College of Veterinary Medicine, Gyeongsang National University, Jinju, Republic of Korea.
Cell Transplant. 2011;20(8):1231-9. doi: 10.3727/096368910X552835. Epub 2011 Feb 3.
Dimethyl sulfoxide (DMSO), a commonly used cryoprotectant in cryopreservation procedures, is detrimental to viability of cells. In this view point, a comparative study was carried out to evaluate the effect of DMSO on porcine mesenchymal stem cells (pMSCs). We compared the viability, colony forming unit-fibroblast (CFU-F) assay, expression of Bak and Bcl2 genes, Bcl2 protein antigen, and CD90 in pMSCs cryopreserved with 5%, 10%, and 20% DMSO. pMSCs isolated from bone marrow were characterized by alkaline phosphatase activity and the expression of transcription factors, such as Oct 3/4, Nanog, and Sox2. The cells were then cryopreserved by cooling at a rate of -1°C/min in a programmable freezer and stored in liquid nitrogen. The results of survival of pMSCs cryopreserved at 5% DMSO were comparable to control group (fresh pMSCs). The survival and the number of colonies formed in cryopreserved pMSCs were inversely proportional to the concentration of DMSO. The number of colonies formed in pMSCs cryopreserved with all concentrations of DMSO was significantly (p < 0.05) lower than the control group. An increased tendency for Bak and Bcl2 gene expression was noticed in cryopreserved pMSCs at 3 h postthawing compared to control group. There was a close resemblance in higher level of expression of CD90 between control and cryopreserved pMSCs. Because there was no considerable difference in the results of pMSCs cryopreserved at 5% and 10% DMSO, this study strongly suggests the use of 5% DMSO in cryopreservation of pMSCs as an alternative to conventional 10% DMSO.
二甲基亚砜（DMSO）是冷冻保存过程中常用的一种细胞保护剂，对细胞活力有害。在这一观点下，进行了一项比较研究，以评估 DMSO 对猪间充质干细胞（pMSCs）的影响。我们比较了 5%、10%和 20%DMSO 冷冻保存的 pMSCs 的活力、集落形成单位-成纤维细胞（CFU-F）测定、Bak 和 Bcl2 基因表达、Bcl2 蛋白抗原和 CD90。从骨髓中分离的 pMSCs 通过碱性磷酸酶活性和转录因子（如 Oct 3/4、Nanog 和 Sox2）的表达来表征。然后，将细胞在可编程冷冻机中以-1°C/min 的速率冷却进行冷冻保存，并储存在液氮中。冷冻保存于 5%DMSO 的 pMSCs 的存活率与对照组（新鲜 pMSCs）相当。冷冻保存 pMSCs 的存活率和形成的菌落数量与 DMSO 的浓度成反比。用所有浓度的 DMSO 冷冻保存的 pMSCs 形成的菌落数量明显低于对照组（p<0.05）。与对照组相比，解冻后 3 小时冷冻保存的 pMSCs 中 Bak 和 Bcl2 基因的表达呈增加趋势。冷冻保存的 pMSCs 和对照组之间的 CD90 表达水平相似。由于 5%和 10%DMSO 保存的 pMSCs 结果没有明显差异，因此本研究强烈建议在 pMSCs 的冷冻保存中使用 5%DMSO 作为传统 10%DMSO 的替代物。
