文献类型：期刊文献

中文题名：PK-15细胞微载体悬浮培养及PCV2增殖工艺研究

英文题名：Study on PK15 cell microcarrier suspension culture and proliferation process of porcine circovirus type 2

作者：梁武[1,2];乔健[1];李亚杰[2];易小萍[3];杨保收[2]

机构：[1]中国农业大学动物医学院,北京海淀100193;[2]天津瑞普生物技术股份有限公司,天津空港300308;[3]华东理工大学生物反应器国家重点实验室,上海徐汇200237

年份：2019

卷号：55

期号：4

起止页码：103

中文期刊名：中国兽医杂志

外文期刊名：Chinese Journal of Veterinary Medicine

收录：北大核心:【北大核心2017】；

语种：中文

中文关键词：PK15细胞;猪圆环病毒2型;微载体;悬浮培养

外文关键词：PK15 cells;Porcine circovirus type 2(PCV2);Microcarriers;Suspension culture

摘要：研究表明,PCV2仅在PK15等少数哺乳动物细胞上增殖,但由于PCV2毒力弱,且不产生细胞病变,获得高滴度病毒难度较大[1]。因此,PCV2的培养滴度高低已成为制约现有疫苗质量的关键瓶颈之一。为建立在生物反应器内微载体逐级放大培养PK-15细胞和增殖PCV2技术,本研究以德国Sartorius14L生物反应器微载体悬浮培养PK-15细胞,对PK-15细胞初始接种密度、搅拌转速、微载体浓度、PCV2接毒时间、接毒剂量、收毒时间等工艺参数进行了摸索和优化[2-3]。结果表明:3g/L的微载体和60r/min的搅拌转速下,采用0.5×106cells/mL的初始接种密度操作工艺可获得最佳PK-15细胞生长效能。细胞生长后6h接毒,采用感染复数(MOI)为0.5的接毒比例,细胞接毒后在微载体上生长96h可获得最高的PCV2增殖滴度108.5TCID50/mL,利用该工艺,经过消化转移将PK-15细胞从14L反应器放大至42L反应器,微载体上细胞贴附均匀、生长旺盛,42L反应器中培养72h细胞密度可达39.0×105cells/mL,病毒滴度108.3TCID50/mL,应用生物反应器培养PCV2滴度较常规转瓶培养工艺提高了近10倍。进一步表明PCV2悬浮培养放大与接毒工艺稳定,为下一步实现工业级规模化生产奠定基础。
Studies have shown that PCV2 only proliferated on a few mammalian cells, such as PK-15 cells,PCV2 has weak virulence and no cytopathic effect, so it is difficult to obtain high titer virus. Therefore, the culture titer of PCV2 has become one of the key bottlenecks to the quality of existing vaccines. In order to develop a technique for scale-up of microcarrier suspension culture of PK-15 cell and propagation of PCV2 in bioreactor, we established the PK-15 cell suspension culture system for PCV2 in 14 L bioreactor. The inoculation density, stirring speed, microcarrier concentration, the time of PCV2 inoculation, the dosage of inoculation and the time of harvesting were explored and optimized. The results showed that the optimal growth efficiency could be achieved through 60 rpm of the stirring rate and 0.5 ×10^6 cells per 1mL inoculation on 3 g/ L microcarriers concentration. When the time of PCV2 inoculation was 6 h, the MOI was 0.5 and the optimal harvesting time was 96h, the highest PCV2 titer was 10 8. 5 TCID 50 / mL. The low serum medium sup- ported the growth of PK-15 cells with favorable cell distributions, and the viable cell density reached 39.0 ×10^5 cells/ mL 72 h after in- oculation in 42L bioreactor,and the highest PCV2 titer was 10 8.3 TCID 50 / mL. Compared with the conventional rolling bottle culture process, the application of bioreactor culture increased the PCV2 titer by nearly 10 times, which indicates that PCV2 suspension culture process is stable.