PK-15细胞在微载体悬浮放大培养及PCV2增殖工艺研究

Scale-up Process of Microcarrier Suspension Culture of PK-15 Cells and Propagation of PCV2

为建立在生物反应器内微载体逐级放大培养PK-15细胞和增殖PCV2技术。采用分批式培养方法,研究了PK-15细胞在微载体胰酶消化放大悬浮培养及猪圆环病毒2型(PCV2)增殖工艺。结果表明,5 g/L的微载体和高糖DMEM下,采用4.0×105cells/m L的初始接种密度及培养至第2天进行换液操作工艺可以获得最佳的PK-15细胞生长效能。胰酶消化转移将PK-15细胞从1 L反应器放大至3 L反应器,微载体上细胞贴附均匀、生长旺盛,培养3 d细胞密度可达34.3×105cells/m L。采用感染复数为0.1的接毒比例,细胞接毒后在微载体上传至第3代收获毒液可获得最高的PCV2增殖效价107.25TCID50/m L。为PCV2疫苗的生物反应器规模化生产奠定了基础。

In order to develop a technique for scale-up of microcarrier suspension culture of PK-15 cells and propagation of PCV2 in bioreae- tor,batch-wise suspension culture of PK-15 cells on microcarriers （ cytodex 1 ） using scale-up of tripsin digest and propagation of porcine cir- covirus type 2 （PCV2） were investigated. The results showed that the optimal growth efficiency could be achieved by 4.0 × 10^5 cells per 1 mL inoculation on 5 g/L concentration, DMEM medium with high glucose and media replacement after 2-day culture. The highest PCV2 titer was achieved 10^7.25TCIDso/mL at the 3^rd passage of PK-15 cells infected with 0.1 multiple of infection （MOI） of PCV2 The optimal processes achieved in this study laid the experimental foundation for mass production of PCV2 vaccines in bioreactor scale.