摘要
为了探索用细胞工厂代替转瓶培养轮状病毒基因重配株LD9及收获高滴度的LD9病毒原液和提高产量的可行性,分别在2层4、层细胞工厂和3L1、5L转瓶培养Vero细胞,比较两种容器内细胞的生长状态。结果显示,以相同活细胞数2.5×104/ml同时接种两种不同培养容器时,细胞工厂培养3d已长成单层,而转瓶培养需5d;对两种容器长满单层时的细胞经胰酶消化后通过细胞仪计数、分析,结果显示,两种容器培养细胞长成单层时的单位面积细胞密度相当;对长成致密单层细胞的两种容器以相同的MOI(MOI=0.1)接种LD9病毒,转瓶培养的病毒于第8d病毒滴度达到高峰,为6.0～6.5 lgCCID50/ml;细胞工厂第5d病毒滴度达高峰,为6.5 lgCCID50/ml,并于第9d病毒滴度再次达到峰值,为6.0～6.5 lgCCID50/ml,实现二次收获病毒。
To explore the possibility of culturing rotavirus reassortant strain LD9 in cell factory.Vero cells were cultured on the cell factory CF2 and CF4,respectively,those on 3L and 15L rolling bottles were used as control.Cells growth state in the two kinds of containers was observed daily under microscope.The results showed that viable cells are inoculated in both kinds of containers at the same concentration of 2.5 104 /ml,and the proliferation rate of cells in cell factory is significantly higher,the surface of cell factory was covered with the monolayer cells at the third day,in contrast the surface of rolling bottle is covered at the 5th day.The grown speed of cells inoculated in the cell factory is almost two folds of that of culturing in the rolling bottle.The monolayer cells in the two kinds of containers are digested and analyzed by cell counter,the result showed that cell density in the two kinds of containers is the same.The monolayer cells on the two kinds of container were infected with the same MOI(0.1) of LD9 virus,and the CPE of virus was observed and the titer of virus was determined daily.The highest titer of virus in rolling bottle is achieved at 8th day with the titer of 6.0～6.25 lgCCID50 and cells are almost shedding,whereas the highest titer of virus in cell factory is achieved at day 5 and cells are nearly not shedding.After the culture supernatant was harvested at the 5th day,fresh maintenance medium is added for the second culture and then the culture supernatant is harvested at the 9th day again.
出处
《微生物学免疫学进展》
2011年第3期10-14,共5页
Progress In Microbiology and Immunology
