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A low-serum medium for high-density suspension culture of bhk-21 cells and its application in the proliferation of foot-and-mouth disease virus

A BHK-21, suspension culture technology, applied in the direction of cell culture active agent, virus, culture process, etc., can solve the problems that cannot meet the high-density culture of cells, can not support the reactor culture mode, etc., to improve the effective utilization rate, Optimizing the nutritional environment of cells and reducing the concentration of metabolic by-products

Active Publication Date: 2019-03-15
CHINA AGRI VET BIO SCI & TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Ordinary medium cannot meet the high-density culture of cells in the reactor, nor can it support the unique culture method of the reactor

Method used

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  • A low-serum medium for high-density suspension culture of bhk-21 cells and its application in the proliferation of foot-and-mouth disease virus
  • A low-serum medium for high-density suspension culture of bhk-21 cells and its application in the proliferation of foot-and-mouth disease virus
  • A low-serum medium for high-density suspension culture of bhk-21 cells and its application in the proliferation of foot-and-mouth disease virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1 BHK-21 Cell Nutritional Metabolism Research and Determination of Medium Formula

[0079] 1 material

[0080] 1.1 BHK-21 cells were obtained from the working cell bank of BHK-21 suspension cells established by Zhongnong Weite Biotechnology Co., Ltd., the batch number is BHK-21 / W / S-201202, and they were used after they passed the scale-up culture test.

[0081] 1.2 Serum Newborn bovine serum was purchased from a qualified supplier and used after passing the internal control quality standard for newborn bovine serum of Zhongnong Weite Biotechnology Co., Ltd.

[0082] 1.3 Reagents and equipment

[0083] Main reagents: American waters amino acid analysis column (5μm, 150×4.6mmol); 20 amino acid standard samples; acetonitrile (chromatographically pure); sodium acetate (analytical pure); acetic acid (analytical pure); triethylamine (TEA); Phenylisothiocyanate (PITC), etc.

[0084] Main instruments: cell analyzer (Innovatis, Germany); American Waters high-performan...

Embodiment 2

[0125] Application of embodiment two personalized culture medium under different culture scales

[0126] 1 material

[0127] 1.1 BHK-21 cells were taken from the working cell bank of BHK-21 suspension cells established by Zhongnong Weite Biotechnology Co., Ltd., the batch number is BHK-21 / W / S-201201, and they were used after they passed the scale-up culture test.

[0128] 1.2 Serum Newborn bovine serum was purchased from a qualified supplier and used after passing the internal control quality standard for newborn bovine serum of Zhongnong Weite Biotechnology Co., Ltd.

[0129] 1.3 Medium

[0130] In the BHK-21 low serum medium ZN-MEM of the present invention, the medium includes amino acid part, balanced salt part, vitamin part, other additives, serum and distilled water, with a pH value of 7.0 to 7.2, wherein the added concentration of serum is 1 %(v / v), the final concentration of other raw materials in the medium is shown in Table 3 below:

[0131]

[0132]

[0133]...

Embodiment 3

[0160] Effect of BHK21 cells cultured in different mediums on virus proliferation in embodiment three

[0161] 1 Materials and methods

[0162] 1.1 Materials

[0163] 1.1.1 Cells

[0164]BHK-21-P cells were purchased from ATCC, source: Hamster Syrian Kidney, preserved by Zhongnong Weite Biotechnology Co., Ltd., and the test cells have been passed to the F18 generation.

[0165] 1.1.2 Medium

[0166] BHK-21 cell commercialized low-serum medium, namely: BHK-21MEM, BHK-21GMEM, SLM BHK-21, respectively named A, B and C medium; the medium ZN-MEM of the present invention, named D culture Base, the formula is the same as the medium in Example two.

[0167] 1.1.3 Medium supplements

[0168] Glutamine, Pluronic F-68, ferric citrate, transferrin, putrescine, hormones, and lipids were purchased from SIGMA Company, and soybean protein hydrolyzate, ultrafiltered plant peptone, yeast extract, etc. were purchased from Beijing Reagent Company.

[0169] 1.1.4 Other reagents and equipment...

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Abstract

The invention discloses a low-serum culture medium for high-density suspension culture of BHK-21 cells and an application of the low-serum culture medium in proliferation of FMDVs (foot and mouth disease viruses). According to researches on the metabolism level and mutual adjustment of nutritional substances such as glucose and the like in the growth process of the BHK-21 cells, the low-serum culture medium for individual BHK-21 cells is provided and comprises an amino acid part, a vitamin part, a balanced salt part and other additive parts. The culture medium is used for high-density suspension culture of the BHK-21 cells, the effective utilization rate of the nutritional substances by the cells can be greatly increased, the concentration of metabolism byproducts is reduced, and accordingly, the cell culture efficiency is improved; besides, the BHK-21 cells cultured by the culture medium have increased sensitivity for different serums of animals suffering from FMDV, the content of effective antigens 146S of the viruses is substantially increased, the purposes of high cell growth efficiency, cell growth controllability and minimization of the concentration of the metabolism byproducts are achieved, the production efficiency of biological products is higher, the production cost is lower, and the low-serum culture medium has remarkable economic benefits.

Description

technical field [0001] The invention relates to the development of a BHK-21 cell full-suspension high-density low-serum individualized culture medium and its application in the proliferation of foot-and-mouth disease virus, belonging to the field of veterinary biopharmaceuticals. Background technique [0002] With the rapid development of biological product technology, the current production process of foot-and-mouth disease inactivated vaccine in my country has developed from the use of traditional spinner bottle adherent cell culture to large-scale cell suspension culture in bioreactors. The use of bioreactors for large-scale cell culture can greatly increase the unit output of large-scale animal cell culture, realize high-density culture and high-density expression of cells, simplify the production process, reduce production costs, and ensure large-scale production of products quality. However, ordinary medium cannot meet the high-density culture of cells in the reactor,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071C12N7/00C12R1/93
CPCC12N5/0686C12N7/00C12N2500/05C12N2500/24C12N2500/30C12N2500/32C12N2500/34C12N2500/38C12N2500/40C12N2500/50C12N2500/74C12N2501/998C12N2501/999C12N2770/32151
Inventor 刘学荣武发菊安芳兰殷宏董文教葛玉凤张荣孙玉霞张云德
Owner CHINA AGRI VET BIO SCI & TECH
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