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Serum-free whole suspension MDCK cell strain and application thereof to production of influenza viruses

An influenza virus, full-suspension technology, applied in the direction of viruses, animal cells, viruses/phages, etc., can solve the problems of reducing the immune effect of vaccines, greatly affecting the quality of viruses, and increasing the pressure of purification, so as to achieve cell density and reduce cultivation volume, to maximize the effect

Active Publication Date: 2017-12-12
兆丰华生物科技(南京)有限公司北京生物医药科技中心 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are some shortcomings in the production of chicken embryos: first, it requires a lot of labor and occupies a large area; second, the quality of the virus is greatly affected by the quality and batch of chicken embryos; third, the supply of SPF chicken embryos; There are more miscellaneous proteins in the vaccine, which reduces the immune effect of the vaccine and increases the pressure of later purification
However, the domestication method of digesting the cells and directly using serum-free medium for adaptive passage without several generations of adaptation in an adherent environment has not been reported yet.
[0006] At present, there are many related reports on the production of avian influenza and human influenza by using MDCK cell microcarrier suspension or full suspension culture, but there is no report on the production of swine influenza virus by using serum-free full suspension culture MDCK cell technology, and there is still no full-suspension virus on the market. Swine Influenza Vaccine Prepared by Suspension Culture MDCK Cells

Method used

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  • Serum-free whole suspension MDCK cell strain and application thereof to production of influenza viruses
  • Serum-free whole suspension MDCK cell strain and application thereof to production of influenza viruses
  • Serum-free whole suspension MDCK cell strain and application thereof to production of influenza viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1 Preparation of serum-free full suspension MDCK cell line

[0036] S1. Passage of adherent MDCK cells:

[0037] Take a T75 square flask to culture MDCK cells covered with a monolayer, digest the cells with 0.25% trypsin, blow and disperse the cells with DMEM / F12 (10% FBS) cell growth medium, add 20ml of cell growth medium, and place the MDCK cells in 37°C, 5% CO 2 Cultivate in a carbon dioxide incubator for 48-72h, and carry out amplified culture when a monolayer of cells is formed. The above cell growth medium is DMEM / F12 culture medium with 10% Jinyuankang fetal bovine serum.

[0038] S2. One-step adaptation method to adapt adherent cells to full suspension cells cultured without serum:

[0039] S21. Cell domestication process:

[0040] The MDCK cells expanded in step S1 were digested with 0.25% trypsin and inoculated into serum-free medium, placed on a shaker for shaking culture, and the initial inoculation density was 0.5×10 6 -2.0×10 6 Cells / ml, e...

Embodiment 2

[0048] The clone screening of embodiment 2MDCK-S cell line

[0049] The MDCK-S cell line in Example 1 was screened for 3 rounds of clones by the limiting dilution method, and 3 cell lines with plump morphology, bright edges, faster growth rate and higher cell viability were obtained. In order to investigate the difference of these 3 clonal cell lines, the 3 lines of cells were divided into 0.5×10 6 Cells / ml were inoculated in 125ml shake flasks with a culture volume of 30 ml, placed at 37°C, 5% CO 2 Culture in a carbon dioxide incubator, take out 1ml of cell solution at 48h, count the cells after staining with trypan blue and calculate the cell viability. The cell density and viability of the three cell lines are shown in Table 1.

[0050] Table 1 Growth of three MDCK-S cell lines

[0051]

[0052] The MDCK-S3 cells with the fastest growth and highest activity were preserved on March 28, 2016 in the General Microbiology Center of the China Committee for the Collection of...

Embodiment 3

[0053] Example 3 Production of H1N1 Subtype Swine Influenza Virus by Suspension Culture MDCK-S Cells

[0054] S1. Cell recovery

[0055] Take out the frozen MDCK-S cells from the liquid nitrogen tank, put them in a 37°C water bath to thaw quickly, add an appropriate amount of medium to the thawed cells and centrifuge to remove dimethyl sulfoxide, and then resuspend the cells with fresh medium and cultured in shake flasks. The revived cells went through an adaptation period of 2-3 passages, and the experiment was carried out when the cell growth state was stable.

[0056] Propagation of S2.H1N1 subtype swine influenza virus cytotoxic species:

[0057] MDCK-S cells were divided into 0.3×10 6 -1×10 6 The cell density of each / ml was inoculated in serum-free medium for suspension culture, when the cell density reached 2×10 6 -3×10 6 Individuals / ml, inoculate the H1N1 subtype swine influenza virus produced by chicken embryos at a ratio of 0.1-0.01% (v / v), cultivate the harvest...

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Abstract

The invention discloses a serum-free whole suspension cultured MDCK cell strain domesticated by a one-step adaptation method and application thereof to preparation of influenza virus vaccines, and belongs to the technical field of veterinary biology. Adherent cells are domesticated into a single dispersed whole suspension MDCK cell line named MDCK-S within two months by the one-step adaptation method, and the strain preservation number is CGMCC No. 12256. The invention further provides a method of culturing influenza viruses using the cell line. The method for producing the H1N1 and H3N2 subtype swine influenza viruses through MDCK cell suspension culture can replace traditional chicken embryo production to significantly reduce the production cost and improve downstream purification efficiency, and can rapidly and steadily increase the production scale.

Description

technical field [0001] The invention relates to a suspended MDCK cell strain domesticated by a one-step adaptation method and its application in cultivating and producing swine influenza virus vaccine. The invention belongs to the technical field of veterinary biology. Background technique [0002] Swine influenza (SI) is an acute, contagious respiratory disease caused by type A influenza virus. The case fatality rate of pigs infected by swine flu alone is low, but it often causes concurrent or stimulates other virus or bacterial infections in pigs, increasing the mortality of pigs. At the same time, pigs are a mixture of human influenza and avian influenza, and there is a risk of infecting humans, so it has important public health significance. Vaccination is by far the best way to prevent swine flu. [0003] The use of chicken embryos to cultivate influenza virus is a classic method for human and avian influenza inactivated vaccines. At present, the only swine flu vacc...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N5/02C12N7/00C12N7/02C12R1/91
CPCC12N5/0686C12N7/00C12N2500/90C12N2760/16051
Inventor 孔文刚王贵华赵亚荣闫林刘飞郎洪彬刘天伦冯鹏
Owner 兆丰华生物科技(南京)有限公司北京生物医药科技中心
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