Objective To create a high density MRC-5 cell culture method by exploring the microcarrierbased cell culture conditions.Methods MRC-5 cells (3.0×104/mL) were cultured in three different media (MEM,M199,DMEM) and their proliferation and passage capacity were observed.In Spinner culture system,MRC-5 cells were cultured at the density of 3.70×105/mL and 2.98×105/mL with 1.0 g/L Cytopore 2 and 3.0 g/L Cytodex 3 microcarriers,respectively.The effect of the two microcarriers on cell growth,metabolism and cell density was recorded to screen out the most appropriate medium and microcarrier.The 5 L CelliGen310 bioreactor was used to explore the MRC-5 perfusion culture condition by the established medium and microcarrier.Results After 96 h cultured in MEM,M199 and DMEM media,MRC-5 cells were proliferated 7.0,4.4,and 3.0 times,respectively.MRC-5 cells cultured in MEM medium generated more than 5 passages and formed dense monolayer after 72 h with passage ratio 1:3,while MRC-5 cells in M199 and DMEM media failed to obtain the ideal cell morphology during the passage.After 96-120 h cultured in Spinner system,the concentration of MRC-5 cells in 1.0 g/L Cytopore2 was higher than that in 3.0 g/L Cydodex3 (2.20×106/mL vs 1.12×106/mL),with more active metabolism of glucose and lactic acid.MEM and Cytopore2 were selected as the MRC-5 perfusion culture system.The concentration of MRC-5 cells reached 1.54×106/mL when cultured with 1.0 g/L Cytopore2 and 2.5 L MEM in 5 L bioreactor for 144 h.Conclusions The initial establishment of cell culture method based on Cytopore 2 microcarrier and MEM medium can obtain high density MRC-5 passage cells with good viability.