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MDCK cell line, method for duplicating viruses and application of method

A cell line, virus technology, applied in the direction of viruses, animal cells, antiviral agents, etc., to achieve the effect of reducing the risk of foreign contamination

Inactive Publication Date: 2018-08-03
吉林冠界生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Despite this, the commonly used cell lines for cultivating influenza virus still have various disadvantages, so it is still unsatisfactory.

Method used

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  • MDCK cell line, method for duplicating viruses and application of method
  • MDCK cell line, method for duplicating viruses and application of method
  • MDCK cell line, method for duplicating viruses and application of method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] This example provides a method for preparing MDCK cells with a deposit number of CCTCC NO: C201857, the steps are as follows:

[0038] Adherent cultured MDCK cells were introduced from ATCC by the Gansu Animal Cell Engineering Technology Research Center (GsACC) of Northwest University for Nationalities. The introduction time: February 2011, ATCC number: CCL-34, generation: P56, preservation number: 58860056 . A cell bank was established after GsACC expansion and culture, and the number of the cell bank is: GsACC2B0000090.

[0039] The MDCK cells were revived with DMEM / F12 medium containing 10% FBS, grown densely and then passaged.

[0040] Select well-grown cells and gradually domesticate and cultivate them, as follows:

[0041] Subcultured with DMEM / F12 medium containing 8% fetal bovine serum for 3 times;

[0042] Subcultured 5 times with DMEM / F12 medium containing 5% fetal bovine serum;

[0043] Subcultured 5 times with DMEM / F12 medium containing 2% fetal bovine s...

Embodiment 2

[0058] This example provides a method for culturing MDCK cells with a deposit number of CCTCC NO: C201857.

[0059] This experimental example provides the culture effect of the GJ-pyj201 medium provided by the present invention on MDCK cells with the deposit number CCTCC NO: C201857.

[0060] 1. Method

[0061] 1.1 Preparation of shake flask cells

[0062] Resuscitate the frozen suspended cells from liquid nitrogen according to the conventional method, add the cell solution into a 500ml Erlenmeyer flask with a pipette, and add filter-sterilized serum-free medium with a pH value of 7.0±0.2 to 100ml. Place the shaker flask in a shaker at 37°C, 120r / min-140r / min for cultivation and propagation. When the number of cells is sufficient, the reactor can be inoculated for culture.

[0063] 1.2 5L reactor cell culture

[0064] Take the suspended MDCK cells cultured in shake flasks, and dilute them with 1.0~2.0×10 6 The density of cells / ml is inoculated into a 5L reactor for cultur...

Embodiment 3

[0088] Cultivate the G01, G02, and G03 cells screened in Example 1 according to the culture conditions of 1.1 to 1.4 in Example 2. The medium used is 3# medium. The only difference is that in step 1.4, the dissolved oxygen is controlled at 25 -35%, thereby simulating the local anoxic environment in the bioreactor.

[0089] Samples were taken to observe the cell viability at 0h and 24h of culture.

[0090] Table 3 Cell viability of different MDCK cells in hypoxic environment

[0091]

[0092]

[0093] Among them, G01 cells obviously have stronger tolerance to hypoxic environment.

[0094] Cultivate the G01 cells screened in Example 1 according to the culture conditions of 1.1 to 1.4 in Example 2, the only difference is that in step 1.4, first use 3# medium to filter them into the corresponding reactor in a sterile manner Pre-incubation for 24 hours to make the initial cell viability in the three reactors consistent. Then during the formal cultivation, the dissolved oxy...

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Abstract

The invention relates to the field of cell culture, in particular to an MDCK cell line, a method for duplicating viruses and application of the method. The cell line is preserved in China Center for Type Culture Collection, the preservation number is CCTCC NO: C201857, and the preservation time is February 10, 2018. The MDCK cell line provided by the invention is not only suitable for the full-suspension culture, but also can adopt animal-source-free serum culture medium to culture, so that the pollution risk of an external source in the virus culture or vaccine production can be reduced.

Description

technical field [0001] The invention relates to the field of cell culture, in particular to an MDCK cell line, a method for replicating a virus and an application thereof. Background technique [0002] The preparation of influenza virus vaccines has undergone a large-scale development process from animal tissues and organs to cell culture, and the cultivation of animal influenza viruses has also experienced large-scale culture from chicken embryos to mammalian cells, from microcarrier suspension culture of mammalian cells to whole-body culture. Suspension culture, the development process from the culture of animal-derived serum to the culture without animal-derived serum or components, combined with centrifugal purification and column chromatography purification, improves the controllability of animal influenza virus or antigen production methods, and also reduces vaccine production. Emergency response to injection. [0003] In spite of this, the commonly used cell lines fo...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N7/00C12N7/02A61K39/145A61P31/16
CPCC12N5/0686C12N7/00C12N2760/16151
Inventor 李莉陈宏金燕斌朱长动杨柳张丽娜杜鑫高晓庆唐东雪宋海岩付春杰石莹张丹孟令伟王琳雅王博
Owner 吉林冠界生物技术有限公司
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