滴为红色。成骨诱导3周,茜素红S染色可见大量橘红色钙结节。4℃和25℃条件下,保存不同时间后,5%人血清清蛋白氯化钠注射液组细胞存活率和增殖能力均高于其余2组。同一组细胞4℃条件下保存不同时间后的增殖能力强于25℃条件下保存的细胞。不同输注液、保存温度及保存时间对细胞表型没有显著影响。结论 4℃条件下,将间充质干细胞保存在5%人血清清蛋白氯化钠注射液中0.5h内回输对细胞活力影响最小。

Objective To investigate the impact of different infusions,storage time and temperature on survival rate,phenotype and proliferative capacity of human bone marrow mesenchymal stem cells （hBMSCs）.Methods hBMSCs,isolated from human bone marrow by using density gradient centrifugation,were cultured and detected for surface markers by flow cytometry and multi-directional differentiation potential by adipogenic and osteogenic induction.The third-generation of hBMSCs was preserved using 3 kinds of common infusion solutions,including 9 g/L sodium chloride injection,9 g/L sodium chloride injection containing 5% hu-man albumin,and 50 g/L glucose and sodium chloride injection.After preserved at 4 and 25 ℃ for 0.5,1.0,2.0,4.0 and 6.0 hours,cell survival rate,phenotype and proliferative capacity were determined.Results hBMSCs were uniform long spindle,with positive expression of CD29 and CD105,and negative expression of CD34.After 2 weeks of adipogenic induction,oil red O staining showed red-stained lipid droplets.After 3 weeks of osteogenic induction,alizarin red S staining showed orange calcium nodules.At 4 and 25 ℃,the survival rate and proliferative capacity of hBMSCs,preserved in infusion solution prepared by human albumin were much higher than hBMSCs preserved in the other two kinds of infusion solutions.Proliferative capacity of hBMSCs preserved at 4℃ was much higher than those preserved at 25 ℃.Different infusion,temperature and storage time had no significant effect on cell phenotype.Conclusion Preservation condition of 4 ℃ and 9 g/L sodium chloride injection containing 5% albumin might be with minimal impact on cell viability of hBMSCs.