马皮肤成纤维细胞的体外培养与冷冻保存

Culture in Vitro and Freezing of Horse Skin Fibroblasts

本研究利用小组织块直接培养法得到了成年马皮肤成纤维细胞的原代培养物,再用酶消化法处理,能够纯化马皮肤成纤维细胞。成纤维细胞的冷冻保存,首先采用手工冷冻法,选用含有不同浓度保护剂如:二甲基亚砜(DMSO)、乙二醇(EG)、甘油(GC))和新生牛血清(NCS)的12种冷冻液对马皮肤成纤维细胞进行冷冻保存;其次用2种冷冻方法对马皮肤成纤维细胞进行冷冻保存,以24h贴壁率评价冻存效果。结果表明,10%DMSO和20%NCS的DMEM冻存液,对马皮肤成纤维细胞的冻存效果好(24h贴壁率84.98%)。从冷冻方法来看,程序冷冻法(86.32%)优于手工冷冻法(79.98%)(P<0.05)。

The primary cultured cells of horse skin tissue were obtained by explant tissue culture and were purified by enzymes digest. Firstly, Horse skin fibroblasts were frozen in 12 different freezing protective media, which contained different concentration of DMSO, EG, GC and NCS. Secondly, Horse skin fibroblasts were frozen in two different cryo-preservation methods. Efficiency of attachment at 24 h was used to evaluate the effects of cryo-preservation. The results of experiment showed that DMEM supplemented with 10 % DMSO and 20 % NCS was the most efficient cryo-preserving media （efficiency of attachment at 24 h after thawing, 84.98%）. The effects of cryo-preservation in programming-cryo-preservation were obviously better than those in manual cryo-preservation （86.32%, 79.98%）（P〈0.05）.