转瓶培养与生物反应器微载体培养重组腺病毒的比较

Comparison of roller bottle and bioreactor microcarrier culture of a recombinant adenovirus containing influenza

目的分别用3L转瓶和7L生物反应器微载体系统培养人胚胎肾细胞（HEK-293细胞）并接种H3型流感重组腺病毒,比较两种方法培养细胞及重组腺病毒的效果。方法分别用3L转瓶和7L生物反应器培养HEK-293细胞。转瓶每24h取样,生物反应器每12h取样,记录细胞总数。待细胞长满表面85%~90%时接种流感重组腺病毒,完全病变后收取病毒液并测定病毒滴度。结果转瓶培养HEK-293细胞3d,细胞增殖增加1.85~2.45倍,为（3.71~4.90）×107个[（2.95~3.90）×104 cells/cm2],生物反应器培养3d,细胞增长4.72~5.00倍,为（132.15~140.09）×107个[（24.88~28.72）×104cells/cm2]。转瓶培养重组腺病毒滴度为6.85TCID50/ml,生物反应器微载体系统培养腺病毒滴度为7.200TCID50/ml。结论生物反应器微载体系统培养流感重组腺病毒数量和滴度均高于转瓶培养,不但提高了疫苗的产量,还减少了细胞分泌物的残留,增强了疫苗接种的安全性。

Objectives To use 3-L roller bottles and a 7-L bioreactor system to culture a recombinant adenovirus containing H3 influenza in human embryonic kidney cells（HEK-293）and to compare the effectiveness of the two methods of culturing cells and a recombinant adenovirus. Methods HEK-293 cells were cultured in 3-L roller bottles and a 7-L bioreactor system.Inoculation density was 1.6×104 cells/cm2.The total number of cells in a roller bottle was recorded every 24 hours and the number in the bioreactor was recorded every 12 hours.When cells covered 85%-90% of the available surface,cells were inoculated with the recombinant adenovirus and the viral titer was determined. Results After 3days of culturing HEK-293 cells in roller bottles,cell growth increased 1.85-2.45-fold,ultimately reaching 3.71-4.90×107cells（2.95-3.90×104 cells/cm2）.After 9days,cells covered the entire wall of the bottle,and the total cell count reached 31.25-36.07×107cells（24.88-28.72×104 cells/cm2）.The cell count increased 4-fold.The growth of HEK-293 cells in the bioreactor system increased 4.72-5.00-fold and the total cell count increased to 132.15-140.09×107cells（24.88-28.72×104 cells/cm2）.The titer of the recombinant adenovirus was 6.85TCID50/ml in roller bottles and7.20TCID50/ml in the bioreactor system. Conclusion After 3days,the density of HEK-293 cells cultured in the bioreactor system was 29 times that in roller bottles.The proliferation of adenovirus in the bioreactor system was 6.4times that in roller bottles.The recombinant adenovirus cell count and the titer were higher in the bioreactor system.The approach used here could increase production of a vaccine,reduce cell secretions,and increase the safety of vaccination.