摘要
目的 探索适合于人骨髓基质细胞 - 196℃冷冻保存条件和方法。方法 以人髂骨区骨髓为材料进行体外人骨髓基质细胞培养 ,传代后以不同浓度二甲亚砜 (DMSO)的冻存液及不同的细胞浓度在液氮中保存 ,2个月后复苏接种培养 ,对其存活率、贴壁率和体外成骨能力进行检测。结果 不同DMSO浓度保存细胞复苏后细胞的存活率从高到低依次为 10 %DMSO组、5 %DMSO组、15 %DMSO组、2 0 %DMSO组 ;在条件培养液中 2周形成多层结构 ,并出现细胞结节 ,碱性磷酸酶染色阳性 ;冻存时细胞浓度为每毫升 (5 .0～ 8.0 )× 10 6个细胞组复苏后细胞存活率高于每毫升 (1.0～2 .5 )× 10 6个细胞组。结论 5 % - 10 %DMSO浓度适合于骨髓基质细胞的长期保存 ,高浓度组骨髓基质细胞的冻存效果优于低浓度组。
Objective To explore the conditions and methods for cryopreservation of human bone marrow stromal cells at -196℃. Methods Human bone marrow stromal cells obtained from human iliac crest was cultured in vitro. The cells were passaged, the passage cells stored in 5%,10%,15%,20% DMSO cryopreseservation medium at different concentrations of cells . After 2 months of cryopreservation,the cells were thawed and inoculated for culture and then viability, attachment rate and osteogenic potential in vitro were detected. Results The cells viability of different DMSO concentrations for the cryopreservative cells form high to low were 10%DMSO group, 5%DMSO group, 15%DMSO group, 20%DMSO group. Cultured in condition medium for two weeks, the cells transformend into multiplayer and gradually congregated into opaque cluster later. Alkaline phosphatase show positive reaction. Conclusions The ideal concentration of DMSO for-196℃ cryopreservation of human bone marrow stromal cells is 5%～10%; The cryoprotective effect of cells in high concentration is better than in low concentration.
