牛皮肤成纤维细胞的体外培养与冻存

In vitro Cultivation and Freezing of Bovine Skin Fibroblast Cells

利用牛皮肤组织块直接培养法 ,得到牛皮肤细胞的原代培养物 ,再用酶消化法和反复贴壁法处理 ,能够纯化成纤维细胞。成纤维细胞的冻存是通过选用 6种分别含有二甲基亚砜( DMSO)、甘油 ( GL)及乙二醇 ( EG)的保护液 ;以相同的冻前处理方法 ,对牛皮肤成纤维细胞进行缓慢冷冻 ,冰箱预冷平衡 1～ 2 h,逐步投入液氮 ( - 1 96℃ )中保存 ,再经 37℃水浴解冻 ,Hanks液脱保护剂 ,以贴壁率评价冻存效果。结果表明 ,2 0 % DMSO保护液对牛皮肤成纤维细胞表现出较好且稳定的冷冻保护效果 ,其平均贴壁率达 87.9%。

Bovine skin tissue was cultivated with direct cultivation methods to get primary culture substance, then it was treated with enzymes digestion and attaching treatments to purify bovine fibroblast cells. Pure bovine fibroblast cells were frozen in the same procedures and with 6 different protective media, which contained DMSO(10%,20%),glycerin(10%,20%) and ethylene glycol(10%,20%),respectively. Fibroblast cells were gradually frozen in refrigerator and balanced for 1 to 2 hours and put into liquid nitrogen for preservation. In order to evaluate the effects of the different freezing protective media, the frozen cells were thawed with water bath ,deprived of protective media with Hanks liquids and cultivated to detect the attachment percentage. The results suggested that the freezing protective effects of the media containing 20% DMSO were the best, the attachment percentage of it reached 87.9 percent.