Objective To balance and optimize four key control parameters,acid-base pH,viral spiking(MOI),TPCK trypsin spiking and cell density in MDCK cell suspension culture of influenza virus H3N2,in order to improve the productivity and virus titer of influenza virus H3N2 in MDCK cell suspension culture.Methods A multi-factorial interaction experimental design was created using MODDE®software of Design of Experiment(DoE)from Sartorius to determine and optimize the infectivity titer of H3N2 virus,considering the interactive effects of four factors:pH,MOI,TPCK trypsin concentration and cell density,aiming to achieve an optimal infectivity titer within a certain range of these four factors.Results The optimal ranges of MDCK cell suspension culture conditions for H3N2 virus were as follows:pH range of 7.88-8.00,MOI of 0.001,TPCK trypsin concentration range of 1.70 to 6.0 μg/mL,and cell density range of(55.70-77.28 × 105)cells/mL.The virus was maintained in Xene-S00l medium at a cultivation temperature of 34.5 ℃ for 72 h,with a shaking speed of 100 r/min and a CO2 concentration of 3％.Under these conditions,the TCID50 of H3N2 reached 6.5,and the CV of repeated experiments for TCID50 was 1.03％,with good reproducibility and stability.Conclusion H3N2 virus in MDCK cell suspension culture can achieve higher virus titer.
MDCK cellsSuspension cultureInfluenza virusDesign of Experiment(DoE)
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