无血清微载体培养Vero细胞和H1N1流感病毒条件的优化

目的优化无血清微载体培养Vero细胞和H1N1流感病毒的条件,为Vero细胞流感疫苗的开发奠定基础。方法在搅拌瓶中采用不同的细胞接种量和不同的微载体浓度培养Vero细胞,制备流感病毒,并检测不同的pH值、TPCK-胰酶含量、病毒接种量及病毒收获时间对流感病毒血凝滴度的影响。结果以(1.0～5.0)×105个/ml的Vero细胞接种至浓度为3mg/ml的无血清微载体中,病毒培养液pH值为7.2～7.4,TPCK-胰酶含量为1.0μg/ml,病毒接种量为1.0MOI,并在感染48h后补加TPCK-胰酶,培养72h后收获上清与细胞内病毒,血凝滴度可达1:512。结论已获得了无血清微载体培养Vero细胞和H1N1流感病毒的适宜条件,为应用生物反应器大规模制备流感病毒奠定了基础。

Optimization of Condition for Serum-free Culture of Vero Cells and Influenza A (H1N1) Virus on Microcarriers

Objective To optimize the conditions for serum-free culture of Vero cells and influenza A(H1N1)virus on microcarriers and lay a foundation of developing Vero cell-based influenza vaccine.Methods Vero cells were inoculated at various concentrations and cultured on microcarriers at various concentrations in stirring flask to prepare influenza virus.The effects of pH value,TPCK-trypsin content,MOI of virus inoculated and time for harvest on hemagglutinatin(HA)titer were evaluated.Results The condition for culture of Vero cells and influenza virus was optimized as follows:(1.0 ～ 5.0)× 105 Vero cells were inoculated onto serumfree microcarriers at a concentration of 3 mg /ml,and cultured in virus medium at a pH value of 7.2 ～ 7.4 and a TPCK-trypsin content of 1.0 μg /ml,then inoculated with influenza virus at a MOI of 1.0.TPCK-trypsin was supplied 48 h,and the virus in supernatant and cells were harvested 72 h after culture.The titer of influenza virus prepared under the optimal condition reached 1:512.Conclusion The condition for serum-free culture of Vero cells and influenza A(H1N1)virus on microcarriers were optimized,which laid a foundation of large-scale preparation of influenza virus by using bioreactor.