摘要
目的应用细胞工厂建立原代地鼠肾(primary hamster kidney, PHK)细胞传代培养工艺,培养狂犬病病毒,为PHK细胞和人用狂犬病疫苗(地鼠肾细胞)的规模化放大奠定基础。方法选用SPF级地鼠,无菌取肾,经消化分散接种到细胞工厂中,在37℃下培养筛选出PHK细胞静置培养的最适细胞接种密度;在最适细胞接种密度下,从0.20%水解乳蛋白MEM培养基和改良的DMEM/F12培养基中筛选出更适宜地鼠肾细胞静置培养的培养基;同时对消化液A(含0.02%EDTA的0.25%胰蛋白酶溶液)和消化液B(含0.30%柠檬酸钠的0.25%胰蛋白酶溶液)的消化效果进行比对,选择适宜的消化液;最后,使用最佳培养基和消化液对PHK细胞进行传代,观察细胞生长情况并分析代谢水平;对同一细胞批不同代次的单层细胞(P0代、P1代和P2代)接种狂犬病病毒aG株,进行多次收获,检测单次病毒收获液病毒滴度。结果 PHK细胞(P0代)在细胞工厂中适宜的接种密度为0.30×10^(6)~0.50×10^(6) cells/cm^(2);改良的DMEM/F12培养基和消化液B更适用于PHK细胞的培养和消化传代;在40层细胞工厂(CF40)中使用最佳培养基和消化液传代地鼠肾细胞至第4代(P4代),随着代次的增加,收获的细胞密度降低、葡萄糖消耗减少和乳酸生成下降;P0代、P1代和P2代细胞均有较佳的细胞状态和增殖能力;用P0代、P1代和P2代细胞培养狂犬病病毒,能有效收获4次,且单次病毒收获液病毒滴度结果符合要求,差异无统计学意义(P>0.05)。结论成功建立了PHK细胞传代培养工艺,为PHK细胞的规模化放大,冻干人用狂犬病疫苗(地鼠肾细胞)生产工艺的改进、变更以及产能的扩大提供了参考。
Objective The cell factory was used to establish the subculture process of primary hamster kidney cells to culture rabies virus, which laid the foundation for the large-scale amplification of primary hamster kidney cells and human rabies virus(hamster kidney cells). Methods The kidney of SPF grade hamster was picked and digested aseptically. The kidney cells were seeded into the cell factory, and the optimal cell seeding density for static culture of primary hamster kidney cells was screened at 37 ℃. Under the optimum cell seeding density, the medium which more suitable for static culture of hamster kidney cells was selected from 0.2% hydrolyzed milk protein MEM culture medium and improved DMEM/F12 culture medium, meanwhile, the digestive effects of digestive solution A(0.25% trypsin solution containing 0.02% EDTA) and digestive solution B(0.25% trypsin solution containing 0.30% sodium citrate) were compared, and the suitable digestive solution was selected. Finally, the primary hamster kidney cells were passaged with the most suitable medium and digestive solution. The cell growth was observed under microscope and the metabolic level was analyzed. The monolayer cells of different generations in the same cell batch(P0 generation, P1 generation and P2 generation) were inoculated with rabies virus aG strain, and the virus titer of single harvest liquid was detected. Results The suitable seeding density of primary hamster kidney cells(P0 generation) in the cell factory was 0.30×10^(6)-0.50×10^(6) cells/cm^(2);modified DMEM/F12 medium and digestive solution B were more suitable for the culture and digestion of primary hamster kidney cells. Hamster kidney cells were passaged to the fourth generation(P4 generation) by using the most suitable medium and digestive solution in 40-layer cell factory(CF40). With the increase of passage times, the harvested cell density, glucose consumption and lactic acid production decreased, and P0, P1 and P2 cells had better cell state and proliferation ability. Rabies virus could be effectively harvested for 4 times when rabies virus was cultured with P0 generation, P1 generation and P2 generation cells.The virus titer of single virus harvest solution met the requirements, and had no significantly difference(P>0.05). Conclusion The subculture process of primary hamster kidney cells was successfully established in this study, which provides a reference for the large-scale enlargement of primary hamster kidney cells, the improvement of the production process of freeze-dried human rabies vaccine(hamster kidney cells) and the expansion of production capacity.
作者
李佳林
张志刚
刘晓巍
肖蕾
徐小明
张艳
李守丽
于潆
李红芳
LI Jia-lin;ZHANG Zhi-gang;LIU Xiao-wei;XIAO Lei;XU Xiao-ming;ZHANG Yan;LI Shou-li;YU Ying;LI Hong-fang(The Second Department of Vaccine,Lanzhou Institute of Biological Products Co.,Ltd.,Center for Gansu Provincial Vaccine Engineering Research,Lanzhou 730046,Gansu Province,China)
出处
《微生物学免疫学进展》
CAS
2021年第5期41-47,共7页
Progress In Microbiology and Immunology
关键词
原代地鼠肾细胞
细胞工厂
培养基
消化液
传代培养
狂犬病病毒
Primary hamster kidney cells
Cell factory
Culture medium
Digestive juices
Subculture
Rabies virus
