Cultivation conditions of influenza vaccine virus strains in MDCK cells

Abstract

Objective To study cultivation conditions in MDCK cells for 4 virus strains in a quadrivalent influenza vaccine. Methods Virus inoculation and amplification were performed in 6-well cell culture plates with different mutiplicities of infection (MOI)(0.100 0, 0.010 0, 0.001 0, 0.000 1)and tosyl-L-phenylalanine chloromethyl-ketone (TPCK)-trypsin concentrations (0, 2, 4, 8 μg/ml). Virus hemagglutinin (HA) titers in cell supernatants 72 h post infection were then detected to determine the optimal virus expansion conditions. MDCK cells were cultivated in stirred bottle with different inoculation cell densities (1.5×105, 2.0×105, 3.0×105 cell/ml) and microcarrier concentrations (3, 5, 10 g/L) to determine the optimal cell expansion conditions. The optimal conditions determined were used to amplify the 4 virus strains in stirred bottles. Results The optimal virus inoculation conditions in 6-well plate were, A/Michigan/45/2015(H1N1) pdm09: MOI 0.010 0, TPCK-trypsin 2 μg/ml; A/Hongkong/4801/2014(H3N2): MOI 0.010 0, TPCK-trypsin 4 μg/ml; B/Brisbane/60/2008: MOI 0.001 0, TPCK-trypsin 4 μg/ml; B/Phuket/3073/2013: MOI 0.010 0, TPCK-trypsin 4 μg/ml. When cultured in stirred bottle with initial density of 3.0×105 cell/ml and 3 g/L microcarrier, MDCK cells amplified well, achieving maximum cell density of 2.1×106 cell/ml. The HA titers of the 4 viruses were 6.75-8.42 log2HA unit/50 μl after inoculation under optimal conditions. Conclusion By exploring the best MOI, TPCK-trypsin concentration and optimizing the suspension culture conditions of MDCK cells, the quadrivalent influenza vaccine virus strains can be effectively expanded in the microcarrier-based MDCK cell suspension culture system. The results lay foundation for future amplification process development. Key words: Influenza vaccines; Quadrivalent; Madin-Darby canine kidney cell; Microcarrier