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Methods for culturing swine testis cell in serum-free manner and producing classical swine fever cell vaccine by using swine testis cell

A serum-free culture, pig testis technology, applied in animal cells, vertebrate cells, artificial cell constructs, etc., can solve the problems of introducing other viruses and pathogens, increasing costs, introducing foreign proteins, etc. Identity, simplified processing, well-defined effects

Active Publication Date: 2014-02-26
CHONGQING AULEON BIOLOGICALS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the use of animal serum in the vaccine production process will increase costs, introduce foreign proteins, and introduce risks of other viruses and pathogens, which is a thorny problem faced by cell vaccines in the production process.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Serum-free culture of porcine testicular cells

[0030] 1) The porcine testicular cells were cultured with a medium containing 10% calf serum. When the cells grew to 60%, the medium containing 5% calf serum was replaced for adaptation and when cells died and suspended, replaced with fresh culture Base, when the cells are congested, use the medium containing 10% calf serum to subculture for 3-5 generations;

[0031] 2) When the cells adhere to the wall, replace the medium containing 5% calf serum for culture, replace the medium containing 2% calf serum when the cells grow to 60% for adaptation and replace when cells die and suspend Fresh medium, when the cells are full, subculture with medium containing 5% calf serum for 3-5 generations;

[0032] 3) After the cells adhere to the wall, replace the medium containing 2% calf serum for culture, replace the medium containing 1% calf serum when the cells grow to 60% for adaptation and replace when cells die and bec...

Embodiment 2

[0042]Example 2: Cell culture and cell batch bank establishment

[0043] The domesticated and screened serum-free cultured porcine testicular cells are collected in DMEM medium, added with gentamicin, cultured in cell factories or cell fermenters, and established cell working seed batch banks, and the foreign aid source factors, pathogenic factors after passage Tumorability and stability were comprehensively tested, and the number of cell generations used was controlled within 40 generations, all of which met the requirements of vaccine production media.

Embodiment 3

[0044] Embodiment 3: cell morphology and stability identification

[0045] The porcine testicular cells were cultured at 37°C under serum-free conditions and continuously passaged. The cells were observed every 24 hours. The adherence and cell morphology did not change.

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Abstract

The invention discloses methods for culturing a swine testis cell in a serum-free manner and producing a classical swine fever cell vaccine by using the swine testis cell. The method for culturing the swine testis cell in the serum-free manner comprises the following steps: culturing the swine testis cell by a culture medium containing 10% calf serum; when the cell grows to 60%, changing a culture medium containing 5% calf serum to carry out adaptive culture; when the cell is dead and is suspended, replacing a new culture medium; when the cell completely grows, carrying out subculture by using a culture medium containing 10% calf serum for 3-5 generations; repeating the steps, and gradually reducing the serum content to 5%, 2% and 1% from 10% till realizing serum-free culture; and finally, screening a pure cell line by using a disappearing dilution method. According to the methods, the swine testis cell can be obtained by serum-free habituated culture; the swine testis cell can enable classical swine fever viruses to be multiplied and is applied to the production of a classical swine cell vaccine, thus not only is the risk caused by use of animal serum avoided, but also the components of the culture mediums are clear, the identity among batches is improved and the downstream purifying process is simplified.

Description

technical field [0001] The invention belongs to the technical field of vaccine preparation, and relates to a serum-free method for cultivating porcine testicular cells and using the same to produce a swine fever cell vaccine. Background technique [0002] Classical swine fever (CSF) is a highly contagious febrile disease caused by classical swine fever virus (CSFV). In 1984, the World Organization for Animal Health (OIE) listed it as a Class A infectious disease, and it is also designated as a Class I infectious disease in my country. In addition to causing sepsis, swine fever can also cause a series of other clinical manifestations, such as abortion in pregnant sows, fetal deformation, chronic nutritional consumption, lymphocyte and thrombocytopenia, etc. At present, most countries in the world have adopted strict sanitation and epidemic prevention measures and eliminated swine fever-infected pigs to purify pig herds to control and eliminate swine fever. Some developed co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071A61K39/187A61P31/14
Inventor 李阳春曹政岳秀阳李春燕
Owner CHONGQING AULEON BIOLOGICALS
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