Study On The Propagation Of Feline Panleukopenia Virus By Using F81 Monoclonal Cell Line After Domestication In Serum-free Suspension

Feline panleukopenia is prevalent worldwide,posing a major threat to pets,the breeding industry and wildlife.The disease has a very high infection rate and fatality rate,and the prognosis is poor.Vaccination is considered to be the most effective means of prevention and control.Antigen preparation is an important link in the vaccine preparation process,which directly affects the quality of the vaccine.At present,most biological products are cultured in roller bottles or microcarriers,resulting in low virus titers.Moreover,due to the use of serum,the product cost remains high,with large batch-to-batch differences,unstable quality,and easy contamination.From the perspective of industry development and product competitiveness,the application of suspension culture cell lines and the control of bioreactor culture parameters to carry out large-scale animal virus culture will help reduce costs and increase efficiency,improve product quality and the core competitiveness of enterprises.In this study,the monoclonal low serum suspension F81 cell line was obtained from the feline kidney cell（F81）line used for the production of feline panleukopenia vaccine by step-down serum method,limiting dilution method and suspension acclimation.Then,the culture parameters such as serum concentration,medium,cell passage density,shaking speed and freezing conditions were optimized.The results showed that the low serum monoclonal F81-18 cell line obtained by domestication,the low serum suspension cell culture serum concentration was 2%,the optimal passage density was 1×10⁶ cells/m L,and the cell culture reached the maximum density of5.83×10⁶ cells/m L after 72 h,and the cell viability was 92%.The optimal culture conditions of serum-free monoclonal strain F81-09 were:serum-free medium M-03-01,cell passage density 1.0×10⁶ cells/m L,shaking speed 120 r/min,and culture temperature 37℃.Under this culture condition,the serum-free monoclonal strain F81-09 reached the maximum growth density of 8.63×10⁶ cells/m L after 96 h of cell culture,and the cell viability was 90%.The optimal cryopreservation conditions for cells were that the cryopreservation density was 1.5×10⁷ cells/m L,and the composition of the cryopreservation solution was serum-free medium,fetal bovine serum and DMSO,and the corresponding ratio was 6:3:1.The key technical parameters of feline panleukopenia virus suspension culture were optimized and analyzed,and parameters such as cell inoculation density,MOI,and culture temperature were screened,and then the culture conditions and collection time were determined according to the change of virus titer.The culture parameters suitable for culturing feline panleukopenia virus by serum-free monoclonal strain F81-09 were screened by the control variable method.The results showed that the optimal culture process of feline panleukopenia virus was as follows:the simultaneous inoculation method was adopted,the cell density was 1.0×10⁶cells/m L,the MOI was 3.5×10^-3,and the shaking speed was 120 r/min.,cultured at 37°C.After culturing for 72 hours after inoculation,the titer of feline panleukopenia virus obtained by culture can reach a maximum of 10^7.6 TCID₅₀/0.1m L,and the antigen production vaccine was prepared,28 days after animal immunization,HI≥1∶1024.The culture parameters p H,dissolved oxygen concentration,stirring speed,and temperature were screened in a 15 L bioreactor.The results showed that the optimal culture conditions of serum-free monoclonal strain F81-09 in the bioreactor were p H=7.1,dissolved oxygen concentration of 50%,stirring speed of 80 rpm and temperature of 37℃.In conclusion,the serum-free suspension culture F81 cells established in this study can grow stably in a bioreactor,and can rapidly multiply viruses on a large scale,and the use of serum-free medium can significantly reduce production costs and improve cost-effectiveness.This paper reports that adherent F81 cells were successfully adapted to serum-free suspension culture,achieving the purpose of reducing costs and increasing efficiency,demonstrating their great potential in the production of related biological products.Using the optimized virus suspension culture process,the obtained virus liquid was used to prepare a vaccine to immunize domestic cats.The preliminary evaluation showed that the vaccine had good immunogenicity and safety.It provides basic data for the next large-scale production of feline panleukopenia vaccine in bioreactors,and provides reference and reference for the development of other biological products using F81 cells as cell substrates.