胰蛋白酶对Madin-Darby犬肾细胞消化方法的研究

Study on detachment by trypsin for Madin-Darby canine kidney cells

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。观察消化过程,记录细胞达到不同状态所需的时间。完全消化后,检测细胞的活率、结团率、直径。以不同浓度的胰蛋白酶消化MDCK细胞,按培养基是否添加胎牛血清分为含血清和无血清组,检测细胞活性。结果重组胰蛋白酶消化时间长于动物源胰蛋白酶。保留和弃去组细胞活率均值都大于94.77%,结团率均值均大于34.56%,直径均值分别为17.33和16.97μm且差异有统计学意义(t=4.632,P<0.001)。细胞活性随胰蛋白酶添加量呈现梯度变化。含血清和无血清组的细胞活性差异有统计学意义(t=12.545,P<0.001)。结论5种胰蛋白酶均可完全解离贴壁MDCK细胞,其中重组胰蛋白酶消化时间较长,作用温和,比较适合生产。弃去胰蛋白酶再继续消化的做法可行且有益,胰蛋白酶浓度对细胞活性有影响,胎牛血清可以减轻胰蛋白酶的潜在毒性。

Objective To evaluate the impact of different trypsins and removal of trypsin after detachment to their detaching effect on Madin-Darby canine kidney(MDCK)cells,and the effect of fetal bovine serum(FBS)in culture medium to reduce the toxicity of trypsin.Methods MDCK cells were detached by 5 kinds of trypsins and divided into 2 groups The trypsin was removed after 5 min in group-discard and not in group-retain.The time of the cells reaching different states was recorded.After the cells were completely detached,cell 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 cell activity was evaluated.Results The detachment time of recombinant trypsin was longer than that of animal trypsin.The mean cell viabilities of group-retain and group-discard were both greater than 94.77%,and the mean cell cluster rates were both greater than 34.56%.The mean diameters of group-retain and group-discard were 17.33 and 16.97μm,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 activities of group-serum and group-non-serum were statistically significantly different(t=12.545,P<0.001).Conclusions All 5 kinds of trypsins 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 affects cell activity.FBS can reduce the potential toxicity of trypsin.