成人骨髓基质干细胞分化来源的肝细胞的冻存与复苏研究

Study of cryopreservation of differentiated hepatocyte derived from human bone marrow stem cells

目的在体外诱导人骨髓基质干细胞向肝细胞分化成熟的基础上,将细胞深冻保存,观察冻存复苏后细胞存活率及功能状况。方法在含有多种细胞因子的条件培养基中体外诱导成人骨髓基质干细胞,当细胞显示成熟肝细胞特征后,将细胞分别冻存于90%胎牛血清(FBS)和10%二甲亚砜(DMSO)(A组)、10%FBS、30%甘油和60%培养基(B组)和10%FBS、10%DMSO和80%威斯康星器官保存液(UW液)(C组)中。冻存4周后复苏细胞,测定各组细胞存活率及白蛋白合成情况。结果成人骨髓基质干细胞分化来源的肝细胞经冻存复苏后可恢复功能细胞形态,A、B和C组冻存液中细胞复苏后细胞存活率分别为(60·0±3·3)%、(91·0±2·6)%和(89·0±1·4)%,培养24h后检测上清液白蛋白浓度分别为(0·210±0·005)g/L、(0·340±0·020)g/L和(0·330±0·030)g/L。结论成人骨髓基质干细胞分化来源的肝细胞冻存复苏后仍保持较高的细胞存活率和功能,可作为临床肝细胞移植及生物人工肝的重要肝细胞来源。

Objective To observe the viability and function of human bone marrow stem cellderived hepatocytes following cryopreservation in vitro. Methods Human bone marrow cells were induced to differentiate into hepatocytes in the presence of multiple factors. Mature hepatocytes were cryopreserved in 90% FBS and 10% DMSO （Group A）, 10% FBS, 30% glycerol and 60% conditioned medium （Group B）, and 10% FBS, 10% DMSO, and 80% UW solution （ Group C）. The cells were thawed after 4 weeks, and the cell viability and the albumin level were determined. Results The human bone marrow derived hepatocytes maintained functional morphology after thawing. The viabilities in Group A, B and C were （60.0±3.3）%, （91.0 ±2.6）%, and （89.0 ± 1.4）%, respectively. After culturing for 24 h, the albumin levels in Group A, B and C were （0.210 ±0.005） g/L, （0.340 ±0.020） g/L and （0.330 ± 0. 030） g/L, respectively. Conclusions Human bone marrow stem cell-derived hepatocytes can maintain the viability and function after cryopreservation. These cells may contribute to an important source of hepatocytes for clinical hepatocyte transplantation and artificial liver support system.