大鲵细胞与大鲵虹彩病毒的微载体规模化培养工艺及优化

摘要

利用Cytodex 3微载体悬浮培养系统规模化培养大鲵肌肉细胞(GSM)和大鲵虹彩病毒(GSIV), 研究了微载体培养GSM细胞的形态和增殖特性, 同时测定了病毒在培养系统中的增殖动态相关指标.结果显示, 在Cytodex 3微载体培养系统中, 将GSM细胞在贴壁期以转速30 r/min, 每静置40 min搅拌2 min的方式间歇搅拌, 10 h后贴壁率可达95%, 培养基中最适血清浓度为10%, 最适微载体浓度为2 g/L, 最适细胞初始接种密度为1.2×105cells/mL; 增殖期以25 r/min的连续搅拌方式可以达到最佳的细胞生长效能.倒置显微镜与扫描电镜观察结果显示, GSM细胞呈长梭形,紧密贴附在Cytodex 3微载体上, 生长良好.采用优化的工艺条件培养GSM细胞, 以感染复数(MOI)为0.5的剂量接种GSIV至规模化培养的GSM细胞, 48 h后GSM细胞出现典型的细胞病变效应, 72 h病毒滴度达到最高TCID50=10–8.50±0.20/mL.本研究为大鲵虹彩病毒病疫苗的规模化生产工艺研究奠定了前期基础.%The Chinese giant salamander Andrias davidianus, a member of the family Cryptobranchidae, is the largest extant amphibian species in the world, which is valued for food, medicine, and research on animal evolu-tion and biodiversity because of its unique phylogenetic position and physiological features. Within the last decade, Chinese giant salamander farming has increased rapidly in China. However, with the rapid development of Chi-nese giant salamander aquaculture, a severe epizootic recently occurred in cultured Chinese giant salamanders in Hubei, Hunan, Sichuan, Shannxi, and Zhejiang Provinces of China, causing tremendous economic losses. The causative pathogen has been identified as the giant salamander iridovirus (GSIV). At present, no effective methods are available for the control of the disease. Immunoprophylaxis is considered the best measure in controlling viral diseases in aquatic animals, and the large-scale cultivation technology of giant salamander cells and GSIV are of significance for the immunoprophylaxis of the disease. In this study, by using Cytodex 3 microcarriers to culture giant salamander muscle cells (GSM) and GSIV at a large scale, the GSM cell morphology, proliferative charac-terization, and GSIV growth dynamics were investigated. The results showed that the attachment efficacy reached 95% after 10 h of cultivation with intermittent agitation of 30 r/min for 2 min followed by 40 min still culture during the cell attachment period in the Cytodex 3 microcarrier culture system. The optimal culture conditions are as follows: serum concentration 5%, microcarrier concentration 2 g/L, and initial inoculation cell density of 1.2 × 105cells/mL. During the cell growth period, the continuous stirring speed was 25 r/min. Under inverted micros-copy and electron microscopy, the GSM cells on the Cytodex 3 microcarriers were long, spindle-shaped, and well adhered. After infection with GSIV at a multiplicity of infection of 0.5, the GSM cells on Cytodex 3 microcarriers showed the typical cytopathic effect at 48 h post infection, and the highest virus titer (TCID50/mL) of 10–8.50±0.20/mL was reached at 72 h post infection. This study established a solid foundation for further investiga-tion on the large-scale technologies of GSIV vaccine production in the future.