Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (6): 1280-1287.doi: 10.3864/j.issn.0578-1752.2021.06.017

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles     Next Articles

Based on PK15 Cell Line for PCV2 Fully Suspension Culture Process

JiaQi WANG1(),YuHong DONG1,JuLing JIANG1,JianNing QIAN1,WenTao WEI1,GuoLiang SONG2,JinBo JIAO2,XinXin GUAN2,GuoBiao JI2,YeXin ZHANG1   

  1. 1Gansu Jianshun Biotechnology Co., Ltd, Lanzhou 730070
    2Luoyang Huizhong Biotech Co.,Ltd., Luoyang 471000, Henan
  • Received:2020-03-20 Accepted:2020-06-30 Online:2021-03-16 Published:2021-03-25

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Abstract:

【Objective】Selected one suspension PK15 cell line which is suitable for PCV2 virus , and then developed the production process for PCV2 vaccines(source of virus, MOI and harvest time ), to provide basic theory and guarantee for large-scale production use suspension culture instead of adherent culture. 【Method】The PK15 primary cells were diluted using the limiting dilution method and seeded in 96-well plates. The cell growth and morphology were observed every 2 days. After 90% of the cells were overgrown, the cells were gradually expanded from 96-well plates to 24-well plates,to 12-well plates, to 6-well plate, and finally to the square flask, three PK15 clones with good morphology that can grow adherently were selected. Three PK15 clones with good morphology that can grow adherently were selected. Three clones (PK15-1C8, 2F11, 1E5) were directly seeding in PK15 serum-free medium at a density of 1×106cells/mL and placed in a shaker incubator at 37 ℃, 5% carbon dioxide, and 120 r/min to continue culture. Monitor the cell density and viability every day, passage every 3 days, make the cells gradually adapt to the suspension environment, the PK15 clone can culture as a fully suspension cell line and can growth well in serum free media. After suspension cells stability passage and cell bank, compared the PCV2 virus content with three suspension clone cells, one clone cell was selected for PCV2 production. For different kinds of viruses (source from adherent cells or suspension cells), explore the infection MOI (0.1, 0.2, 0.5) and harvest time (48, 72, 96, 120h), determine the best production process of PCV2. 【Results】(1)The results showed that when the adherent cells passage to second generation in serum media CD PK15 259, the cells can suspended growth, continuous passage for eleven generation, suspension cells can growth stable, when seeding with 1×106 /mL cells, cells can reach 10×106 /mL cells when growth at 72h, viability rate is above 95%, and the doubling time is about 20h; (2) The three suspension cells use the same condition to infection PCV2 virus, the virus content of PK15-1C8 cloned cells can reach 106.4TCID50/mL, clone PK15-2F11 (105.5TCID50/mL), PK15-1E5 (105.6TCID50/mL), the virus content of the three cloned cells was higher than that of the original clone (104.7TCID50/mL), however, the virus content of PK15-1C8 cloned cells is higher and more stable, so it is determined as a cell for follow-up research; (3) After infecting PK15-1C8 cloned cells with seed virus (106.4TCID50 /mL) from adherent cells, the optimal process is 1×106/mL cell density with 0.1MOI and harvest at 72h the virus content can reach 106.5TCID50 /mL. After infecting PK15-1C8 cloned cells with seed virus (106.3TCID50 /mL) from suspension cells, the optimal process is 1×106/mL cell density with 0.2MOI and harvest at 72h the virus content can reach 107.3TCID50 /mL. 【Conclusion】Through the monoclonal screening of PK15 cells, adapted to suspension cells compared the PCV2 virus content of 3 suspension cells, a suspension cell with the highest virus content was determined, and based on this suspension cell, explored the process of PCV2 virus, and then established the PCV2 fully suspension, serum-free culture process. The SV15-1C8 cell proliferation PCV2 process of full suspension serum-free culture was established. This process used suspension cells to amplify PCV2 virus for seed poisoning for the first time. The highest virus content can reach 107.3TCID50/mL, which can be used for factory vaccine production. According selection sensitive clones and optimized the process for PCV2, can increase the virus titer, and realizes full suspension culture without serum, improved the production process of PCV2, improve the production efficiency, reduce the cost and improve the quality of the production. This process first use suspension cells to amplify the PCV2 virus. The highest virus content can reach 107.3TCID50/mL, which can be used for large-scale PCV2 virus production.

Key words: PK15 cells, PCV2, clone selection, suspension culture

Table 1

Experiment parameters for optimization of infection process"

试验
Experiment		 感染培养基
Infection medium		 感染时细胞密度
Infection cell density（cells/mL）		 感染量
MOI		 收获时间
Harvest time
（h）		 重复次数
Repeat times
贴壁种毒
Virus from adherent cell		 感染MOI摸索
Infection MOI explore		 接毒培养基A
Infection medium A		 1.0×106 0.1、0.2、0.5 72 3
收获时间摸索
Harvest time explore		 1.0×106 0.1 48、72、96、120 3
悬浮种毒
Virus from suspension cell		 感染MOI摸索
Infection MOI explore		 接毒培养基A
Infection medium A		 1.0×106 0.1、0.2、0.5 72 5
收获时间摸索
Harvest time explore		 1.0×106 0.2 48、72、96 5

Fig. 1

Adaption suspension growth curves of four monoclonal cells A. PK15 cells adapted growth curve. B. Acclimation growth curve of PK15-2F11 cells. C. Acclimation growth curve of PK15-1E5 cells. D. Acclimation growth curve of PK15-1C8 cells. Among them, the solid line is the cell growth density curve, and the dashed line is the cell viability curve. The light-colored line is the growth curve of 3 kinds of monoclonal and PK15 adherent cells, and the dark line is the growth curve of the acclimated suspension cells of 3 kinds of monoclonal and PK15 cells."

Fig. 2

TCID50 after PCV2 infection in PK15 cells A. TCID50 of PK15, PK15-2F11, PK15-1E5, PK15-1C8 cells infected with PCV2. B. PK15-2F11, PK15-1E5, and PK15-1C8 were infected with PCV2 virus harvest from Figure A. Among them, C1 is the experimental group infected with inoculation medium A, and C2 is the experimental group infected with inoculation medium B."

Table 2

The content of PK15-1C8 virus infected with PCV2 at different MOI and different time"

参数
Parameter		 条件
Condition		 实验中病毒含量（lgTCID50/mL）
Virus content（lgTCID50/mL）
1 2 3
感染病毒量
MOI		 0.1 6.5 6.6 6.0
0.2 6.7 6.4 6.2
0.5 6.3 6.5 6.2
取样时间/h
Sample Time/h		 48 6.4 5.8 6.4
72 6.5 6.3 6.3
96 6.7 6.4 5.6
120 6.7 6.7 5.6

Table 3

The content of PK15-1C8 virus infected with PCV2 at different MOI and different time"

参数
Parameter		 条件
Condition		 实验中病毒含量（lgTCID50/mL）
Virus content（lgTCID50/mL）
1 2 3 4 5
感染病毒量
MOI		 0.1 6.5 6.1 6.2 6.0 6.0
0.2 7.0 6.7 6.5 6.3 6.3
0.5 6.8 6.7 6.3 5.5 5.7
取样时间
Sample time (h)		 48 6.5 6.2 6.4 6.3 6.0
72 7.2 7.3 6.9 6.5 6.3
96 6.7 7.2 6.4 6.5 6.3
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