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ST cell adapting to full-suspension culture, and application thereof, and vaccine virus culturing method

A full suspension, cell technology, applied in the biological field, can solve problems such as inability to industrialize large-scale production, inability to linearize amplification, and product availability, achieve good industrial application prospects, meet the requirements of large-scale industrialization, and have a high degree of automation. Effect

Active Publication Date: 2015-12-02
郑州爱科生物科技有限公司
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  • Abstract
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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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  • ST cell adapting to full-suspension culture, and application thereof, and vaccine virus culturing method
  • ST cell adapting to full-suspension culture, and application thereof, and vaccine virus culturing method

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Embodiment 1 adapts to the expansion culture of full suspension culture ST cell line

[0032] The porcine testis cell suspension-adapted strain ST-2014S preserved in China Typical Culture Collection Center with the preservation number CCTCCC201567 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:

[0033] 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;

[0034] 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;

[0035] ③Determination of cell density in ...

Embodiment 2

[0037] Embodiment 2 Utilizes the method for bioreactor suspension culture ST cell culture classical swine fever virus

[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, an...

Embodiment 3-5

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

[0040] The ST-2014S cell suspension culture method is the same as in Example 2. When cultivating for 72 hours, the cell growth medium is replaced with a virus culture maintenance medium. The virus culture conditions are appropriately adjusted according to the difference of the inoculated virus in the specific embodiment. Referring to Table 2, all viruses The determination of the titer was carried out according to the routine test method, and the test results are shown in Table 2.

[0041] Example 3 4 5 Bioreactor working volume (liter) 9 10 10 Cell seeding density (10 5 pcs / ml) 8.2 8.7 9.0 72h cell density (10 5 pcs / ml) 32.9 33.4 34.5 Vaccination PRV Bartha-k61 TGEV Hua strain PPV HN14 Serum content of virus maintenance medium % 0.5 1.0 1....

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Abstract

The invention relates to an ST cell adapting to full-suspension culture, and an application in vaccine virus culturing, and belongs to biotechnical field. The ST cell adapting to full-suspension culture is named as a swine testicle cell suspension adaptation strain ST-2014S, and is preserved in China Center for Type Culture Collection on May 13, 2015 with the preservation number of CCTCC C201567. The invention also discloses a method for culturing a vaccine virus through using the full-suspension adaptation ST cell. Compared with vaccine viruses in the prior art, the full-suspension adaptation ST cell cultured vaccine virus has the advantages of high automation degree, no need of vector intervention, realization of suspension culture in a serum-free or low-serum medium, meeting of large-scale industrial requirements of virus culture, development of a large-scale production method meeting GMP production technology requirements, and very good industrial application prospect.

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 (swinetestis, referred to as ST) cells are engineered cell lines transformed from primary cells of porcine testis through passage cloning. They grow adherently in vitro and can be continuously subcultured. Comparison of ST cells against various swine disease viruses 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 suspension...

Claims

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Application Information

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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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