Production, Passaging Stability, and Histological Analysis of Madin–Darby Canine Kidney Cells Cultured in a Low-Serum Medium

Madin-Darby canine kidney (MDCK) cells are commonly used to produce cell-based influenza vaccines. However, the role of the low-serum medium on the proliferation of MDCK cells and the propagation of the influenza virus has not been well studied. In the present study, we used 5 of 15 culture methods with different concentrations of a mixed medium and neonatal bovine serum (NBS) to determine the best culture medium. We found that a VP:M199 ratio of 1:2 (3% NBS) was suitable for culturing MDCK cells. Furthermore, the stable growth of MDCK cells and the production of the influenza virus were evaluated over long-term passaging. We found no significant difference in terms of cell growth and virus production between high and low passages of MDCK cells under low-serum culture conditions, regardless of influenza virus infection. Lastly, we performed a comparison of the transcriptomics and proteomics of MDCK cells cultured in VP:M199 = 1:2 (3% NBS) with those cultured in VP:M199 = 1:2 (5% NBS) before and after influenza virus infection. The transcriptome analysis showed that differentially expressed genes were predominantly enriched in the metabolic pathway and MAPK signaling pathway, indicating an activated state. This suggests that decreasing the concentration of serum in the medium from 5% to 3% may increase the metabolic activity of cells. Proteomics analysis showed that only a small number of differentially expressed proteins could not be enriched for analysis, indicating minimal difference in the protein levels of MDCK cells when the serum concentration in the medium was decreased from 5% to 3%. Altogether, our findings suggest that the screening and application of a low-serum medium provide a background for the development and optimization of cell-based influenza vaccines.