Development Of Serum-Free Single-Cell Suspension Culture System Of MDCK Cells For The Production Of Influenza Vaccines

In recent years, world health and animal products industry suffered huge losses every year because of influenza viruses. Traditionally, influenza virus isolation and production often use chicken embryonic eggs. However, chicken embryonic eggs may contain various microbiological contamination and residual endotoxin resulting in the disadvantages. Anymore, this production process has poor influenza pandemic emergency response capability. So it's very important to develop satisfactory mammalian cell systems for vaccines production. Since MDCK cell line multiplies fast and is easy to be scaled up, and influenza virus can infect MDCK cells with high efficiency, it is considered to be one of the most important cell lines for influenza vaccines production.In this work, a serum-free medium MDCK-SFM for culturing of MDCK cells was developed successfully after exploring cell growth characteristics and screening serum-free additives. With this serum-free medium had the similar effects on cell specific growth rate, viability and density to the serum-containing medium. Next, we developed a serum-free medium MDCK-SHP and a low protein containing serum-free medium MDCK-SLP, which support high-density single-cell suspension culture of MDCK cells. And then, a cell line ssf-MDCK suitable for single-cell suspension culture in serum-free media was obtained. In MDCK-SHP, cell growth density and maximum specific growth rate could reached 38.05×105 cells/mL and 0.056 h-1, respectively. And for MDCK-SLP, their values were 22.94×105 cells/mL and 0.050 h-1, respectively. Further, the production conditions were optimized preliminarily with the MDCK cell suspension culture system. During the process of influenza virus production, medium changing was not necessary, but the addition of TPCK-trypsin was indispensable. The optimal TOI and MOI were 50 h and 0.1 respectively, and peak TCID50 titers of 1010.85±0.81 TCID50/mL and 1011.43±0.73 TCID50/mL reached in this process at 48 h post infection in MDCK-SHP and MDCK-SLP respectively. These results were important to provide technique and theory basis for developing serum-free single-cell suspension MDCK cell culture and large-scale influenza vaccine production processes.