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

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 results 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 min stilling culture.The optimal serum concentration for cells attaching to the Cephodex microcarrier 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 multiplicity of infection(MOI),the typical cytopathic effect appeared after infected for 3 days,the GCRV titer(LgTCID50/mL) reached about 8.5.This study established a solid basis for the large-scale preparation of vaccine against the grass carp hemorrhage.