Method for preparing MDCK (Madin-Darby canine kidney) cell line adaptive to serum-free full-suspension culture and MDCK cell line

A serum-free medium and full-suspension technology, applied in the cultivation process, microbial-based methods, biochemical equipment and methods, etc., can solve the problems of not meeting high-density growth requirements, cumbersome steps, and increased production costs, and achieve The effect of shortening the domestication time, high cell density, and improving efficiency

Active Publication Date: 2016-08-17
ZHAOQING DAHUANONG BIOLOGIC PHARMA +2
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, a microcarrier adherent culture production process has been developed that utilizes MDCK cell lines instead of chicken embryos to cultivate influenza virus. Although this process has a certain production scale that can be achieved, it still has certain defects: 1. Microcarriers are difficult to multiply 2. The culture density of adherent cells is limited by the adherent area, resulting in a decrease in virus production; 3. Serum is usually added to help cell attachment and growth in adherent culture, and mediu

Method used

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  • Method for preparing MDCK (Madin-Darby canine kidney) cell line adaptive to serum-free full-suspension culture and MDCK cell line
  • Method for preparing MDCK (Madin-Darby canine kidney) cell line adaptive to serum-free full-suspension culture and MDCK cell line
  • Method for preparing MDCK (Madin-Darby canine kidney) cell line adaptive to serum-free full-suspension culture and MDCK cell line

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preparation example Construction

[0047] A preparation method for MDCK cell lines adapted to serum-free full suspension culture, comprising the following steps:

[0048] 1) Cultivate MDCK cells that require serum adherent culture with DMEM medium with 10% newborn bovine serum until the confluence of the cells reaches 80-90%, discard the medium for culturing MDCK cells, and wash the cell layer with trypsin solution Discard the solution; add trypsin solution again to cover the MDCK cells and digest for 5-15 minutes; after the cells become round, add medium containing 10% fetal bovine serum to stop the digestion; then collect the cell suspension and centrifuge the cell suspension Discard the supernatant to obtain cell pellets;

[0049] 2) Resuspend the cell mass obtained in step 1) with serum-free medium to a cell density of 1.3-1.6×10 6 cells / mL, obtain the cell resuspension;

[0050] 3) Add the cell resuspension obtained in step 2) into the square flask, and incubate at a speed of 30 rpm, a temperature of 37°...

Embodiment 1

[0080] A preparation method for MDCK cell lines adapted to serum-free full suspension culture, comprising the following steps:

[0081] 1) Cultivate MDCK cells that require serum adherent culture with DMEM medium with 10% newborn bovine serum until the confluence of the cells reaches 80-90%, discard the medium for culturing MDCK cells, and wash the cell layer with trypsin solution Discard the solution; add trypsin solution again to cover the MDCK cells and digest for 5-15 minutes; after the cells become round, add medium containing 10% fetal bovine serum to stop the digestion; then collect the cell suspension and centrifuge the cell suspension Discard the supernatant to obtain cell pellets;

[0082] 2) Resuspend the cell mass obtained in step 1) with serum-free medium to a cell density of 1.3-1.6×10 6 cells / mL, obtain the cell resuspension;

[0083] 3) Add the cell resuspension obtained in step 2) into the square flask, and incubate at a speed of 30 rpm, a temperature of 37°...

Embodiment 2

[0116] A preparation method for MDCK cell lines adapted to serum-free full suspension culture, comprising the following steps:

[0117] 1) Cultivate MDCK cells that require serum adherent culture with DMEM medium with 10% newborn bovine serum until the confluence of the cells reaches 80-90%, discard the medium for culturing MDCK cells, and wash the cell layer with trypsin solution Discard the solution; add trypsin solution again to cover the MDCK cells and digest for 5-15 minutes; after the cells become round, add medium containing 10% fetal bovine serum to stop the digestion; then collect the cell suspension and centrifuge the cell suspension Discard the supernatant to obtain cell pellets;

[0118] 2) Resuspend the cell mass obtained in step 1) with serum-free medium to a cell density of 1.3-1.6×10 6 cells / mL, obtain cell resuspension;

[0119] 3) Add the cell resuspension obtained in step 2) into the square bottle, and mix at a speed of 30 rpm, a temperature of 37°C, and 5...

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Abstract

The invention discloses a method for preparing an MDCK (Madin-Darby canine kidney) cell line adaptive to serum-free full-suspension culture and the MDCK cell line. The method includes steps of 1), discarding culture media for cultivating MDCK cells, cleaning cell layers by the aid of pancreatin solution, then discarding the pancreatin solution, adding pancreatin solution into the MDCK cells again, digesting the MDCK cells, stopping digesting the MDCK cells after the cells are rounded, centrifuging cell suspension and then discarding supernatant to obtain cell clusters; 2), re-suspending the cell clusters obtained at the step 1) by the aid of serum-free culture media to obtain cell re-suspension; 3), arranging the cell re-suspension obtained at the step 2) in a shaking table, cultivating the cell re-suspension in culture tanks, diluting the cell re-suspension by the aid of the serum-free culture media and carrying out passage on the cell re-suspension to obtain the MDCK cell line. The method and the MDCK cell line have the advantages that the MDCK cell line is high in cell density and activity and uniform in size, individually grows in a dispersion manner, is plump in form and is suitable for serum-free suspension culture by the aid of bioreactors.

Description

technical field [0001] The invention relates to the field of cell line culture and domestication, in particular to a preparation method of an MDCK cell line adapted to serum-free full suspension culture and the MDCK cell line obtained by the preparation method. Background technique [0002] Canine kidney epithelial cells (Madin-Darby canine kidney cells, MDCK) are regarded as one of the most suitable cell lines for the production of influenza A and B influenza virus vaccines, and can be used to replace chicken embryos to culture influenza viruses. At present, a microcarrier adherent culture production process has been developed that utilizes MDCK cell lines instead of chicken embryos to cultivate influenza virus. Although this process has a certain production scale that can be achieved, it still has certain defects: 1. Microcarriers are difficult to multiply 2. The culture density of adherent cells is limited by the adherent area, resulting in a decrease in virus production;...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N5/02C12R1/91
CPCC12N5/0686C12N2500/30C12N2500/90C12N2501/90
Inventor 赖汉漳陈瑞爱刘玉鹏詹烜子刘旭平麦康聪汤钦盘伟岚许东蕾陈华坚
Owner ZHAOQING DAHUANONG BIOLOGIC PHARMA
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