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Domestication method of full suspension culture-type MDCK cell line

A full-suspension, cell line technology, applied in the field of cell culture, can solve the problems of inability to meet the needs of virus vaccine production, unsuitable for large-scale culture, and decreased virus yield, etc., to facilitate large-scale production, reduce site and labor input, reduce The effect of the chance of contamination

Pending Publication Date: 2020-09-18
ZHAOQING DAHUANONG BIOLOGIC PHARMA
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

However, there are some defects in this culture method. For example, the virus yield is reduced due to the limitation of the surface area of ​​the substrate in the adherent culture; Serum culture technology is an effective tool to elucidate the basic issues of cell generation, proliferation, differentiation and gene expression regulation, but even serum-free adherent culture is only suitable for experimental research, not for large-scale culture, and cannot meet the actual production of virus vaccines need

Method used

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  • Domestication method of full suspension culture-type MDCK cell line
  • Domestication method of full suspension culture-type MDCK cell line
  • Domestication method of full suspension culture-type MDCK cell line

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0030] 1. Experimental materials Frozen adherent cultured MDCK cell line, DMEM, F12, RPMI1640 medium, 0.25% EDTA trypsin, trypan blue staining solution, cell square flask, cell shaker flask, pipette.

[0031] 2. Instruments and equipment CO 2 Incubators, orbital shakers, inverted microscopes, Countstar cell counters, low speed refrigerated centrifuges.

[0032] 3. Method

[0033] 3.1 Acclimatization of MDCK cells in serum-free suspension

[0034] Step 1. Mix DMEM, F12 and RPMI1640 according to the ratio of 3:1:1, and use this as the basal medium, add 12%, 8%, 5%, 3%, 1%, 0% fetal bovine serum respectively Configured as DMEM-F12-RPMI1640 medium with serum or DMEM-F12-RPMI1640 medium without serum;

[0035]Step 2. Resuscitate an adherent cultured MDCK cell, inoculate and culture it with DMEM-F12-RPMI1640 containing 12% fetal bovine serum as described in step 1. The culture condition is 37°C, 5% CO 2 , pH7.2; after the cells adhered to the wall and the confluence of the monol...

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Abstract

The invention relates to the field of cell culture, in particular to a domestication method of a full suspension culture-type MDCK cell line. According to the domestication method, adherent MDCK cellsare domesticated into a low-serum full suspension culture-type MDCK cell line and a serum-free full suspension culture-type MDCK cell line, so that traditional production of an avian influenza virusthrough chicken embryos can be replaced, the subsequent separation and purification efficiency is improved, further upgrade of a process is promoted, a culture process is also simplified while the cost is reduced, and the domestication method is easier to industrialize.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a domestication method of a full suspension culture type MDCK cell line. Background technique [0002] Bird Flu (Bird Flu or Avian Influenza) is an acute infectious disease caused by a subtype of influenza A virus (also known as bird flu virus), which can infect humans, and the mortality rate of humans after infection with bird flu is over 30%. . Up to now, no ideal treatment drug has been found for bird flu, and vaccination is still the most effective means to prevent the occurrence and spread of the disease. [0003] The traditional production process of avian influenza vaccine is chicken embryo culture method. This method of using chicken embryos as the production substrate has been used for many years, and the technology is mature. There is a big problem in the supply of chicken embryos due to the large-scale outbreak. Using cells as the medium is the current trend of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N5/02A61K39/145A61P31/16
CPCC12N5/0686A61K39/12A61P31/16C12N2500/90C12N2760/16134
Inventor 施维松卢少华詹烜子刘德城郑彩丽孔凤英谢玉红
Owner ZHAOQING DAHUANONG BIOLOGIC PHARMA
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