| Influenza caused by influenza virus, is a highly contagious acute respiratory illness of global importance. In a seasonal epidemic, it can spread worldwide and lead to a huge impact on the economy and society. The global burden of inter-pandemic influenza may be on the order of 1 billion cases, including 3~5 million cases of severe illness and 300,000~500,000 deaths annually. Vaccination is considered the most effective and cost-effective method to lower the mortality and morbidity associated with influenza.The use of continuous cell lines for influenza virus antigen production can be considered an attractive alternative to embryonated chicken eggs for several reasons. Firstly, influenza viruses propagated in animal cells usually resemble the original isolate more closely than do viruses propagated in eggs. Secondly, cell cultures are maintained easily and can be expanded in a relatively short time and therefore will allow initiation and scaling up of antigen production at any time. Finally, the use of animal cell derived influenza vaccines would overcome allergic reactions in vaccinated individuals sensitive to egg proteins. WHO encourages developing the technology of cell culture for influenza virus production.According to the present development of cell culture and virus production, we brought up a novel close and continuous process of cell culture for influenza virus production, containing cell culture, virus production and virus inactivation stages. By continuous operation mode, the process can produce large quantities of influenza virus in a relatively small space. Furthermore, the processes of cell culture and virus production are carried out in two different bioreactors, so the virus production process can be run in a relatively small space with a high safety level. That is to say, the process can be characterized by its high level of operational safety and low risk of virus leak, which is especially suitable for highly pathogenic and dangerous virus production.Some key techniques related to the close and continuous process were investigated by the application of H9N2 avian influenza virus with Vero cells, such as the susceptibility of cell to influenza virus, virus production with cell microcarrier culture method, cell bead-to-bead transfer, virus production through bead-to-bead transfer, cell culture and virus production with serum free medium, metabolism analysis, and repetitiously intermittent bead-to-bead transfer of cell for virus production to simulate the close and continuous process.Firstly, the susceptibility of Vero cell to influenza virus was investigated. After several subculture of H9N2 virus in Vero cells, Vero-adapted strain was obtained. HA titer was 2.2~2.3logHA unit/100μL with the addition of 1.5μg/mL TPCK-trypsin while pH 7.0~7.6.Microcarrier cell culture also was carried out for the scale up production of influenza virus. The optimal cell culture condition was found: inoculation density of 2.0×105cells/mL, Cytodex 3 concentration of 2g/L and stirring speed of 35rpm. Cell density increased to 9.7×105cells/mL 3 days after inoculation. Microscopic observation showed that most microcarriers were confluent with cells. Then the cells were infected with H9N2 virus with a MOI of 0.1 in DMEM medium. A maximum virus titer of 5.8 logPFU/mL was reached after 48h.p.i,when was suitable to collect virus for the live vaccine production. While maximum HA titer of 2.1~2.2logHA unit/100μL was reached after 60~72h.p.i, showing a suitable virus collection time for the inactivated vaccine production.The bead-to-bead transfer of Vero cells mainly relied on the formation of cellular bridges between confluent and fresh microcarriers, and 5μg ml-1 trypsin couldn't improve the cell bead-to-bead transfer between cytodex-3. In this experiment, Taguchi's method was firstly applied to identify critical parameters affecting the efficiency of bridge formation. It was found that two factors (stirring time and rest time) and two interactions (stirring time / rest time and stirring speed / rest time) had significant effects on the formation of cellular bridges between confluent and fresh microcarriers. To 2: 1 expansion (the ratio of fresh and confluent microcarriers), the percentage of bridged fresh microcarriers (PBFM) increased to about 21% after 8 h intermittent agitation, and the percentages of bare microcarriers (PBM) decreased to approximately 2% 6 day after the confluent and fresh mictrocarrier was mixed, with a cell density of 1.3×106 cells/mL. Then the Vero cells were infected with H9N2 virus, the HA titer increased to 2.1~2.2logHA unit/100μL 4d.p.i. It was shown that influenza virus production could be scaled up through simple bead-to-bead cell transfer.When Vero cells were cultured in EX-CELLTM Vero serum free medium, a higher seeding efficiency was obtained by Cytodex 3 more than Cytodex 1. The seeding efficiency increased to 53% after 8 h intermittent agitation (stirring intermittently at 35 rpm for 3 min every 30 min) with the addition of 200 mg/L CaCl2. Vero cells grew more slowly in this serum free medium than in serum containing DMEM, and reached stationary phase after 4 day. While there was no significant difference in HA titer between the two kinds of media. Furthermore, the HA titer still increased to 2.1logHA unit/100μL when medium exchange was omitted before infection, which was meaningful for the simplification of influenza virus production.In this experiment, repetitiously intermittent bead-to-bead transfer of cell was chosen to simulate the close and continuous process. During the transfer of 1:4 expansion (the ratio of fresh and confluent microcarriers) for 4 times, it was found that cell density remained stable, while the HA titer showed a slight decrease probably due to the bad culture environments.Finally, a close and continuous process of suspended cell culture for influenza virus was also supposed as well as the calculation models of corresponding kinetics parameters, which could be used as a reference for other cell lines and highly pathogenic and dangerous virus continuous culture and production.
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