大鼠肾细胞分离制备及培养方法的比较

目的比较胶原酶消化法、胰蛋白酶消化法和胶原酶、胰蛋白酶两步消化法(简称两步消化法)对大鼠肾细胞分离效果及细胞产量的影响,以及不同培养基(MEM、RPMI1640)、鼠龄(乳鼠、成年鼠)对细胞产量、质量、增殖率和乳酸脱氢酶(LDH)漏出量的影响。方法分别对两组Wistar大鼠(乳鼠和成年鼠)用三种不同的消化方法制备大鼠肾组织细胞悬液,分别在MEM培养液和RPMI1640培养液中原代培养,培养48小时之后第一次传代,观察细胞质量,计算细胞产量,MTT法检测传代细胞24~48小时之间的增殖率,生化分析仪检测传代细胞第1~7天培养液中LDH漏出量。结果胶原酶消化法和两步消化法的肾细胞产量显著高于胰蛋白酶消化法(P<0.01),两步消化法的细胞质量优于胶原酶消化法,MEM培养液和RPMI1640培养液对细胞产量、增殖率和LDH漏出量的影响均无显著性差异,乳鼠的肾细胞产量显著高于成年鼠(P<0.01)。结论两步消化法对于肾细胞的分离制备尤为适合,MEM培养液和RPMI1640培养液均适于肾细胞的培养,乳鼠比成年鼠更适合肾细胞的制备。

Comparison of isolation, preparation and culture methods of rat kidney cells

Objective To compare the isolation effects and yields of rat kidney cells by three different digestion methods (collagenase, trypsinase, and the two step digestion method), the yields, proliferation rate, and LDH leakage in two different media (MEM and RPMI1640 media) in rats in two different ages (suckling and adult). Methods The renal cortex from the suckling and the adult rat group was digested by three different methods, respectively, and the suspension was separately cultured in MEM and RPMI1640 media. After 48 hours, the cells were digested and counted to calculate the yields, and the proliferation rate between 24h and 48h was examined by MTT examination, and the leakage of LDH in the media was measured by biochemical analyzer every day for 7 days. Results The yields of kidney cells by collagenase and the two_step method were significantly higher than by trypsinase method (P<0.01), the suckling group were significantly higher than the adult group. The isolation effect of the two_step method was better than the collagenase method. The yields, proliferation rate, and LDH leakage of cells in MEM and RPMI1640 media had no significant difference. Conclusion The two_step digestion method was the most appropriate method for the preparation of kidney cells. Both MEM and RPMI1640 media were suitable for the culture of kidney cells. The suckling rat was proper than the adult for kidney cell preparation.