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A method for domesticating vero cells suitable for serum-free culture system and its application

A serum-free culture and cell technology, applied in biochemical equipment and methods, animal cells, vertebrate cells, etc., can solve problems such as unclear components of bovine serum, rising prices, and limited sources

Active Publication Date: 2018-12-18
GUANGDONG WENS DAHUANONG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because bovine serum is a natural medium, its source is limited, and its price has been rising year after year, which increases the cost of raw materials for vaccine production; the composition of bovine serum is unclear and unstable, and the differences between batches affect the stability of vaccine products; in addition, There are certain risks in the serum supply chain, and there is a potential risk of foreign pathogen contamination

Method used

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  • A method for domesticating vero cells suitable for serum-free culture system and its application
  • A method for domesticating vero cells suitable for serum-free culture system and its application
  • A method for domesticating vero cells suitable for serum-free culture system and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] The invention discloses a vero cell acclimation method applicable to a serum-free culture system. After the frozen vero cells are revived, they are cultured in a serum-containing culture solution, and the vero cells are acclimated by gradually reducing the serum content.

[0034] Specifically include the following steps:

[0035] (1) resuscitating the frozen vero cells;

[0036] (2) Place the resuscitated Vero cells in DMEM medium containing 8% fetal bovine serum for culture. When the Vero cells grow to a single layer, add digestive solution Ⅰ to digest the volume of Vero cells before digestion and after digestion. The volume ratio of the vero cells was 1:3 for digestion and subculture, and cultured for 2-3 generations;

[0037] (3) The Vero cells obtained in step (2) are placed in the mixed culture solution I consisting of 80% serum culture solution and 20% serum-free culture solution by volume percentage and cultivated until the Vero cells grow When the monolayer is f...

Embodiment 2

[0044] The propagation culture method of porcine epidemic diarrhea virus (PEDV virus) in serum-free culture system, the DMEM that adopts is purchased from Hyclone, and batch number is NZH1194; VP-SFM is purchased from gibco company, and batch number is 1637720; The L-glutamine Amide was purchased from Sigma Company, batch number WXBB6280v.

[0045] Specifically include the following steps:

[0046] (1) Culture the Vero cells obtained by the domestication method described in Example 1 in VP-SFM culture medium, and after the Vero cells adapted to VP-SFM grow into a monolayer, pour off the supernatant of the VP-SFM culture medium , insert porcine epidemic diarrhea virus at a dose of 1-3% by volume, add maintenance solution I after adsorption for 1 hour; then place in 5% CO at 37°C 2 Cultivate in an incubator, observe the lesions of the monolayer cells every day, and when more than 70% of the monolayer cells have lesions, freeze and thaw 2-3 times to harvest the virus liquid, and...

Embodiment 3

[0050] The propagation culture method of porcine epidemic diarrhea virus (PEDV virus) in serum-free culture system, the DMEM that adopts is purchased from Hyclone, and batch number is NZH1194; VP-SFM is purchased from gibco company, and batch number is 1637720; The L-glutamine Amide was purchased from Sigma Company, batch number WXBB6280v.

[0051] (1) Culture the Vero cells obtained by the domestication method described in Example 1 in VP-SFM culture medium, and after the Vero cells adapted to VP-SFM grow into a monolayer, pour off the supernatant of the VP-SFM culture medium , insert porcine epidemic diarrhea virus at a dose of 1-3% by volume, add maintenance solution Ⅱ after adsorption for 1 hour; then place in 5% CO 2 Cultivate in an incubator. When more than 70% of the monolayer cells have lesions, freeze and thaw 2-3 times to harvest the virus liquid, and record it as the virus liquid II F1 generation;

[0052] (2) The virus liquid II F1 generation was inserted into the...

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Abstract

The invention discloses a vero cell domestication method suitable for a serum-free culture system. The vero cell domestication method comprises the following steps: reviving frozen vero cells; cultivating in a serum culture medium; and domesticating the vero cells by virtue of a domestication method of gradually reducing the serum content. The vero cells are domesticated and cultivated by virtue of the domestication method of gradually reducing the serum content, so that the vero cells can adapt to the serum-free culture environment; the vero cells which can adapt to the serum-free culture are obtained; and the domesticated vero cells can be continuously subcultured for at least 50 generations in a VP-SFM (serum-free medium).

Description

technical field [0001] The invention relates to the field of veterinary biological products, and more specifically relates to a vero cell domestication method suitable for a serum-free culture system and its application in a porcine epidemic diarrhea virus propagation and culture method. Background technique [0002] At present, in the production process of domestic animal vaccines, the proliferation and culture of passaged cells and viruses mostly contain bovine serum. Because bovine serum is a natural medium, its source is limited, and its price has been rising year after year, which increases the cost of raw materials for vaccine production; the composition of bovine serum is unclear and unstable, and the differences between batches affect the stability of vaccine products; in addition, There are certain risks in the serum supply chain, and there is a potential risk of foreign pathogen contamination. The serum-free (VP-SFM) culture system has no animal-derived components...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071C12N7/00
Inventor 何玲陈瑞爱唐满华梁桂益
Owner GUANGDONG WENS DAHUANONG BIOTECH
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