Abstract
Animal cell culture is one of key technologies for the modern biotechnology. Microcarrier system provides the ability to scale up the culture of the anchorage-dependent animal cells in stirred-tank bioreactor for producing viral vaccine and gene therapy viral vector. The shear stress and the serum concentration in microcarrier culture system would influence the cell physiology and morphology, so these factors may effect viral vaccine and vector production. In this study, stationary and microcarrier culture system were used for producing Japanese encephalitis virus (JEV), adenoviral vector (AV) and retroviral vector (RV) and investigated the effects of shear stress, serum concentration, multiplication of infection (MOI) and microcarrier types on the yield of viral titers. The results demonstrated that the stationary culture produces the higher titers of JEV and AV, but less titers of RV compared with the microcarrier culture. Shear stress reduced the productivity of AV and JEV strain CH2195LA, but enhanced the productivity of JEV strain Beijing-1. Reducing serum concentration resulted in decreasing the productivity of AV and JEV. The solid microcarriers were preferred to the AV and RV production. High MOI is needed for gaining the high titer of AV, but not for JEV. This study compares the production of JEV, AV and RV in the stationary and microcarrier systems and investigates the factors effected production during culture, contributing the industrial production of vitral vaccine and gene therapy viral vector.頁次第一章 緒論1-1哺乳動物細胞培養 11-2病毒疫苗 21-3基因治療病毒載體 41-4微載體哺乳動物細胞培養系統 91-5微載體動物細胞培養的影響因素 10第二章 材料與方法2-1日本腦炎病毒 152-2重組腺病毒 152-3細胞株 162-4細胞繼代與培養基 162-5靜態培養法 182-6微載體懸浮瓶培養法 182-7微載體上細胞數之測定 212-8靜態培養細胞數之測定 212-9日本腦炎病毒感染力價之測定 212-10腺病毒感染力價之測定 222-11反轉錄病毒感染力價之測定 22第三章 結果3-1 日本腦炎病毒疫苗3-1-1 靜態培養與微載體培養中Vero細胞與BHK細胞生長的情形 243-1-2 靜態培養與微載體培養中日本腦炎病毒的生產情形 253-1-3 剪力對於日本腦炎病毒繁殖的影響 263-1-4 血清濃度對於日本腦炎病毒繁殖的影響 283-1-5 使用無血清培養基培養Vero細胞 293-1-6 使用無血清培養基生產日本腦炎病毒 303-2 腺病毒載體3-2-1微載體培養與靜態培養中HEK-293細胞的生長情形 303-2-2微載體培養與靜態培養中腺病毒載體的生產情形 313-2-3 MOI對腺病毒載體生產的影響 323-2-4 流體剪力對於腺病毒載體生產的影響 333-2-5 血清濃度對於腺病毒載體生產的影響 343-3 反轉錄病毒載體3-3-1 微載體培養與靜態培養中PA317-RCM1細胞的生長情形 353-3-2 微載體培養與靜態培養中反轉錄病毒載體的生產情形 353-3-3 比較細胞生長速率與反轉錄病毒載體生產的關係 36第四章 討論4-1 日本腦炎病毒疫苗4-1-1 流體剪力對於日本腦炎病毒生產的影響 384-1-2 血清濃度對於日本腦炎病毒生產的影響 394-2 腺病毒載體4-2-1 微載體種類對於腺病毒載體生產的影響 404-2-2 血清濃度對於腺病毒載體生產的影響 414-2-3 MOI對於腺病毒載體生產的影響 414-3 反轉錄病毒載體4-3-1 微載體種類對於反轉錄病毒載體生產的影響 424-3-2 反轉錄病毒載體生產與日本腦炎病毒及腺病毒載體的差異 43第四章 結論 45參考文獻 46圖表 54附錄 78