Effect of bioreactor scale-up culture of micro-carrier Vero cells after extra-tank trypsinization on virus-producing ability of rabies virus CTN-1V strain
Objective To investigate the effect of amplification culture of micro-carrier Vero cells from 30 L bioreactor to 300 L bioreactor after extra-tank trypsinization on the virus-producing ability of rabies virus(RABV)CTN-1 Ⅴ strain.Methods The 140-passage of Vero cells were cultured at 37 ℃ for 72-120 h,then amplified by passaging at a cell density ratio of 1:4 into the 10 x cell factory.After incubation at 37 ℃ for 72-120 h,the monolayer cells were detached and inoculated into the 30 L bioreactor with micro-carriers 7-10 g/L,culture temperature 37 ℃,pH 7.0-7.4,dissolved oxygen 30％-80％,stirring speed 10-50 r/min,and continuous perfusion culture 72-120 h.Total three batches of micro-carrier Vero cells were cultured,which were amplified to the 300 L bioreactor after extra-tank trypsinization,with microcarrier 5-8 g/L,culture temperature 37 ℃,pH 7.0-7.4,dissolved oxygen 30％-80％,stirring speed 30-80 r/min,and perfusion culture 72-120 h.RABV CTN-1 Ⅴ was inoculated at the MOI of 0.05,and the virus solution was harvested every 24 h and detected for the virus titer and antigen content.Results The cell density was about 1 × 107 cells/mL after culture for 96 h in the 30 L bioreactor,and was about 7.4 × 106 cells/mL after culture for 96 h in the 300 L bioreactor.At 96 h after virus inoculation,the virus harvest solution reached the peak potency,with the average virus titer of 6.8 lgLD50/mL and the average antigen content of 2.58 IU/mL.Conclusion The scale-up culture process of micro-carrier Vero cells after extra-tank trypsini-zation from 30 L bioreactor to 300 L bioreactor is stable and feasible,with no significant effect on the virus-producing ability of RABV CTN-1Ⅴ strain,which provides a reference for the large-scale production of inactivated RABV vaccine.
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