低温冻存基质细胞特性的研究

Study of Biological Properties of Cryopreserved Stromal Cells

目的 探讨骨髓细胞低温冻存后贴壁基质细胞培养生长的基本特性 ,为低温冻存基质细胞的应用提供实验依据。方法 取新鲜骨髓和经Dexter长期培养法培养 14d的骨髓贴壁基质细胞 (称“新鲜基质细胞”) ,用 5 %二甲基亚砜 6 %羟乙基淀粉为保护剂 ,- 80℃冰箱降温 ,- 196℃液氮冻存 (前者称“冻存骨髓” ,后者称“冻存基质细胞”) 2周 ,复温后 ,再用Dexter法培养这些细胞 ,检测细胞增殖、细胞形态、细胞化学染色、细胞表面及基质细胞支持另一骨髓造血细胞形成的鹅卵石造血区、长期培养起始细胞等为指标 ,比较它们的生长特性、成分及功能。结果 生长特性 :冻存骨髓比新鲜骨髓出现贴壁细胞、细胞丛和细胞融合成片迟 1～ 2d ,冻存基质细胞融合成片比新鲜基质细胞迟 12～ 18h ;冻存骨髓与新鲜骨髓 ,冻存基质细胞与新鲜基质细胞增殖数比明显低。细胞成分 :冻存骨髓比新鲜骨髓形成的成纤维细胞、内皮细胞比率下降 ,而巨噬细胞和脂肪细胞比率升高 ,冻存基质细胞上述现象更明显 ;内含凋亡小体的细胞冻存基质细胞多于新鲜基质细胞 ,冻存骨髓细胞多于新鲜骨髓 ;冻存骨髓和冻存基质细胞台盼蓝拒染率分别为 95 2 %、89 5 %;冻存骨髓、冻存基质细胞CD14、人白细胞DR抗原表达百分率比新鲜骨髓、新鲜基质细胞高 ,CD45。

Objective To observe the basical properties of adherent stromal cells in culture derived from cryopreserved bone marrow cells (BMCs), and to provide laboratory evidences for clinical application of cryopreserved stromal cells.Methods Fresh BMCs and adherent stromal cells cultured for 14 days in Dexter long-term culture system (fresh stromal cells) plus 5% DMSO-6% HES cryopreservatives were frozen in -80℃ refrigerator, and cryopreserved in -196℃ liquid nitrogen for 2 weeks (the former is called cryopreserved BMCs, the latter called cryopreserved stromal cells). These cells were cultured in Dexter long-term culture system after they were thawed. We have examined the growth features, contituents and stimulating functions of these cells. Results Growth features: cryopreserved BMCs produced adherent stromal cells, cell clusters and cell layer 1～2 days later than fresh BMCs. Cryopreserved stromal cells formed cell layer in 2nd day of culture, and were 12～18 hrs later than fresh stromal cells. Cryopreserved BMCs and stromal cells proliferated significantly less than fresh BMCs and stromal cells. Constituents: The ratio of fibrocytes and endothelial cells were lower, and the ratio of macrophages and fat cells were higher in cultutre of cryopreserved BMCs than that in fresh BMCs. The measurements in culture of cryopreserved stromal cells were much more significant compared with that in fresh stromal cells. Numbers of cells containing apoptic bodies in culture of cryopreserved BMCs and stromal cells were more than that in fresh BMCs and stromal cells. TBRR of cryopreserved BMCs and stromal cells were 92.5% and 89.5% respectively. The expression rates of CD14 and HLA-DR in culture of cryopreserved BMCs and stromal cells were higher than that in fresh BMCs and stromal cells, and just in contrast with the expression rates of CD45 and CD33. Stimulating fuctions: CAFC and LTC-IC on the stromal cell layer derived from cryopreserved BMCs, cryopreserved stromal cells, fresh BMCs and fresh stromal cells were all growing well, and there were no significant differences among these groups. Conclusion Biological properties of adherent stromal cells derived from BMCs and stromal cells were injured slightly and still maintained completely after cryopreservation with 5% DMSO-6% HES cryopreservatioves.