Application of bovine kidney cells capable of being subjected to suspension culture to virus culture and vaccine production

A technology of suspension culture and virus culture, which is applied in the field of cell engineering, can solve the problems of complex large-scale production process, small batch of culture, and difficulty in production and application, so as to save repeated domestication work of cells, simple production scale-up process, convenient and reliable application Effect

Active Publication Date: 2017-09-26
JINYUBAOLING BIO PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

MDBK can usually be cultured with Kjeldahl bottles, spinner bottles or even reactor carriers, but the essence has not changed. The cells are all adherent culture techniques. The adherent culture technique has small batches, large batch-to-batch differences, low production efficiency, and labor-intensive Large, takes up a lot of space, low cell unit yield, complex large-scale production process
[0003] Virus culture or vaccine production such as bovine viral diarrhea / mucosal membrane, bovine infectious rhinotracheitis, and pseudorabies is usually amplified by static culture or spinner bottle culture of MDBK adherent cells, but the scale-up of the adherent culture process is complicated, There are many problems such as small batch size, large batch-to-batch variation, low production efficiency, high labor intensity, and large space occupation, low cell unit yield, and complex large-scale production process
In CN104162154A, it is proposed to use suspension culture technology to produce bovine viral diarrhea and bovine infectious rhinotracheitis dual inactivated vaccine. It uses domesticated MDBK cells and has not formed a suspension cell strain that can be stably passed on. Due to the uncertainty of cell suspension domestication Factors, not every time can be successfully replicated, there are great difficulties in production applications

Method used

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  • Application of bovine kidney cells capable of being subjected to suspension culture to virus culture and vaccine production
  • Application of bovine kidney cells capable of being subjected to suspension culture to virus culture and vaccine production
  • Application of bovine kidney cells capable of being subjected to suspension culture to virus culture and vaccine production

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Embodiment 1

[0045] Embodiment 1, acquisition and preservation of suspension bovine kidney cell line (suspension MDBK cell line)

[0046] The suspension MDBK cell strain in the present invention is a MDBK cell strain capable of suspension culture obtained by screening ordinary adherent MDBK cells through procedures such as low-serum adherent culture, low-serum suspension culture, and serum-free suspension culture.

[0047] The specific screening process is as follows:

[0048] 1. Adherent MDBK Cell Culture

[0049] Usually, the adherent MDBK cells are cultured at 37°C with 10% (volume percentage, V / V) newborn bovine serum (purchased from: Jinyuankang Bioengineering Co., Ltd.) in commercially available DMEM medium (purchased from: GIBCO), and the cells are After the monolayer is overgrown, digest and disperse with 0.25% (V / V) trypsin, dilute and subculture according to the ratio of 1:3 to 1:5, and expand the culture.

[0050] 2. Low serum adherent culture

[0051] Take the adherent MDBK ...

Embodiment 2

[0074] Embodiment 2, the acquisition of bovine kidney cell MDBK-S CGMCC No.11795 kinds of cell suspensions

[0075] The method for obtaining bovine kidney cell MDBK-S CGMCC No.11795 kinds of cell suspensions comprises the following steps:

[0076] 1) Recovery of bovine kidney cells MDBK-S CGMCC No.11795

[0077] Take out a suspension of frozen bovine kidney cell line MDBK-S CGMCC No.11795 from liquid nitrogen, thaw quickly in a water bath at 37°C-40°C, centrifuge at 1000rpm for 5min, discard the supernatant, and add the bovine kidney cell suspension above Resuspend the cells in the culture medium, transfer them to the Erlenmeyer shaker flask, add special suspension medium for bovine kidney cells, and adjust the cell density to 0.3-0.5×10 6 Cells / mL, placed in a shaker at 37°C, 100rpm for suspension culture.

[0078] 2) Passaging of bovine kidney cells MDBK-S CGMCC No.11795

[0079] Step 1) Sampling and counting after 48-72 hours of cell culture, if the cell density is ≥1.0×...

Embodiment 3

[0080] Example 3. Reactor fed-batch culture of bovine kidney cells MDBK-S CGMCC No.11795

[0081] Fed-batch culture of bovine kidney cells MDBK-S CGMCC No.11795, including the following levels and scale-up culture:

[0082] 1) Fed-batch culture in 5L reactor

[0083] First use bovine kidney cell-specific suspension medium to dilute the seed cell suspension (prepared in Example 2) of bovine kidney cell MDBK-S CGMCC No.11795 to a density of 0.3-0.5×10 6 cells / mL, then transfer 1000-2000mL of diluted cell suspension (according to the reactor conditions) to a 5L reactor, and set the cell culture conditions: temperature 37°C, dissolved oxygen 40-60% (saturation percentage), pH value 7.0-7.2, rotation speed 40-60rpm, until the cell density reaches 0.8×10 6 When cells / mL (judging that the cells are growing vigorously), start feeding the special suspension medium for bovine kidney cells, the flow rate is 1000mL / day, stop feeding when the culture volume reaches 5L, and take it out to...

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Abstract

The invention discloses application of bovine kidney cells capable of being subjected to suspension culture to virus culture and vaccine production. The application has the advantages that a large quantity of manual operation for cell digestion, liquid change, frequent seed separation and the like can be omitted by bovine kidney cell (MDBK) suspension culture methods, the suspension culture methods are simple, convenient and speedy, processes can be automated by the aid of reactor culture, the cell yield of the unit volume can be increased, and the technical difficult problem of difference between adherent culture cell batches can be solved.

Description

technical field [0001] The invention belongs to the technical field of cell engineering, in particular to the application of suspensible cultured bovine kidney cells (MDBK) in virus culture and vaccine production after suspension culture. Background technique [0002] The bovine kidney cell (MDBK) passage cell line is derived from bull kidney cells, which can be used for virus culture, raw material inspection, and nutrient metabolism research. MDBK can usually be cultured by Kirschner flasks, spinner bottles or even reactor carriers, but the essence has not changed. The cells are all adherent culture processes. The adherent culture process has small batches, large batch-to-batch differences, low production efficiency, and labor intensity. It is large, takes up a lot of space, has a low yield per unit of cells, and has a complex large-scale production process. [0003] For virus culture or vaccine production of bovine viral diarrhea / mucosal membrane, bovine infectious rhinot...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N5/02C12N7/00A61K39/245A61K39/275A61K39/12A61K39/265A61K39/155A61P31/22A61P31/20A61P31/14C12R1/93
CPCA61K39/12C12N5/0686C12N7/00A61K2039/5252A61K2039/5254C12N2710/16034C12N2710/16051C12N2710/16063C12N2710/16734C12N2710/16751C12N2710/16763C12N2710/24263C12N2710/24234C12N2710/24251C12N2760/18534C12N2760/18551C12N2760/18563C12N2770/24351C12N2770/24363C12N2770/24334
Inventor 郝鹏刘国英温谢张燕红羡东堡杨波孔彩平田志辉路荣李超宋志刚范秀丽张贵刚
Owner JINYUBAOLING BIO PHARMA CO LTD
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