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

Abstract

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.

Keywords: MDCK cells; RNA sequencing; flu vaccine; low-serum medium; proteomics.

Figure 1

Growth curves of MDCK cells in 15 cultures; VP:M199 = 1:2 (3%NBS, 2% NBS), VP:M199 = 1:3 (4% NBS, 3% NBS), and VP:M199 = 1:4 (3% NBS) cultures were selected for the validation of cell factory scale-up cultures (blue: control, red: experimental).

Figure 2

MDCK cell factories (CF1, CF2, CF10) enlarged culture validation results. ns Not Significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 3

MDCK cell culture continuous passaging stability ((A) cell growth status, (B) cell metabolism). ns Not Significant.

Figure 4

Growth curves of MDCK cells of different generations (A–D) Solid line: cell density, dashed line: cell viability and cell colony formation (E,F); numbered as serum content—cell generation—inoculum density, example: 3 (3% NBS)—65 (Cell generation)—1 (Inoculated at a density of 5 × 10⁴ cells/mL). ns Not Significant.

Figure 5

Effect of low-serum medium on the viral susceptibility of different generations of MDCK cells ((A) H1N1 virus susceptibility, (B) H3N2 virus susceptibility, (C) BV virus susceptibility, (D): BY virus susceptibility). (3% NBS)—65: (Serum content)-cell generation. ns Not Significant.

Figure 6

Results of transcriptomics and proteomics analyses. (A) Volcano plot showing DEGs of 3-uninfected group vs. 5-uninfected group. Red, blue, and black dots stand for genes with upregulation, downregulation, and non-differentiation, respectively. (B) Volcano plot showing DEGs of 3-infected group vs. 5-infected group. (C) Dot plot of 3-uninfected group vs. 5-uninfected group KEGG enrichment. (D) Dot plot of 3-infected group vs. 5-infected group KEGG enrichment. (E) Heatmap showing differentially expressed proteins between the 3-uninfected group and the 5-uninfected group. (F) Heatmap showing differentially expressed proteins between the 3-infected group and the 5-infected group. (G) Venn diagram showing the results of transcriptomic and proteomic correlation analysis between the 3-uninfected group and the 5-uninfected group. (H) Venn diagram showing the results of transcriptomic and proteomic correlation analysis between the 3-infected group and the 5-infected group.