骺板软骨细胞的冻存及复苏

Frozen reservation and recovery of epiphyseal plate chondrocyte

目的 研究骺板软骨细胞冻存的方法。方法 取对数生长期的骺板软骨细胞 ,加入配制好的冻存液 (冻存液A :培养液与DMSO ;冻存液B :含 5 %小牛血清的培养液与DMSO ;冻存液C :含 2 0 %小牛血清的培养液与DMSO ;冻存液D :小牛血清与DMSO混合 ;其中DMSO终浓度均为 10 %。) ,冻存。细胞复苏后用台盼蓝拒染试验检测细胞存活率。然后接种于培养皿中 ,采用苏木 伊红染色和甲苯胺蓝染色 ,观察细胞形态及合成糖胺多糖的能力。结果 细胞活率为 :A冻存液 70 % ,B冻存液 75 % ,C、D两种冻存液无明显差别 ,分别为 89%和 92 %。复苏的细胞贴壁时间延迟 ,但培养 72小时以后 ,细胞活力明显增强。甲苯胺蓝异染提示 ,经冷冻保存后软骨细胞仍能保持合成异染基质 (糖胺多糖 )的能力。结论 本实验冻存、复苏的方法结果稳定 ,可用于保存骺板软骨细胞。

Objective To study frozen reservation method of epiphyseal plate chondrocyte.Methods Cell at logarithmic growth phase of 5×10 6/ml and frozen stock solution were subpackage into frozen stock tube to perform frozen reservating(frozen stock solution A:culture solution and DMSO; frozen stock solution B:culture solution contained 5%calf serum and DMSO; frozen stock solution C:culture solution contained 20%calf serum and DMSO; frozen stock solution D:calf serum and DMSO; the concentration of DMSO was 10% in all frozen stock solution). After recovered, the cell survival rate was detected by the method of trypan blue strain. Then the cells were plated in culture dish. The morphology of cell and the ability to synthesizing glycamine and polygluose were observed by HE stain and toluidine blue stain.Results The cell motility rate arrived at 70% at A frozen stock solution, 75% at B frozen stock solution, and there were no significant difference about the cell motility rate in between C and D frozen stock solution, which was 89%, 92%, respectively. The activity of frozen cell was lower than fresh chondrocyte in the first 24h, but the activity was obviously enhanced after 72h. Toluidine blue dye showed that the frozen chondrocyte still could keep the ability to synthesizing matrices(glycamine and polyose). Conclusion The result of this method of frozen and recovery is stable and it can be used to preserve chondrocyte of epiphyseal plate.