Suspension Domestication of LMH Adherent Cells and Their Application on Culture Process for Newcastle Disease Virus
邵营格 1晁锦 1朱晓玮 1周永银 1汪爱芬 1尹秀凤 1何召庆1
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作者信息
1. 兆丰华生物科技(南京)有限公司,南京 211102
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摘要
为建立鸡新城疫病毒La Sota株在LMH细胞上的全悬浮培养工艺,以获得高滴度的新城疫疫苗抗原,采用LMH贴壁细胞悬浮培养驯化方法,获得了形态良好、能稳定传代的LMH悬浮细胞株;以LMH悬浮细胞在摇瓶培养NDV La Sota株为基础,对接毒细胞密度、接毒剂量、胰酶添加浓度、收获时间等工艺参数进行了摸索和优化,并在14 L生物反应器中进一步进行3个批次的培养验证.结果显示:LMH悬浮细胞以初始密度1.5 ×106/mL左右接种,培养72 h可增殖到8.0 ×106～9.0 ×106/mL,细胞活率达97％以上.确定NDV La Sota株在LMH悬浮细胞株上的培养工艺:LMH细胞悬浮培养至不低于4.5 × 106/mL按照感染复数不低于0.2接种病毒,并添加终浓度为5 μg/mL的胰酶,于37 ℃培养72 h左右,细胞活率在30％左右收获病毒液,NDV HA均可达1:2048～1:4096,病毒含量≥107.63EID50/0.1 mL.结果表明成功建立了 LMH细胞全悬浮培养工艺,并能在生物反应器扩大培养,为ND相关疫苗研发提供了技术支撑.
Abstract
In order to establish a suspension culture process of Newcastle disease virus La Sota strain in LMH cells to obtain high-titer Newcastle disease vaccine antigen,in this experiment,a well-shaped and stably passaged LMH suspension cell line was obtained through the acclimation of adherent LMH cells for suspension culture;Based on the flask culture of NDV La Sota strain in LMH suspension cells,the process parameters such as the density of virus-infected cells,the dose of virus infection,the addition concentration of trypsin,and the harvest time were explored and optimized,and three batches of culture verification were further carried out in a 14 L bioreactor.The results showed that LMH suspension cells were inoculated at an initial density of about 1.5 x 106 cells/mL,and could proliferate to 8.0 x 106～9.0 x 106 cells/mL after 72 h,with a cell viability of more than 97％.The culture process of NDV La Sota strain on LMH suspension cell line was determined:LMH cells were suspension-cultured to no less than 4.5 x 106 cells/mL,and the virus was inoculated at a multiplicity of infection of no less than 0.2,and trypsin with a final concentration of 5 μg/mL was added.The cells were cultured at 37 ℃ for about 72 h,and the virus liquid was harvested when the cell viability was about 30％.NDV HA could reach 1:2048～1:4096,and the virus content was ≥ 107 63 EID50/0.1mL.The results indicated that the suspension culture process of LMH cells was successfully established and could be expanded in the bioreactor,providing favorable technical support for ND-related vaccines Research and Development.
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