PK15 cell acclimation suspension method and two-stage virus production process

A cell and virus technology, applied in the field of PK15 cell domestication suspension and second-stage virus production technology, can solve the problems of inability to achieve serum-free culture, weak nutrient composition, small surface area/volume, etc.

Pending Publication Date: 2018-09-25
上海健士拜生物科技有限公司 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The defects of the existing PK15 cell acclimatization and suspension are: 1. Some cannot achieve complete serum-free culture; 2. Generally, a two-step method is adopted, that is, first reduce to serum-free and then suspend culture, because the medium used for adherent culture Serum is usually added to the basal medium. The nutrient content of the basal medium is weak, and the surface area/volume ratio of the adherent culture during the domestication process is usually small, which limits the intake of nutrients. The entire domestication process takes a long time; 3. Af

Method used

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  • PK15 cell acclimation suspension method and two-stage virus production process
  • PK15 cell acclimation suspension method and two-stage virus production process
  • PK15 cell acclimation suspension method and two-stage virus production process

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0100] Embodiment 1 PK15 cell serum-free acclimation suspension method and medium development

[0101] (1) Experimental materials

[0102] Cell line: Adherent PK15 adherent PK15 cell line derived from ATCC (ATCC, CCL-33, Lot: 61284198)

[0103] Medium: BD004(JSB-DP049)

[0104] (2) Serum-free acclimation suspension method

[0105] Include the following steps:

[0106] 1) Recovery and passage of adherent cells:

[0107] a. Preparation of medium: prepare 10L liquid medium according to the instructions BD004(JSB-DP049), after sterilizing and filtering, store in the dark at 4°C;

[0108] b. Recovery of cells: Take out the frozen cell line from the liquid nitrogen tank, melt it at 37°C and add it to 15mL Place BD004 (JSB-DP049) and 5% newborn calf serum (NBCS) in a T75 culture flask at 37°C, 5% CO 2 incubator for cultivation;

[0109] c. Subculture: The newly recovered cells are subcultured every 3 days, according to 1.0x10 6 Cells / T75 were inoculated, subcultured t...

Embodiment 2

[0114] Second stage culture process test of embodiment 2 suspension PK15 cells

[0115] (1) Experimental materials

[0116] a. Cell line: PK15 cells that have been domesticated in suspension and can grow stably at high density in Example 1;

[0117] b. Culture medium: prepare 10L growth medium according to the instructions CD PK15 (JSB-LM17019), after sterilizing and filtering, store in the dark at 4°C;

[0118] (2) Experimental steps

[0119] a. If figure 2 As shown, on the third day of cell growth, the cell density reached 8.0 × 10 6 For cells / mL, add growth medium 1 times the original working volume to dilute the cell density to 4.0×10 6 cells / mL, inoculated with porcine circovirus at a MOI of 0.5, the cells began to grow again after inoculation, and after 2 days the cell density grew to 10.0×10 6 cells / mL;

[0120] b. by figure 2It can be seen that the superiority of the second-stage culture technology of the PK15 cell production process: high-density growth, l...

Embodiment 3

[0121] The test of embodiment 3PCV on PK15 cell

[0122] (1) Experimental materials

[0123] a. Cell line: PK15 cell line derived from ATCC, which can be grown in suspension without serum or low serum (such as 0.5% newborn calf serum) after being independently domesticated by Jianshun Biotechnology. The domestication conditions are as above, and can be briefly described as follows: The serum of the wall PK15 cells was reduced to 0.5% for 3 passages, and then the suspension acclimation culture was carried out, and the acclimatization condition was 5% CO 2 , the shaker speed was 120rpm / min, and the PK15 cell line capable of high-density suspension growth was screened out;

[0124] b. Medium: CD PK15 (JSB-LM17019).

[0125] c. Virus: PCV (provided by Huanong (Zhaoqing) Institute of Bioindustry Technology)

[0126] (2) Experimental method

[0127] Include the following steps

[0128] a. Preparation of medium: prepare 10L liquid medium according to the instructions CD PK...

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Abstract

The invention relates to a PK15 cell acclimation suspension method and a two-stage virus production process. According to the PK15 cell acclimation suspension method, PK15 cells are acclimated to adapt to serum-free suspension culture and used as host cells for producing viruses which can have cytopathic effect or sensitivity on suspension PK15 cells by two-stage culture virus inoculation; when the cells grow to 2.0-20.0*10<6> cells/mL, diluting with a production culture medium by 1-5 times is performed to reach a density range of 1.0-10.0*10<6> cells/mL, and then virus inoculation is carriedout or virus inoculation is performed before dilution. The suspension PK15 cells are adaptive to suspension growth in the serum-free chemical-defined culture medium, and the two-stage virus productionprocess is simple and easy in amplification; liquid changing is avoided, and high culture medium utilization efficiency is achieved; production and growth culture media can be identical or not and can be mixed proportionally to realize cell secondary growth and viral expression promotion.

Description

technical field [0001] The invention relates to a PK15 cell acclimation suspension method and the field of production of suspension PK15 cell-related vaccines, in particular to a technique for cultivating suspension PK15 cells in a bioreactor of 30 L or above and cultivating the suspension PK15 cells by a second-stage culture method to produce vaccines. Background technique [0002] Mammalian cell large-scale culture technology is one of the downstream common technologies of biopharmaceutical companies, and it plays an extremely important role in this industry. The key to this technology is to achieve large-scale culture of mammalian cells in suspension serum-free, thereby increasing production capacity, reducing costs, and facilitating scale-up. The usual practice in culture of adherent cells is to add a certain amount of serum to the culture medium to allow the cells to grow on the wall. However, the chemical composition of the serum is uncertain, and the quality is unstab...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N7/00
CPCC12N5/0686C12N7/00
Inventor 蔡仕君王嘉琪
Owner 上海健士拜生物科技有限公司
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