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Method for large-scale high-density suspension culture of cell 293-high yielding adenovirus

A suspension culture and high-density technology, applied in the field of biomedicine, can solve the problems of low toxin production, low cell density and yield, and achieve the effects of reducing cell culture loss, simplifying operations, and improving substances required for metabolism

Active Publication Date: 2020-03-20
GUANGXI WUZHOU PHARMA GRP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the production of adenoviruses and adeno-associated virus gene carrier viruses in this technology is limited by low cell density and low cell toxin production, resulting in low yield. Low cell density and cell culture technology are affected by nutrient supply, oxygen, and metabolism. It is difficult to cultivate high-quality cells. Density of cells, in addition to itself, infected cells compete with cell growth, metabolism, etc., resulting in low toxin production

Method used

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  • Method for large-scale high-density suspension culture of cell 293-high yielding adenovirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] 1. The 293 parietal cells were taken and affixed to the serum-free 293SFM+serum gradient descending culture method, and finally became serum-free suspension cells.

[0028] 2. Suspension culture with serum-free 293SFM medium, first cultured to a certain concentration in a shaker flask, then transferred to a tank for cultivation, inoculated into a bioreactor at a certain cell density to adjust the culture conditions: culture temperature at 36 ° C; pH Maintained at 7.0; dissolved oxygen concentration at 30%; stirring speed at 80rpm, the stirring speed was set to 80rpm on the first day of cultivation, and the stirring speed was gradually increased at 120rpm from the second day of cultivation to continue culturing until the virus was infected.

[0029] 3. At the 45th hour of culture in the tank, perfusion was added to increase fresh serum-free 293SFM, and the required culture speed was 3L / d. At the same time, the cells in the excreted liquid were intercepted in the tank with...

Embodiment 2

[0032] 1. The 293 parietal cells were taken and affixed to the serum-free 293SFM+serum gradient descending culture method, and finally became serum-free suspension cells.

[0033] 2. Suspension culture with serum-free 293SFM medium, first cultured to a certain concentration in a shake flask, then transferred to a tank for cultivation, inoculated to a bioreactor at a certain cell density to adjust the culture conditions: culture temperature at 38 ° C; pH Maintained at 7.4; dissolved oxygen concentration at 50%; stirring speed at 160rpm, the stirring speed was set to 100rpm on the first day of cultivation, and the stirring speed was gradually increased at 160rpm from the second day of cultivation to continue culturing until the virus was infected.

[0034] 3. On the 52nd hour of culture in the tank, start perfusion to increase fresh serum-free 293SFM, and the required culture speed is 8L / d. At the same time, use a cell interceptor to trap the cells in the excreted liquid in the t...

Embodiment 3

[0037] 1. The 293 parietal cells were taken and affixed to the serum-free 293SFM+serum gradient descending culture method, and finally became serum-free suspension cells.

[0038] 2. Suspension culture with serum-free 293SFM medium, first cultured to a certain concentration in a shaker flask, then transferred to a tank for cultivation, inoculated to a bioreactor at a certain cell density to adjust the culture conditions: culture temperature at 37 ° C; pH Maintained at 7.2; dissolved oxygen concentration at 40%; stirring speed at 120rpm, the stirring speed was set to 90rpm on the first day of cultivation, and gradually increased at 140rpm from the second day of cultivation to continue culturing until virus infection.

[0039] 3. At the 48th hour of culture in the tank, start perfusion to increase fresh serum-free 293SFM, and the required culture speed is 5L / d. At the same time, use a cell interceptor to trap the cells in the excreted liquid in the tank, and start to infect the v...

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Abstract

The invention discloses a method for large-scale high-density suspension culture of a cell 293-high yielding adenovirus. The method comprises the steps of firstly, carrying out serum-free 293SFM culture, then, starting CD culture medium culture to produce a virus, combining a perfusion technology and a cutoff technology, and meanwhile, controlling temperature changes of cell culture and viral infection. According to the method, the enlarging of cell culture density is achieved, the volume of cell culture is reduced, subsequent labor intensity is lowered, the yield of the virus is increased, the cost is reduced, the cell death loss of a cutoff process is reduced, the quality is uniform and stable during culture, a production process is close in link and easy in control, and thus, stable quality of batches is guaranteed.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a method for large-scale high-density suspension culture of 293 cells with high yield of adenovirus. Background technique [0002] HEK293 cells are the most commonly used adenovirus vector packaging cells. They are transformed with type 5 adenovirus 75 strains and contain human embryonic kidney cells containing the Ad5 E1 region. They were developed by F.L Graham and J.s.Miley of McMaster University in Canada in 1976 with DNA constructed by transfection technology. HEK293 cells are anchorage-dependent epithelial-like cells that exhibit a phenotype typical of adenovirus-transformed cells that allow Ad5 and other serotype adenoviruses to proliferate within them. HEK293 cells are a human hypotriploid cell line that can be grown in the absence of Ca 2+ or with Ca 2+ It can grow in culture medium, also can grow in medium with lower serum concentration, and can also be used for ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12N5/073C12N5/02C12R1/93
CPCC12N7/00C12N5/0603C12N5/0686C12N2710/10051C12N2500/90
Inventor 潘钊林赖树生黄运琦徐翔宇
Owner GUANGXI WUZHOU PHARMA GRP
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