鲫细胞与病毒微载体规模化培养工艺研究

本文研究了在Cephodex微载体悬浮培养系统中规模化培养鲫脑组织细胞(Gi CB)和鲤疱疹病毒Ⅱ型(Cyprinid herpesvirusⅡ,CyHV-2)的工艺。结果表明,Cephodex微载体适合GiCB细胞的贴壁培养,细胞贴壁期培养基中血清浓度为10%,微载体浓度为6 g/L;细胞初始接种密度为2.5×10~5cells/m L时,以转速35 r/min,每静置30 min搅拌2 min的间歇搅拌条件贴壁率最佳,8 h后贴壁率可达90%以上;增殖期以45 r/min的连续搅拌速度可获得最佳的细胞生长效能。采用优化的工艺条件,以感染复数为0.2的CyHV-2病毒接种规模化培养的GiCB细胞5 d后,出现典型的细胞病变效应,病毒滴度(TCID_(50)/m L)可达10~(6.50±0.30)。本项研究为鲫造血器官坏死症疫苗的规模化制备技术研究奠定了前期基础。

Studies on the technology for large-scale cultivation of gibel carp brain cells and cyprinid herpesvirus 2 by microcarrier

In this study, the technology for the large-scale cultivation of gibel carp brain cells and CyHV-2 by Cephodex microcarrier in suspension system was investigated. The results showed that the Cephodex microcarrier is suitable for the growth of anchorage-dependent GiCB cells. The optimal serum concentration for cells attaching to the Cephodex microcarrier was 10%, the microcarrier concentration was 6 g/L and the density of cells inoculated was 2.5×10⁵ cells/mL. With an intermittent agitation for 2 min at 35 rpm after 30 min stilling culture, the attachment efficacy reached 90% after 8 h cultivation; while in growing period, the optimal speed was 45 r/min for continuous agitation. After infection of GiCB cells on Cephodex microcarrier with CyHV-2 at a multiplicity of infection (MOI) of 0.2, the typical cytopathic effect (CPE) appeared after 5 days post-infection and the virus titer (TCID₅₀/mL) reached 10^6.50±0.30. This study established a solid basis for the large-scale preparation of vaccine against the crucian carp hematopoietic necrosis.