应用细胞工厂建立人用狂犬病疫苗连续培养多次收获工艺

目的为提高生产效率、增加原代地鼠肾细胞单产量及狂犬病病毒产量，建立人用狂犬病疫苗（地鼠肾细胞）连续培养多次收获工艺。方法选用12-14日龄SPF地鼠，无菌取肾经消化，制备成细胞悬液，分装到40层细胞工厂并培养细胞成单层；接种狂犬病病毒固定毒aG株，连续培养病毒并多次收获。分别对同一细胞批制备的多个单次病毒收获液的免疫原性、病毒滴度和地鼠肾细胞蛋白质含量进行检测。结果用40层细胞工厂培养原代地鼠肾细胞和狂犬病病毒，细胞接种浓度为1．0×10。～1．5x10。cells／mL，（36±1）℃培养72h成致密单层；按0．1MOI病毒接种，可进行6次收获病毒；多个单次病毒收获液病毒滴度均不低于6．0lgLD50／mL；免疫原性检查保护指数不低于100；地鼠肾细胞蛋白质残留量随着收获次数的增加而不断降低。结论用细胞工厂建立了人用狂犬病疫苗连续培养多次收获工艺，能显著提高地鼠。肾单产量，增加产能。

Establishment of a new continuous culture and multiple harvests process using cell factory for rabies vaccine for human use

Objective To improve the production efficiency and increase primary hamster kidney cells （ PHKC ） and rabie virus yield. To establish a new continuous culture and multiple harvests process using cell factory for rabies vaccine { Ham ster kidney cells） for human use. Methods PHKC suspensions were prepared by asepsis digesting 12 to 14 day old pri mary hamster kidney. The cells suspension was packed into cell factory and cultured to cell monolayer. Rabies fixed strain aG was inoculated and cultured , and then multiple harvests were taken. Immunogenicity test, virus titer test and PHK（ matrix proteins tset were taken for every single virus harvest from one cell batch. Result Cells cultures were carried out i~ CellSTACK~-dO{40-1ayers cell factory） with cell density of 1.0x106 -1. 5xl06cells /mL at （ 36±1 ） °C for 72 h. Cell were infected with rabies aG strains at 0.1 MOI and six virus harvests were manufactured from one cell batch . The titer o] / single virus harvests was not less than 6.01gLDs0/mL, and immunogenicity protection index was not less than 100. Cell ma trix protein value reduced with the increase of virus harvest times. Conclusion The continuous culture and multiple har vests process using cell factory for rabies vaccine （ primary hamster kidney cells } for human use was established. It im proved the production efficiency and increased PHKC and rabies virus yield.