Method for culturing porcine circovirus type 2 by microcarrier bioreactor

A bioreactor, porcine circovirus technology, applied in the biological field, can solve the problems of insufficient virus reproduction, low antigen titer, easy cell death, etc. The effect of increasing cell density

Active Publication Date: 2018-06-01
兆丰华生物科技(福州)有限公司 +3
View PDF5 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] This method has the following disadvantages: due to the limited surface area of ​​the cells cultured in the spinner bottle, the cell density is low when the cells are cultured, and the titer of the virus cultured after inoculation is also relatively low
[0006] There are following deficiencies in this method: 1, the cells are digested from the spinner bottle, and there are many cell clumps, and the cells are unevenly adhered to the wall in the microcarrier, and many clumps are attached to the microcarriers, and the clumps in the process of cultivating Cells are easy to die or fall off early, resulting in insufficient cell density and low titer of antigen harvested
2. The culture of circovirus is generally inoculated synchronously. After 48 hours of inoculation, the cells begin to shed a large number of cells. After 48 hours, due to the large number of shedding cells and insufficient virus reproduction, the titer of the antigen finally harvested is not high.
3. When harvesting the antigen, only the supernatant is harvested. Since there are still some infected cells on the carrier that cannot be shed during harvesting, a part of the virus is lost, resulting in a low titer of the harvested antigen.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for culturing porcine circovirus type 2 by microcarrier bioreactor
  • Method for culturing porcine circovirus type 2 by microcarrier bioreactor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] A method for cultivating porcine circovirus type 2 in a microcarrier bioreactor, comprising the following steps:

[0043] 1) Spinner bottle cell digestion: select spinner bottle PK-15 cells with good cell state, digest the cells with trypsin digestion solution with a mass fraction of 0.25%, and shake the digested cells well;

[0044] 2) Cell filtration: Use a 15-micron cell filter to filter cells to obtain cell fluid. Most of the filtered cells are single, and a small number of 2-3 cells are in the state. Take a small amount of filtered cell fluid and use maintenance solution for 5 Then take a small amount of diluted cell solution and add it to the cell counting plate for cell counting, and calculate the cell density of the filtered cell solution;

[0045] 3) Cell culture by inoculation with poison: add microcarriers into the bioreactor at 4 g / liter, and at the same time add nutrient solution containing serum containing 8 times the volume of microcarriers, turn on the a...

Embodiment 2

[0054] A method for cultivating porcine circovirus type 2 in a microcarrier bioreactor, comprising the following steps:

[0055] 1) Spinner bottle cell digestion: select spinner bottle PK-15 cells with good cell state, digest the cells with trypsin digestion solution with a mass fraction of 0.25%, and shake the digested cells well;

[0056] 2) Cell filtration: Use a 10-micron cell filter to filter the cells to obtain the cell fluid. The filtered cells are mostly single, and a small number of 2-3 cells. Take a small amount of the filtered cell fluid and use the maintenance solution for 4 Then take a small amount of diluted cell solution and add it to the cell counting plate for cell counting, and calculate the cell density of the filtered cell solution;

[0057] 3) Cell culture in tanks with poison: add microcarriers into the bioreactor at a rate of 3 g / liter, and at the same time add nutrient solution containing serum containing 7 times the volume of microcarriers, turn on the...

Embodiment 3

[0065] A method for cultivating porcine circovirus type 2 in a microcarrier bioreactor, comprising the following steps:

[0066] 1) Spinner bottle cell digestion: select spinner bottle PK-15 cells with good cell state, digest the cells with trypsin digestion solution with a mass fraction of 0.25%, and shake the digested cells well;

[0067] 2) Cell filtration: Use a 20-micron cell filter to filter the cells to obtain the cell fluid. The filtered cells are mostly single, and a small number of 2-3 cells. Take a small amount of the filtered cell fluid and use the maintenance solution for 6 Then take a small amount of diluted cell solution and add it to the cell counting plate for cell counting, and calculate the cell density of the filtered cell solution;

[0068] 3) Cell culture by inoculation with poison: Add microcarriers into the bioreactor at a rate of 5 g / liter, and at the same time add nutrient solution containing serum containing 9 times the volume of microcarriers, turn ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses a method for culturing porcine circovirus type 2 by a microcarrier bioreactor. The method comprises the following steps: (1) cell dissociation in a spinner bottle; (2) cell filtration; (3) cell virus receiving and tank charging culture; (4) 24 h liquid exchange; (5) gaining of a primarily received antigen; (6) second-time cell dissociation and tank charging; (7) gaining ofa secondarily received antigen and liquid exchange culture; (8) gaining of a thirdly received antigen; and (9) melting of the primarily received antigen, the secondarily received antigen and the thirdly received antigen, filtration to remove a microcarrier to obtain an antigen liquid for titer detection. By adoption of the method, the cell wall-adhering efficiency can be improved, cell aggregatesare reduced, the cell density is improved, the yield of single-batch antigens can be increased, the titer of the antigens is improved, and the production cost is lowered.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for cultivating porcine circovirus type 2 in a microcarrier bioreactor. Background technique [0002] The culture method of porcine circovirus type 2 is generally a spinner bottle culture and a microcarrier bioreactor culture. [0003] The spinner bottle culture process is generally as follows: use spinner bottle culture cells, after a period of culture in spinner bottle cells, use enzyme digestion solution to digest, subculture and amplify the culture, and at the same time carry out synchronous inoculation. Connect it to a spinner bottle for cultivation, change the medium after cultivating for a period of time, and continue to cultivate until 72-96h to harvest the virus liquid. [0004] This method has the following disadvantages: due to the limited surface area of ​​the spinner bottle cultured cells, the cell density is low when the cells are cultured, and the...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12N5/071C12N5/02C12R1/93
CPCC12N5/0686C12N7/00C12N2531/00C12N2750/10051
Inventor 江兴华张小兰阮鹭静陈丽萍崔龙萍朱鸿杰徐丽云
Owner 兆丰华生物科技(福州)有限公司
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap