贴壁及悬浮培养的Trex-293细胞生长及其重组腺病毒制备产率的比较分析

摘要

为了提高病毒产率并避免制备的重组腺病毒产物中残留的动物血清,将贴壁生长的Trex-293细胞用无动物血清的CD293培养基在搅拌式培养瓶中悬浮培养.分析比较其生长特征及不同条件下的病毒产率.结果表明悬浮培养的细胞生长为稳定状态的时间比贴壁培养时稍长,但每次以(4-6)×105 cells/mL密度传代.(3-4)d后基本达到2×106 cells/mL,21 d达3×106 cells/mL以上,比贴壁细胞密度约高3倍.培养初期,悬浮细胞存活率稍低,培养14 d开始维持90％以上,细胞倍增时间约32 h.而贴壁细胞存活率是基本维持在95％以上.重组腺病毒以200,500和1000 VP/cell的比例感染悬浮细胞所获产率均值各为18000,14000和990 VP/cell,而1000 VP/cell的比例感染贴壁细胞所获产率均值为10500 VP/cell.结论为Trex-293细胞用无动物血清的培养基在搅拌式培养瓶中可悬浮培养,当重组腺病毒感染比例为200 VP/cell,感染后48 h,细胞存活率50％时收获有利于提高病毒产率,且高于感染贴壁细胞所获产率.%In order to avoid detection of residual animal serum and improve recombinant adenovirus productivity in the preparation of recombinant adenovirus production, Trex-293 human embryonic kidney adherent cells adapted to growth in suspension using animal serum-free CD293 medium in spinner flask and observed the growth patterns and virus yields in different conditions. The experimental results show that adherent cultured Trex-293 cell is able to be adapted to suspension culture with serum-free CD293 medium in spinner flask. Initial growth time to a stable state is longer than that of adherent cell culture, but since then they rapidly growth. The cell viability was low at early stage, maintained 90% viability at 14 d after culture. The suspension cells seeded at (4—5) x 105 cells /mL reached 2 X 106 cells/mL by (3—4) d of culture, and reached 3 X 106 cells/mL at 21 d after culture, doubling time was about 32 h. The yield of recombinant adenovirus infected in suspension cells with ratio of 200 VP/cell and cell densities of 2 x 106 cells / mL is higher than that of infection ratio of 500 VP/cell and 1 000 VP/ cell, and it also higher than that of in adherent cells. These results conclude that adherent Trex-293 can be adapted to growth in suspension conditions with serum-free CD293 medium in spinner flask. For recombinant adenovirus infects in suspension cells, it helps to improve the yield of virus and the yield also higher than adherent cells under the condition of an infection ratio of 200 VP/cell, harvest at 48 h post-infection and cell viability of 50%.