Studies On Proliferation Of Influenza A(H3N2) Virus By MDCK Culture In Bioreactor

Influenza,an acute respiratory infectious disease caused by influenza virus.Influenza virus belongs to Orthomyxology virus and can be divided into three types:A,B and C.Influenza A H3N2 virus is the most common seasonal influenza virus and is also the main component of multivalent influenza vaccines.Influenza vaccines are mainly traditional vaccines produced by chicken embryo culture method.Although this traditional production process is mature,it has the disadvantages of unstable yield,long production cycle,easy variation of virus,easy to cause allergic reactions,etc.Compared with chicken embryos,large-scale bioreactor culture technology is simple to operate and easy to control,has short production cycle and high degree of automation,can ensure vaccine quality and safety.MDCK cells are currently recognized as sensitive to influenza virus.In this study,we used the MDCK cells which are stored in GansuEngineering and technology research center for animal cell.In accordance with the general requirements of the pharmacopoeia of the People's Republic of China(three parts) for the production of host cell lines,a qualified MDCK cell bank is established through identification and selection of growth characteristics,exogenous pollution and genetic characteristics.The resuscitation activity of MDCK cells was 96%,and the cells cultured for 48h after resuscitation grew into dense monolayer with triangular and irregular polygon shapes,which were basically consistent with the morphology before cryopreservation.The growth curve presented an‘S'shape,with the maximum proliferation density of 5.45×10~5 cells/mL and the doubling time of 23.2 h,and the morphology and growth rate keep the same for 10 generations of continuous culture.Not contaminated by fungi,bacteria,mycoplasma and internal and external viral factors.Chromosomes number are between 78 and 82.The most suitable medium and carriers for MDCK cells was selected through growth characteristics.The results showed that,the multiplication time of MDCK cells cultured in DMEM medium was 20.2 h,and the maximum proliferation density was 9.58×10~5 cells/mL.The cells cultured for 10 generations grew well,which was superior to DM/F12,MEM and M199 medium;MDCK cells were cultured with Cytodex-1 microcarriers and flaky carriers.The results showed that both the Cytodex-1 microcarriers and the flaky carriers could support the growth of MDCK cells.However,in the same volume of bioreactor,the highest cell densities of MDCK cells were culturedwith the flaky carriers were 6.18×10~6 cells/mL were obviously higher than the highest cell densities of MDCK cells were culturedwith the Cytodex-1 microcarriers.Therefore,the flaky carriers was selected to culture MDCK cells in this study for later experimental research.The optimal TPCK trypsin concentration and MOI of MDCK cells cultured with bioreactor flaky carriers for high density proliferation of influenza A H3N2 virus are 2.5?g/mL and 0.01 respectively.When the harvested virus liquid is used to produce influenza vaccine,the appropriate time for virus collection is 72 hours.When the obtained virus liquid is used as the seed virus for further amplification of influenza virus,the appropriate time for virus collection is 60h.Summarizing the research work,an efficient process for influenza vaccine production was established.It lays a foundation for industrial production of influenza vaccine based on the large-scale animal cell culture.