生物反应器内微载体培养细胞的胰酶消化放大技术

A technique for scale-up of trypsinization of cells cultured on microcarrires in bioreactor

目的建立生物反应器内微载体上Vero细胞球转球胰酶消化放大技术。方法用500 ml搅拌瓶培养Vero细胞,待细胞浓度达1.8×106个/ml时,加入25和37℃消化液（0.25%胰酶＋0.02%EDTA）消化不同时间,计算细胞回收率及细胞活率,筛选微载体细胞最佳培养温度及消化时间。将3 L反应器内微载体及T25方瓶培养的Vero细胞消化后接种T25方瓶,比较两种传代方式下的细胞形态及比生长速率。将3 L反应器内微载体培养的Vero细胞用消化液消化后,按1∶3、1∶6、1∶8、1∶10、1∶12的比例接种至15 L反应器,确定最适接种比例。将转瓶及3 L反应器内微载体培养的Vero细胞消化后接种至15 L反应器,比较两种放大方式下的细胞形态、密度及比生长速率。结果微载体上培养的Vero细胞经25℃消化15 min可有效保证细胞的回收率及活率。两种传代方式下的细胞形态及比生长速率无明显区别。细胞比生长速率在1∶6~1∶10比例放大时较高。两种放大方式下的细胞形态、密度及比生长速率无明显区别。结论初步建立了生物反应器微载体内细胞的胰酶消化球转球放大技术,Vero细胞存活率较高,贴壁良好,空珠率较低,可应用于生物反应器微载体培养系统的规模放大。

Objective To develop a technique for scale-up of beads-to-beads trypsinization of Vero cells cultured on microcarriers in bioreactor. Methods Vero cells were cultured in 500 ml stirring until a concentration of 1. 8 × 106 cells / ml was reached, and digested with 0. 25% trypsin ＋ 0. 02% EDTA at 25 and 37 ℃ for various minutes, based on which the recovery and survival rates of cells were calculated, and the temperature for culture and time for digestion were optimized.The Vero cells cultured on microcarriers in 3 L bioreactor and in T25 flask were digested and inoculated to T25 flask, of which the morphologies and specific growth rates were compared. The Vero cells cultured on microcarriers in 3 L bioreactor were digested and inoculated to 15 L bioreactor at ratios of 1 ∶ 3, 1 ∶ 6, 1 ∶ 8, 1 ∶ 10 and 1 ∶ 12 respectively to determine the optimal ratio. The Vero cells cultured in spinner and in 3 L bioreactor were digested and inoculated to 15 L bioreactor, of which the morphologies, densities and specific growth rates were compared. Results The recovery and survival rates of Vero cells cultured on microcarriers were ensured effectively after digestion at 20 ~ 25 ℃ for 15 min. No significant difference was observed between the cells subcultured by two methods. The specific growth rates of cells were high when they were inoculated to 15 L bioreactor at ratios of 1 ∶ 6 ~ 1 ∶ 10. No significant differences were observed in the morphologies, densities or specific growth rates of cells of which the trypsinizatons were scaled up by two modes.Conclusion A technique for scale-up of beads-to-beads trypsinization of cells cultured on microcarriers in bioreactor was preliminarily developed. The Vero cells showed high survival rate, good adherence and low empty bead rate, indicating that the technique might be used for the scale-up of microcarrier culture system in bioreactor.