流感疫苗病毒株在MDCK细胞中的培养条件

Cultivation conditions of influenza vaccine virus strains in MDCK cells

目的 研究4价流感疫苗所用病毒株在MDCK细胞中的扩增条件.方法 在6孔细胞培养板中以不同感染复数(multiplicity of infection,MOI)(0.1000、0.0100、0.0010、0.0001)和对甲苯磺酰-L-苯丙氨酸氯甲基酮(TPCK)-胰蛋白酶(胰酶)浓度(0、2、4、8μg/ml)进行病毒接种和扩增,感染后72 h收获细胞上清液,检测病毒血凝素效价,确定病毒最佳扩增条件.随后,在搅拌瓶中进行MDCK细胞微载体悬浮培养,研究不同起始细胞接种密度(1.5×105、2.0×105、3.0×105个/ml)和微载体浓度(3、5、10 g/L)对MDCK细胞生长的影响,确定细胞最佳扩增条件.根据确定的最佳扩增条件,在搅拌瓶培养体系中扩增4种病毒.结果 6孔板中4种流感病毒的最佳接种条件分别是,A/Michigan/45/2015(H1N1)pdm09:MOI 0.0100、TPCK-胰酶浓度2μg/ml;A/Hongkong/4801/2014(H3N2):MOI 0.0100、TPCK-胰酶浓度4μg/ml;B/Brisbane/60/2008:MOI 0.0010、TPCK-胰酶浓度4μg/ml;B/Phuket/3073/2013:MOI 0.0100、TPCK-胰酶浓度4μg/ml.在搅拌瓶中以3.0×105个/ml初始细胞密度接种,3 g/L微载体浓度培养,MDCK细胞能够实现较好的扩增,最高密度可达2.1×106个/ml;搅拌瓶悬浮培养,以最佳接种条件接种后,4种病毒的血凝素效价为6.75~8.42 log2血凝素单位/50μl.结论 通过摸索病毒接种最佳MOI、TPCK-胰酶浓度及优化MDCK细胞微载体悬浮培养条件,能够在MDCK细胞微载体悬浮培养体系中有效扩增4价流感疫苗用病毒株,为后期工艺放大研究奠定基础.

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 × 10^5,2.0×10^5,3.0×10^5 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 2μg/ml;A/Hongkong/4801/2014 (H3N2):MOI 0.010 0,TPCK-trypsin 4μg/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 × 10^5 cell/ml and 3 g/L microcarrier,MDCK cells amplified well,achieving maximum cell density of 2.1 ×10^6 cell/ml.The HA titers of the 4 viruses were 6.75-8.42 log2 HA unit/50μl 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.