骨髓细胞液氮保存21-25年后的细胞活力体外研究  

In Vitro Activity of Human Bone Marrow Cells after Cryopreservation in Liquid Nitrogen for 21-25 Years

作　　者：黄友章[1] 沈建良[1] 宫立众[1] 郑培浩[1] 刘毅[1] 尹文杰[1] 岑坚[1] 王宁[1] 赵德峰[1] 

机构地区：[1]海军总医院血液科,北京100048

出　　处：《中国实验血液学杂志》2010年第1期224-229,共6页Journal of Experimental Hematology

摘　　要：为探讨适合临床应用的骨髓细胞保存方法,揭示细胞长期低温保存效果,将20人份骨髓加入DMSO-AuP(10%二甲亚砜、10%自体血浆)或DMSO-HES-HuA(5%DMSO、6%羟乙基淀粉、4%人血白蛋白)保护剂,分装2毫升/管,600-800管/人份,自控程序降温仪或-80℃低温冰箱预冻降温、液氮中保存21-25年。取冷冻骨髓细胞于38℃复温,检测细胞相关指标。结果表明:加保护剂的细胞标本在-80℃低温冰箱中为低速降温,-30℃前,与自控程序降温仪设定1℃/min的低降温速率接近。DMSO-HES-HuA与DMSO-AuP比较,红细胞形态畸形率分别为(3.5±1.5)%和(12.6±4.8)%;溶血率分别为(3.3±1.6)%和(23.1±5.1)%(p<0.05);前者渗透性脆性不变,后者减低;红细胞、血小板、粒单系造血祖细胞、长期培养起始细胞回收率,前后者对比分别为(96.1±1.8)%、(70.0±9.5)%、(49.2±10.9)%、(54.2±13.8)%vs(76.3±5.6)%、(52.7±8.1)%、(43.5±12.3)%、(47.2±13.6)%。用上述保护剂配合上述降温法保存后,细胞在间充质干细胞培养上,生长特性良好。同一保护剂用上述两种降温法保存后,细胞各种回收效果差异不显著(p>0.05)。结论:细胞在液氮中保存21-25年,其细胞形态和回收活力(率)良好;保存如骨髓这种细胞成分并非单一的标本时,用5%DMSO-6%HES-4%HuA为保护剂的-80℃冰箱预冻降温后液氮保存,方法简便、经济、易操作,适合临床应用。The aim of this study was to investigate the best method to preserve human bone marrow cells and the effectiveness of long term cryopreservation at - 80℃. The human bone marrow cells in 20 samples were firstly frozen by a programmed freezer or -80℃ refrigerator, and then were preserved in liquid nitrogen with DMSO-AuP （10% dimethylsulfonamide, 10% autologous plasma） or DMSO-HES-HuA （5% dimethylsulfonamide, 6% hydroxyethyl starch, 4% human serum albumin） as cryoprotectant for 21 to 25 years. They were thawed in 38℃. The cell sample frozen in -80℃ refrigerator was frozen at a low frozen speed of 1℃/min which was the same as the programmed freezer before -30℃. Before detection the bone marrow cells were taken from liquid nitrogen and were thawed in 38℃, then the suspension of bone marrow cells was prepared for detection. The cell morphology and recovery rate of erythrocytes, nucleocytes and pJatelets; the recovery rate of hematopoietic stem progenitors cells, as well as mesenchymal stern cells were determined. The results showed that the protective effectiveness of DMSO-HES-HuA was better than DMSO-AuP. The mature erythrocytes were destroyed lightly （3.5 ± 1.5 ） % versus （ 12.6 ± 4.8 ） % ], the hemolysis rate was lowerL （3.3 ± 1.6） % versus （23.1 ± 5.1 ） % , Osmotic fragility of erythrocytes in the former was not changed, but was droped in the latter. The recovery rates of red cell, platelet, granulocyte-macrophagc colony forming units and long term culture-initiating cells were higher in the former than that in the latter （96.1 ± 1,8） %, （70.0±9.5）%, （49.2±10.9）%, （54.2±13.8）% versus （76.3 ±5.6）%, （52.7 ±8. 1）%, （43.5±12.3）%, （47.2 ± 13.6）% respectively. With each kind of cryoprotectant or frozen method, the frozen MSC could keep the original growth properties. With the same cryoprotectant and different frozen method, the cryopreservative effectiveness was not different. The influence of the cryoprotectant prescriptions and the froz

关 键 词：骨髓细胞 低温保护剂 程控降温 -80℃低温冰箱降温 

分 类 号：R318.52[医药卫生—生物医学工程]