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A st cell adapted to full suspension culture and its application and method for cultivating vaccine virus

A full-suspension and cell-based technology, applied in the biological field, can solve problems such as inability to scale up linearly, inability to industrialize large-scale production, and product release to the market, and meet the requirements of large-scale industrialization, good industrial application prospects, and high degree of automation Effect

Active Publication Date: 2018-07-24
郑州爱科生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Conventional ST cells also grow in a strict anchorage-dependent manner, so most of the viruses such as CSFV, PPV, PRV, and TGEV are cultured in roller bottles, and only a few of them are cultured in carrier suspension, and full suspension culture of cells has not yet been achieved. Cell carrier suspension culture is still anchorage-dependent culture in essence, needs spinner bottle to provide seed cells, needs expensive microcarriers, needs to increase steps such as microcarrier culture and digestion, and cell growth still requires medium with high serum content, the most important of which is It is because the cell carrier suspension culture technology cannot be scaled up linearly like full suspension culture, and cannot be industrialized and mass-produced, which restricts the industrial application of this technology. Although there are many researches or patents on carrier suspension culture, there are few. The reason why the product is on the market

Method used

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  • A st cell adapted to full suspension culture and its application and method for cultivating vaccine virus
  • A st cell adapted to full suspension culture and its application and method for cultivating vaccine virus

Examples

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Effect test

Embodiment 1

[0030] Example 1 Adaptation to the Expansion Culture of Full Suspension Culture ST Cell Lines

[0031] The porcine testis cell suspension adaptation strain ST-2014S preserved in the China Typical Culture Collection Center with the preservation number CCTCC C201567 was revived and subcultured, and then inoculated into a bioreactor for suspension culture to obtain ST cell suspension culture medium. The specific steps are as follows:

[0032] ①Take the ST-2014S cell line cryopreserved in liquid nitrogen, quickly thaw it, add it to a shaker flask filled with nutrient solution, culture it at 36-37°C for 60-72 hours, and subculture and enlarge it according to the ratio of 1:3-1:5;

[0033] ② Dilute the ST-2014S cell line amplified in step ① to 5-10×10 5 Cells / ml density, inoculated into bioreactor, culture temperature is 36-37℃, pH value is 6.8-7.2, dissolved oxygen is 40%-60%, ST-2014S cell suspension growth curve is as follows: figure 1 shown;

[0034] ③Determination of cell den...

Embodiment 2

[0037] Embodiment 2 The method for cultivating classical swine fever virus by using bioreactor suspension culture ST cells

[0038] Cultivate and expand suspension ST cells in shake flasks, press 7.5×10 5 Cells / ml density, inoculated into bioreactor, cell culture conditions: reactor working volume 10 liters, cell culture temperature 37 ℃, pH value 7.0, dissolved oxygen 50%, culture 72 hours, cell density 32.7 ×10 6 Each / ml, replace with virus culture maintenance medium containing 1.0% newborn bovine serum, and inoculate CSFV strain C according to 6% of the volume of virus culture maintenance medium. The virus culture conditions are: temperature 36°C, pH value 7.0 , the dissolved oxygen was 45%, and the first batch of virus liquid was harvested after 72 hours of cultivation, and the virus was continued to be injected with the virus maintenance liquid to continuously cultivate the virus. A total of 3 batches of virus liquid were harvested. Determine the heat reaction standard,...

Embodiment 3-5

[0039] Embodiment 3-5 Utilize bioreactor suspension culture ST cell to cultivate the method for pseudorabies virus, porcine transmissible gastroenteritis virus, porcine parvovirus

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Abstract

The invention relates to ST cells adapted to full suspension culture and its application in cultivating vaccine viruses, belonging to the field of biotechnology. The invention discloses a ST cell adapted to full suspension culture, which is named as the pig testicular cell suspension adapted strain ST-2014S, which has been preserved in the China Center for Typical Culture Collection on May 13, 2015, and the preservation number is CCTCC C201567. The invention also discloses a method for cultivating vaccine virus by using ST cell suspension adapted strain. Compared with the prior art, the ST cell culture vaccine virus disclosed by the present invention has a high degree of automation, does not require carrier intervention, and can be cultured in suspension in serum-free or low-serum medium, solving the problem of large-scale virus culture. In order to meet the requirements of industrialization, the development of large-scale production methods that can meet the requirements of GMP production technology has a good prospect for industrial application.

Description

technical field [0001] The invention relates to a ST cell suitable for full suspension culture and its application in cultivating vaccine virus and a method for cultivating vaccine virus, belonging to the field of biotechnology. Background technique [0002] Porcine testis (swine testis, referred to as ST) cells are engineered cell lines transformed from primary cells of swine testis through passage cloning. They grow adherently in vitro and can be continuously subcultured. ST cells are resistant to various swine disease viruses. Relatively sensitive, such as swine fever virus (CSFV), porcine parvovirus (PPV), pseudorabies virus (PRV), porcine transmissible gastroenteritis virus (TGEV), etc., are currently widely used in the preparation of these swine disease vaccine viruses . [0003] Since 1962, Capstick et al. have domesticated BHK21 cells to achieve suspension culture and applied them to vaccine production. After half a century of development, the application of cell su...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071C12N7/00C12R1/93
Inventor 李少英徐树兰吴华伟徐光科
Owner 郑州爱科生物科技有限公司
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