胰蛋白酶对Madin-Darby犬肾细胞消化方法的研究

目的:考察不同胰蛋白酶及消化后是否弃去胰蛋白酶对Madin-Darby犬肾(Madin-Darby canine kidney，MDCK)细胞消化效果的影响，并评价培养基中添加胎牛血清对减轻胰蛋白酶细胞毒性的作用。方法:选用5种胰蛋白酶分别消化MDCK细胞，消化5 min后弃去胰蛋白酶为弃去组，不弃去为保留组。观察消化...

Comparison of detachment effects by 5 trypsin for Madin-Darby canine kidney cells#br#

Objective  To evaluate the impact of different trypsins and removalt of trypsin after detachment to their detaching effect on Madin-Darby canine kidney(MDCK) cells, and the effect of fetal calf serum (FBS) in culture medium to reduce the toxicity of trypsin.  Methods  MDCK cells were detached by 5 kinds of trypsin and divided into 2 group. The trypsin was removed after 5 min in group-discard and not in group-retain. The time of the cells reaching different states were recorded. After the cells were completely detached, cells viability, cluster rate and diameter were measured. MDCK cells were detached by trypsin with different concentrations and divided into group-serum and group–non-serum according to whether FBS was added to culture medium, and the cells activity was evaluated.  Results The detachment time of recombinant trypsin was longer than that of animal trypsin. The mean cell viability of group-retain and group-discard were both greater than 94.77%, and the mean cell cluster rate were both greater than 34.56%. The mean diameter of group-retain and group-discard were 17.33 and 16.97 μm , respectively, with statistically significant difference (t=4.632,P<0.001) . There was a gradient change in cell activity with the concentration of trypsin. The cell activity of group-serum and group–non-serum was statistically significantly different (t=12.545,P<0.001).  Conclusions All the 5 kinds of trypsin can completely dissociate the adherent MDCK cells. The recombinant trypsin has a long detachment time and mild effect, and is suitable for production. Removing trypsin is feasible and beneficial. Concentration of trypsin residue effects cell activity. FBS can reduce the potential toxicity of trypsin.