摘要
目的利用Vero细胞生物反应器微载体培养技术规模化培养轮状病毒重配株LH9(G4型)。方法用3 L生物反应器,以4 g/L载体浓度培养Vero细胞,待细胞浓度达1.8×106个/ml时,分别用含2、1、0.75、0μg/ml胰酶的DMEM培养液,35℃连续培养21 d,观察细胞生长状态。分别按0.5、0.1、0.05、0.01 MOI接种轮状病毒重配株LH9(G4),培养过程中每日取样,检测病毒滴度以及葡萄糖、乳酸含量。根据培养过程中葡萄糖、乳酸含量的变化,确定病毒培养过程中的收获时间,优化培养工艺。结果在反应器中用含不同浓度胰酶的DMEM培养Vero细胞,细胞形态良好,与不含胰酶的DMEM培养液培养的细胞无明显差异;按0.05 MOI接种后的病毒收获液滴度较高,可达7.0 lg CCID50/ml,且持续时间较长;通过对病毒培养液中葡萄糖、乳酸含量的监测,调整了培养过程中的收获时间,收获次数增加至4次,可收获病毒液4个工作体积,病毒高峰滴度可达8.5 lg CCID50/ml。结论采用生物反应器微载体系统培养轮状病毒重配株LH9(G4),可显著提高病毒滴度,通过对培养过程的优化,增加了病毒收获量,为进一步规模化生产轮状病毒疫苗奠定了基础。
Objective To culture rotavirus reassortant strain LH9（type G4）by using microcarriers in bioreactor. Methods Vero cells were cultured on 4 g / L microcarriers in a 3 L bioreactor until a concentration of 1. 8 × 10^6 cells / ml. The cells were further cultured in DEME containing 2, 1, 0. 75 and 0 μg / ml trypsin at 35 ℃ for 21 d, and observed for growth,then inoculated with LH9 strain at MOIs of 0. 5, 0. 1, 0. 05 and 0. 01, from which samples were taken daily and determined for virus titer as well as glucose and lactic acid contents. The harvest time of virus was determined according to the changes of glucose and lactic acid contents during culture, and the culture procedure was optimized. Results The Vero cells cultured in DMEM containing various concentrations of trypsin in bioreactor showed normal morphology, which showed no significant difference with that in trypsin-free DMEM. The titer of LH9 strain inoculated at a MOI of 0. 05 reached 7. 0 lg CCID50/ ml and last for a long time. The harvest time of virus was adjusted by monitoring the glucose and lactic acid contents in culture medium, by which four working volumes of virus were harvested and reached a titer of8. 5 lg CCID50/ ml. Conclusion Culture by microcarriers in bioreactor increased the titer of rotavirus reassortant strain LH9（type G4）significantly, and the harvested virus increased by optimization of culture procedure, which laid a foundation of further large-scale production of rotavirus vaccine.
出处
《中国生物制品学杂志》
CAS
CSCD
2015年第5期528-530,535,共4页
Chinese Journal of Biologicals
基金
2014年甘肃省战略性新兴产业创新支撑项目
