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Low-serum culture method of PK15 cells, PK15 cells obtained by method and application thereof

A technology of cell culture and culture method, which is applied in the field of PK15 cells, can solve the problems of no application instructions, etc., and achieve the effects of reducing the risk of pollution, simplifying the virus culture process, and reducing the amount of use

Inactive Publication Date: 2016-09-21
GUANGDONG WENS DAHUANONG BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] There are domestic research reports on the low-serum culture and domestication of PK15 cells, such as patent application number 201510098627.X "Method for producing porcine circus type II DBN-SX07 strain virus by culturing PK-15 cells with low serum", which discloses a PK15 cells were domesticated by gradually reducing serum to adapt to low serum culture, but there is no instruction on its application in the cultivation and propagation of porcine transmissible gastroenteritis virus (TGEV)
[0004] And about the report of porcine transmissible gastroenteritis virus (TGEV) low-serum culture, such as patent application number 201510098147.3 "low-serum culture ST cell production method of porcine transmissible gastroenteritis virus strain virus", but did not adapt to low serum Systematic PK15 cell culture report of porcine transmissible gastroenteritis virus

Method used

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  • Low-serum culture method of PK15 cells, PK15 cells obtained by method and application thereof
  • Low-serum culture method of PK15 cells, PK15 cells obtained by method and application thereof
  • Low-serum culture method of PK15 cells, PK15 cells obtained by method and application thereof

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Experimental program
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Embodiment 1

[0058] A low serum culture method for PK15 cells, comprising the following steps:

[0059] 1) The adherent cultured PK15 cells were recovered, and then placed in DMEM cell culture medium containing 8% bovine serum, digested and passaged with EDTA-trypsin digestion solution, and cultured until the cells covered a single layer; then the volume Use EDTA-trypsin digestion solution at a ratio of 1:3 to digest and passage the cells, and subculture for 3 generations;

[0060] 2) Place the PK15 cells treated in step 1) in MD611 cell culture medium containing 3% bovine serum at a volume ratio of 1:2.5 to digest and passage the cells with EDTA-trypsin digestion solution, and culture until the cells are confluent Monolayer; then use EDTA-trypsin digestion solution at a volume ratio of 1:3 for digestion and passage until the cells are confluent in the monolayer;

[0061] 3) Place the PK15 cells treated in step 2) in MD611 cell culture medium containing 1-2% bovine serum at a volume ratio...

Embodiment 2

[0066] The PK15 cell that obtains by embodiment 1 is applied to the proliferation culture of TGEV Chinese virus strain, comprises the following steps:

[0067] Ⅰ) Prepare PK15 cells: After culturing PK15 cells until the monolayer is confluent, pour off the culture supernatant;

[0068] Ⅱ) Inoculation strain: insert the TGEV Hua strain into PK15 cells with a volume ratio of 1%, add MD611 cell culture medium containing 0.5% bovine serum; then place at a temperature of 37°C, 5% CO 2 cultivated in an incubator;

[0069] Ⅲ) Collect the virus liquid: culture for 48-72 hours until more than 80% of the PK15 monolayer cells have lesions, freeze and thaw repeatedly to harvest the virus liquid, and use this virus liquid as the first generation seed virus aF1;

[0070] Ⅳ) Repeat steps Ⅰ) to Ⅲ) to inoculate aF1 into new PK15 cells and collect the virus liquid to obtain the second generation seed virus aF2;

[0071] Ⅴ) Continuously repeat steps Ⅰ)~Ⅲ) to domesticate the seed poison of the...

Embodiment 3

[0077] The characteristic of this embodiment is that: in step II), MD611 cell culture medium containing 0.3% bovine serum is added; other steps and parameters are the same as in embodiment 2. Obtain seed viruses bF1, bF2, bF3, bF4, bF5, measure the virus content values ​​of bF3, bF4, bF5, use MD611 cell culture medium containing 0.3% bovine serum as virus diluent, and the cells used for virus titer measurement are for virus culture For PK15 cells, the average value was measured 3 times, and the virus content values ​​are shown in Table 2:

[0078] Table 2 Example 3 virus content value

[0079]

[0080]

[0081] The diseased cells of PK15 cells are as follows Figure 5 As shown, all PK15 cells can undergo pathological changes.

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Abstract

The invention relates to a low-serum culture method of PK15 cells, PK15 cells obtained by the method and application thereof. The low-serum culture method of the PK15 cells comprises the following steps: (1) taking adherent culture PK15 cells, recovering the PK15 cells, then carrying out digestion passage; (2) placing the cells in an MD611 cell culture solution containing 3% bovine serum for passage; (3) passaging the cells till the cells fully spread into a single layer according to a volume ratio of 1 to 2.5; and (4) passaging the cells till the cells fully spread into a single layer according to a volume ratio of 1 to 3 to obtain PK15 cells adapted to low-serum culture. The application is characterized by carrying out propagation culture of TGEV strains via the PK15 cells and comprising the following steps: preparing the PK15 cells; inoculating the virus strains; and collecting virus liquid. The TGEV strains can be subjected to continuous passage culture for at least five generations under the low-serum condition; through the TGEV strains, the PK15 cells have significant pathological changes.

Description

technical field [0001] The present invention relates to the technical field of biological cells, more specifically, to a method for culturing PK15 cells in low serum, the PK15 cells obtained by the method and applications thereof. Background technique [0002] At present, in the large-scale production of animal vaccines in China, the culture of passaged cells mostly contains high concentration of 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. Therefore, in vaccine production, the cost of bovine serum is the highest, and the quality of serum is also the most critical. Usin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N1/36C12N7/00
CPCC12N5/0602C12N1/36C12N7/00C12N2770/20051
Inventor 何玲陈瑞爱唐满华梁桂益
Owner GUANGDONG WENS DAHUANONG BIOTECH
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