LMH贴壁细胞悬浮驯化及其在新城疫病毒培养工艺中的应用

Suspension Domestication of LMH Adherent Cells and Their Application on Culture Process for Newcastle Disease Virus

关键词:  LMH悬浮细胞  新城疫病毒  生物反应器  悬浮培养工艺

Key words:  LMH Suspension cells  Newcastle Disease Virus  Bioreactor  Suspension culture technology

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8.0～9.0×106/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 1.5×106 cells/mL, and could proliferate to 8.0~9.0×106 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 4.5×106 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 °C 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 EID50/0.1 mL. 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.