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Mdck-derived cell strain suspension-cultured in protein-free medium and method for proliferating virus using cell strain

A technology of protein-free medium and suspension culture, which is applied in the field of cell lines, can solve the problems that egg protein cannot be completely removed, vaccines are not suitable for vaccination, and high tumorigenicity, etc., and achieve low tumorigenicity, uniform productivity, and high productivity Effect

Inactive Publication Date: 2016-03-02
MOGAM BIOTECH RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the use of fertilized eggs has the following disadvantages: chicks suitable for production should be raised and managed; influenza vaccine production should be adjusted to the production range of fertilized eggs; allergic person
[0004] The Madin Darby canine kidney (Madin Darby canine kidney (hereinafter referred to as "MDCK") cell line established from canine kidney exhibits strong surface adhesion; has been cultured using animal serum; and requires a large space for mass production, thereby high cost
Additionally, adherent MDCK cell lines have been reported to display high tumorigenicity

Method used

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  • Mdck-derived cell strain suspension-cultured in protein-free medium and method for proliferating virus using cell strain
  • Mdck-derived cell strain suspension-cultured in protein-free medium and method for proliferating virus using cell strain
  • Mdck-derived cell strain suspension-cultured in protein-free medium and method for proliferating virus using cell strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1: Selection process for candidate cell lines

[0056] 1.1. Thawing Adherent MDCK Cells

[0057] Cryopreserved adherent MDCK cells (obtained from: ATCC, CCL-34) were thawed and cultured in 15 mL of serum medium (EMEM medium supplemented with 10% FBS) (obtained from: Lonza). Place cells stored in LN2 tanks in a 37 °C water bath and thaw. Add the resuspended cell solution to a T75 flask containing serum medium, then incubate at 37 °C and 5% CO 2 cultured in an incubator.

[0058] 1.2. Adhesive culture

[0059] After 3 to 4 days, the cells obtained from 1.1 were subcultured at a ratio of 1:4 to 1:30. The parental cells were passed to passage 55. Subculture was carried out by the following procedure.

[0060] Take the T75 bottle out of the incubator in 1.1, remove the medium and wash. Cells were detached by treatment with 0.25% trypsin-EDTA and resuspended to the desired ratio by pipetting the serum medium. Subculture was performed every 3 to 4 days at a...

Embodiment 2

[0081] Example 2: Comparative analysis of candidate cell lines and selection of production cell lines

[0082] 2.1. Comparison of cell line stability by long-term subculture

[0083] 2.1.1. Candidate cell line A

[0084] To determine the cell growth of candidate cell line A at each passage, cell line A was subcultured by centrifuging the cells and removing the medium, then adding new medium to adjust the cell number to 5e+05 cells / mL every 3 to 4 days for 100 days. Candidate cell line A subculture results in Figure 5 shown in .

[0085] Such as Figure 5 As shown, even in long-term culture for 35 passages or more (p85+35: 85 passages before suspension culture and 35 passages after suspension culture), candidate cell line A showed almost the same level of cell growth in each passage.

[0086] 2.1.2. Candidate cell line B

[0087] To determine cell growth at each passage of candidate cell line B that had completed the suspension culture adaptation of Example 1, cell l...

Embodiment 3

[0134] Example 3: Comparison of Viral Genetic Stability

[0135] Influenza viruses are amplified according to the altered gene sequence if genetic information is altered during amplification in the host; such mutated portions may be major surface antigens such as HA or NA antigens. As a result, antibodies induced by altered surface antigens may not exhibit the expected vaccine effect.

[0136] Therefore, the viruses passaged in the candidate cell line A and the MDCKS-MG cell line were examined for any changes in the amino acid sequences of the major surface antigens HA and NA of the virus; and the effect of any changes on antibody formation was examined.

[0137]Specifically, the gene sequence of influenza virus produced in cell line A and MDCKS-MG cell line was compared with that of influenza virus produced in fertilized eggs with reference to the basic gene sequence registered in NCBI ("Ref.").

[0138] First, viruses derived from fertilized eggs were obtained from NIBSC; a...

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Abstract

The present invention relates to a novel MDCK-derived cell strain capable of being suspension-cultured in a protein-free medium and a method for proliferating virus using the MDCK cell strain to produce a vaccine. The novel MDCK-derived cell strain according to the present invention exhibits high and uniform productivity with respect to various viruses, and has a low tumorigenic capacity while causing less viral antigenic variation, and thus can be useful in producing vaccine viruses.

Description

technical field [0001] The present invention relates to novel MDCK-derived cell lines capable of suspension culture in protein-free media, and methods for amplifying viruses using such MDCK cell lines for the production of vaccines. Background technique [0002] Typically, fertilized eggs are used in influenza vaccine production. However, the use of fertilized eggs has the following disadvantages: chicks suitable for production should be raised and managed; influenza vaccine production should be adjusted to the production range of fertilized eggs; people with allergies. [0003] From a long time ago, studies have been conducted on producing influenza vaccines by using cell culture to replace fertilized eggs having the above-mentioned disadvantages. Because animal cells for cell culture are in unlimited supply, the use of cell culture allows mass production in a short period of time. In addition, it has the advantages that the vaccine can be administered to people who are ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N5/071C12N5/02A61K39/12
CPCA61K39/145C12N2760/16134C12N2760/16152C12N2760/16234C12N2760/16252A61K39/12C12N5/0686Y02A50/30C12N5/10C12N5/0602C12N5/00C12N7/00C12N2500/92C12N2760/16151C12N2760/16251
Inventor 黄美嬉朴国辰辛德香李贤金秀仁赵恩莹金美淑安东浩
Owner MOGAM BIOTECH RES INST
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