微载体规模化培养草鱼细胞与病毒的工艺及优化

Technique and optimization for large-scale culture of grass carp cells and reovirus by microcarrier

研究了在悬浮培养系统中使用微载体Cephodex规模化培养草鱼肾脏组织细胞CIK和草鱼呼肠孤病毒(GCRV)的条件并进行了优化。结果表明,Cephodex是一种适合CIK细胞贴壁生长的微载体,搅拌方式(搅拌时间/静止时间)及血清浓度(0%、5%、10%、15%)对CIK细胞在Cephodex微载体上的贴附效率有影响。贴附期以转速35 r/min、每静置45 min搅拌2 min的间歇搅拌方式最佳,8 h后细胞贴附率可达90%以上;细胞贴附期培养基中的血清浓度为5%。当Cephodex微载体用量10 mg/mL、细胞初始接种密度2.5×105cells/mL、连续搅拌速度40 r/min时可获得最佳的细胞生长效能。用优化的最佳工艺条件大规模培养CIK细胞,接种感染复数为0.1的GCRV病毒3 d后,培养细胞出现典型细胞病变效应,病毒滴度(lgTCID50/mL)为8.5。本项研究为草鱼出血病疫苗规模化制备技术研究奠定了基础。

The techrique for large - scale culture of the grass carp kidney cells （CIK） and the grass carp reovirus （GCRV） were investigated and optimized in suspension culture system with Cephodex microcarrier. The resuhs showed that the Cephodex is a kind of microcarrier suitable for the growth of anchorage - dependent CIK cells, the agitation procedure （stirring time / stilling time） and the different serum concentrations （0% , 5%, 10% , 15% ）affected the efficacy of cell attachment on Cephodex microcarrier significantly. The attachment efficacy reached 90% after 8h cultivation with an intermittent agitation for 2 min at 35 rpm after 45 rain stilling culture. The optimal serum concentration for cells attaching to the Cephodex microearrier was 5%. The optimal growth result could be achieved while the cell seeding concentration was 2.5×105cells/ml and the microcarrier concentration was 10 mg/ml, and continuous stirring speed of 40rpm, respectively. After infection of the CIK cells on Cephodex microcarrier with GCRV at 0.1 of muitiplicity of infection （MOI）, the typical eytopathie effect appeared after infected for 3 days, the GCRV titer （LgTCIDs0/mL） reached about 8.5. This study established a solid basis for the large - scale preparation of vaccine against the grass carp hemorrhage.