Development And Optimization Of ST Cells-derived Classical Swine Fever Vaccine Based On Low Serum Microcarrier Suspension System

Classical swine fever is a highly contagious disease caused by swine fever virus, vaccinating is the main measure to prevent swine fever. The traditional CSFV production process is hard to meet the stable quality and huge demand in a short period for the reason of process complexity and long production cycle. Thus, there is an urgent to develop and optimize low serum microcarrier suspension culture system based on ST cells, which is an promising approach to laid the foundatio for further large-scale production of cell source classical swine fever vaccine for its high efficiency and low cost.In this paper, six backup serum-free medium with extra 1% FBS were mixed according to Design of Experiment. ST cells could continuous and stably passage in the LSM at higher growth rate (0.626 d^-1) than in the original medium. Further, studying the influence of microcarrier concentration and cell seeding density on cell growth. The results showed that the adequated adherent area could be supplied using 3×10⁵ cells/ml ST cell seeding density and 3 g/L Cytodex 1 congcentration, while the microcarriers utilization reached 44.4%. Maximum cell density rose to （2.94±0.30）×10⁶ cells/ml,which was 1.77 times of that using original medium, amplifying 8.80-fold. The cell density reached to 27.7×10⁵ cells/ml in 3 L Sartorius bioreactor which agitation speed was set to 50 rpm after calculating the reasonable scope.Secondly, the production process of CSFV based on LSM in T-flask was studied. The results showed that the virus gene concentration reached to （26.68±0.73）×10⁷ copies/ml, which was 1.90 times of DMEM according to RT-qPCR detection method. However, the results of the rabbit immune detection method showed the titer was not as high as the RT-qPCR method showed, some defects in the structure of CSFV might be the cause. Aiming to maintain the rapid changes of pH during the CSFV production process, different pH regulation methods were applied and significantly optimized the pH while the virus production was not significantly improved. Besides, the CSFV content only showed 5.6% difference after shifting temperature from 37℃ to 35℃. And the amount of CSFV would decrease by 50% after repeating freezing and thawing for 5 times.Thirdly, the influence of MOI, TOI and intermittent agitation modes on virus production were investigated. The results showed that infected ST cells at 120 h with 15%（v/v） MOI could reach the highest CSFV yield which was （4.55±0.46）×107 copies/ml, and titer could also meet the rabbit immune detection method. Below 15% MOI, CSFV could not effectively take advantage of the high cell density for replication and amplification, and 30%MOI would be harmful. In addition, the yield of CSFV under different MOI condition increased with the delay of TOI, indicateing that inoculating CSFV during plateau period would be more favorable to generate offspring virus in short time.Stopped stirring for 10 mins every 20 mins during the 12 hous post infection had a promoting effect on the proliferation of CSFV. Finally, only the first harvest of CSFV was up to the standard of both rabbit immune detection method and RT-qPCR method under the operation of changing all the medium every three and four days, all the other harvests’virus content decreased with the increasing of harvest times.Oxygen-supplying ability might be the cause under the condition of 50 rpm by theoretical analysis.Taken together, a low serum medium suitable for ST cell adherent growth was established and some key factors in connection with cell growth and virus proliferation were investgated, which lays foundation for large-scale low serum microcarrier suspension system producing ST cell source CSFV vaccine.