Development And Application Of Maintenance Medium For H9N2 Influenza Virus Production Based On MDCK Cell Suspension Culture

Avain influenza pandemic is a serious threat to poultry and human health,and causes enormous economic losses to society.Currently,vaccination is the most effective method to prevent avain influenza.The suspension cell culture in serum-free medium has significant advantages in large scale production,process stability and process control,being the main development trend of influenza vaccine production.Facing the current situation of the avain influenza vaccine market in short supply,how to apply the serum-free single-cell suspension culture technology to improve the production of influenza virus is particularly important.In this culture process,serum-free medium is one of the most critical factors revelant to the production of influenza virus.However,the serum-free medium currently relies on foreign imports,the cost is expensive.Moreover,the formula is confidential,thus it is not conducive to optimize the process.In addition,the virus production characteristics are different from the cell growth,while in research or industrial production,the maintenance medium used in the virus production stage is mostly the cell growth medium,this imbalance in nutrient components limits the efficient amplification of influenza viruses.Therefore,it is urgent to develop a maintenance medium suitable for influenza virus production in the specific virus production stage.Therefore,this research took MDCK cells and H9N2 avian influenza virus as the research object.Firstly,based on MDCK cell suspension culture process,three mathematical statistical methods,including Simplex Lattice,Placektt-Burman and central composite design experiments were applied in order.As a result,Opt-MM medium that supported the efficient amplification of H9N2 influenza virus in MDCK cells was obtained.Next,in order to establish the virus amplification process suitable for large scale production in the bioreactor,a 1:1 dilution process was optimized,and then the process was employed in a 2 L bioreactor.Finally,the maximum virus titer of Opt-MM medium was 214 55 HAU/100?l,and the cell specific virus yield was 51888.11 virions/cell,which were about 1.22 and 1.26 times higher than that of cell growth medium(XP medium),respectively.The cellular metabolic characteristics of this high-yield process were further studied.Compared with XP medium,cells under the high-yield medium not only enhanced the glycolysis,but improved the specific consumption rate of some amino acids such as Ser,Arg,Val,Ile,Leu,His,Cys,Phe,Lys,Met,His,Tyr,Thr and Gly,and reduced the specific generation rate of Ala.Finally,the maintenance medium was well verified in different culture systems,different trypsin,different strains and cell passage.In conclusion,a maintenance medium based on MDCK cell suspension culture to amplify H9N2 influenza virus was developed,which laid a foundation for the industrial production of avian influenza vaccine;the cell metabolism during virus infection based on the medium was further studied,which provided guidance for the development of viral vaccines for other cell lines.