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MDCK-derived cell lines adapted to serum-free culture and suspension culture and method for preparing vaccine virus using the cells

A serum-free medium and suspension culture technology, which can be used in biochemical equipment and methods, medical preparations containing active ingredients, viruses, etc., and can solve the problems of tumorigenicity danger and complex culture process.

Inactive Publication Date: 2013-06-12
SK CHEM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The second method has the defect that the cultivation process is relatively complicated
Therefore, there is a risk of potential tumorigenicity when using the original MDCK cell line for vaccine production

Method used

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  • MDCK-derived cell lines adapted to serum-free culture and suspension culture and method for preparing vaccine virus using the cells
  • MDCK-derived cell lines adapted to serum-free culture and suspension culture and method for preparing vaccine virus using the cells
  • MDCK-derived cell lines adapted to serum-free culture and suspension culture and method for preparing vaccine virus using the cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Prepare the cell line derived from MDCK by serum-free culture and suspension culture

[0034] CCL-34 in MDCK cell line provided by ATCC. In T-25 flasks, in EMEM medium supplemented with 10% serum at 37°C and 5% CO 2 CCL-34 was cultured under. After cell expansion, cells were cultured in a medium consisting of EMEM medium and serum-free medium (50%). During the culture process, confirm whether the growth of the cells is normal. When normal cell growth was confirmed, the growing cells were cultured in medium containing serum-free medium (75%). Repeat the process to obtain cell lines adapted to serum-free medium (100%). EX-CELL MDCK (Sigma), UltraMDCK (Lonza) and VP-SFM (Invitrogen) can be used as medium for serum-free culture.

[0035] Cell lines adapted to serum-free media are fully expanded in T-flasks. Then, in a spinner bottle (Corning) at 37 °C and 5% CO 2 The expanded cell line was adapted to suspension culture with agitation at a rate of 40-80 rpm. When th...

Embodiment 2

[0036] Evaluation of Cell Line Proliferation Potential

[0037] The prepared MDCK-derived cell lines cultured in a serum-free medium were cultured under the conditions shown in Table 1. The proliferative potential of MDCK-derived cell lines was assessed. MDCK cell line (ATCCCCL-34) was cultured in medium supplemented with 10% serum as a negative control.

[0038] Table 1

[0039] [Table 1]

[0040] [surface]

[0041]

[0042] At the beginning of the culture, the cell culture concentration was about 1.0×10 5 cells / ml. When the cell concentration reaches about 1×10 6 cells / ml or after 3-4 days of culture, the cells were passaged. Adjust the cell concentration at the initial stage of subculture to 1×10 5 cells / ml.

[0043] Each of the MDCK-derived cell lines grown in serum-free media exhibited comparable cell growth rates to MDCK cells grown in serum media.

Embodiment 3

[0044] Evaluation of proliferation curve and passage stability of cell lines

[0045] After adaptation to serum-free culture and suspension culture, the three cell lines were further cultured in spinner flasks, and their proliferation curves and passage stability were evaluated. Adjust the concentration of cells at the initial stage of culture to approximately 4×10 5 cells / ml. After about 3-4 days of culture, the cell concentration reached about 2×10 6 cells / ml or more. Culture was performed under the following conditions. The result is as figure 1 shown.

[0046] Starting cell concentration: 4 x 10 5 cells / ml

[0047] Culture scale: 50ml spinner bottle

[0048] Spinner rotation rate: 60rpm

[0049] Culture conditions: 37°C, 5% CO 2 , wet

[0050] Subculture conditions: 3-4 days after culture

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Abstract

Disclosed is a Madin-Darby canine kidney (MDCK)-derived cell line. The MDCK-derived cell line is derived from MDCK cells deposited under accession number ATCC CCL-34. The MDCK-derived cell line can be prepared by serum-free culture and suspension culture. Preferably, the MDCK-derived cell line has low or no tumorigenicity. The MDCK-derived cell line is preferably selected from MDCK Sky1023, MDCK Sky10234 and MDCK Sky3851. Further disclosed are a culture method for growing the MDCK-derived cells and a method for producing a vaccine virus using the MDCK-derived cells.

Description

technical field [0001] The present invention relates to a new MDCK-derived cell line (MDCK-derived cell line), the MDCK-derived cell line can be prepared by serum-free culture and suspension culture and does not need to be attached to carriers (carriers), the preparation of the A method for MDCK-derived cell lines, and a method for preparing vaccine viruses using said MDCK-derived cell lines. Background technique [0002] Fertilized eggs, mouse brain, primary cells, and established cell lines are commonly used as sources for vaccine preparation. However, there are many problems with this traditional source of vaccines. For example, when trying to use fertilized chicken eggs for vaccine preparation, there are obstacles in raising chickens, managing fertilized eggs that rely on vaccine production programs, and purifying components derived from egg proteins. [0003] Fetal bovine serum is often added as a growth factor for cell culture. However, serum is susceptible to prion...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N5/02A61K39/145
CPCC12N2760/16134C12N2760/16234C12N2760/16152C12N2760/16252A61K39/12A61K2039/5254C12N5/0686C12N2500/90C12N7/00A61P31/12A61P31/16Y02A50/30A61K39/00C12N5/06C12Q1/02A61K39/145A61K2039/525C12N2760/16151C12N2760/16171C12N2760/16251C12N2760/16271
Inventor 朴容郁李建世李峰镛朴万勋金勋金允熙李受陈
Owner SK CHEM CO LTD
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