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ST culture medium and application thereof

A culture medium and vitamin technology, applied in the cultivation process, tissue culture, microorganisms, etc., can solve the problems of easy batch-to-batch differences in cell culture results, exogenous viruses, and unstable sources of serum imports

Inactive Publication Date: 2018-11-13
SHANGHAI OPM BIOSCI CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] (1.) The concentration of serum in the culture stage is high, and the results of cell culture are prone to batch-to-batch differences; moreover, the imported supply of serum has been unstable and expensive in recent years;
[0005] (2.) The serum-containing process may bring safety problems such as exogenous virus and mycoplasma contamination, such as bovine viral diarrhea virus (Bovine Viral diarrhea-mucosal Virus, BVDV) brought by serum may affect the expression of swine fever virus, so Higher requirements for serum quality;
[0006] (3.) The culture process requires a complex digestion process. Even if microcarriers are used for culture, it is difficult to turn the ball because the ST cells are firmly attached to the wall.

Method used

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] In this embodiment, the ST medium used is a serum-free medium, which can be used for the suspension culture of ST cells. The composition and content of the ST medium are shown in Table 1 below:

[0089] Table 1

[0090]

[0091]

[0092] Using the ST medium described in Table 1, the adherent ST cells were gradually and successfully domesticated into suspension ST cells, such as figure 1 shown. The resulting suspended ST cells were treated with the ST medium described in Table 1 at 1x10 6 The density of cells / mL was inoculated in a 125mL shake flask, the working volume was 30mL, and the rotation speed was set at 110rpm. The result: the cell density can reach 1x10 7cells / mL. Under the same experimental conditions, after adding CSFV at a ratio of 0.5% (volume ratio), the cell growth was relatively low, and the cell density reached 0.8×10 7 cells / mL, the expression of the virus was 30% higher than that of the adherent ST cells using the same inoculation strategy. ...

Embodiment 2

[0094] In this embodiment, the ST medium used is a serum-free medium, which can be used for the suspension culture of ST cells. The composition and content of the ST medium are shown in Table 2 below:

[0095] Table 2

[0096]

[0097]

[0098] Using the ST medium described in Table 2, the adherent ST cells were gradually and successfully domesticated into suspension ST cells, such as figure 2 shown. The resulting suspended ST cells were treated with the ST medium described in Table 2 at 0.5x10 5 The density of cells / mL was inoculated in shake flasks, the working volume was 45mL, and the rotation speed was set at 160rpm. After 3 days, the cell density reached 1.8x10 6 cells / mL. Under the same experimental conditions, after adding CSFV at a multiplicity of infection (MOI) of 0.1, the virus expression level was equivalent to that of adherent ST cells.

Embodiment 3

[0100] In this embodiment, the ST medium used is a serum-free medium, which can be used for the suspension culture of ST cells. The composition and content of the ST medium are shown in Table 3 below:

[0101] table 3

[0102]

[0103]

[0104] Using the ST medium described in Table 3, the adherent ST cells were gradually and successfully domesticated into suspension ST cells, such as image 3 shown. The resulting suspension ST cells, using the ST medium described in Table 2, with 5x10 5 The density of cells / mL was inoculated in a 125mL shake flask, the working volume was 30mL, and the rotation speed was set at 150rpm. As a result, the cell density reached 0.8x10 7 cells / mL. Under the same experimental conditions, after adding CSFV strain C according to 0.5MOI, the cell density reached 0.8x10 7 cells / mL, the virus expression reached 6.2±0.3LogTCID50 / mL, which was equivalent to the virus titer of ST adherent cells under the same conditions.

[0105] In summary, using...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to a ST culture medium and application thereof. The ST culture medium does not contain serum, fully-suspended serum-free culture of ST cells can be realized by adopting the ST culture medium, and high-density culture can be realized without using a microcarrier. The serum is not needed in the process of suspended culture of the ST cells, and the culture medium does not contain animal-derived components, so that batch-to-batch difference and the safety problem caused by the animal-derived components like the serumare eliminated, production cost is lowered, and the downstream separation and purification step is simplified. In addition, components in the culture medium are determined, so that repeatability andstability from supply of the culture medium to cell culture to product production.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an ST culture medium and its application. Background technique [0002] Pig testicular cells (Swine Testicle, ST cells) are fibroblastic diploid cells and can be continuously passaged. ST cells are usually used to produce vaccines against classical swine fever virus, which is one of the main infectious diseases of pigs and belongs to acute contact infectious diseases. CSFV can proliferate in porcine-derived cells, such as porcine kidney cells (PK-15) and porcine testicular cells (ST). Most of the newly synthesized viruses are adsorbed or bound to the cells, and only a very small amount of viruses exist in the culture medium. [0003] ST cells are suitable for adherent monolayer culture. The commonly used culture conditions are traditional basal medium such as MEM supplemented with 10% fetal bovine serum (or calf serum). ST cells are relatively firm and difficult to diges...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/0683C12N2500/12C12N2500/16C12N2500/20C12N2500/22C12N2500/24C12N2500/30C12N2500/32C12N2500/34C12N2500/35C12N2500/38C12N2500/46
Inventor 肖志华刘智张淑香
Owner SHANGHAI OPM BIOSCI CO LTD
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