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Suspension culture domestication method of MDCK cell line

A technology of suspension culture and cell line, which is applied in the field of production of biological products, can solve the problems that the cell density cannot meet the large-scale production, frequent cell clumping, and cannot support high-density growth, etc., to improve cell clumping, chemical The effect of definite ingredients and full form

Active Publication Date: 2020-11-17
SHANGHAI RONGSHENG BIOLOGICAL PHARM CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Many cases have shown that the gradual reduction of serum method takes a long time to acclimatize cells, the steps are cumbersome, and there are many defects such as cell clumping, which cannot support high-density growth.
CN107460156A discloses a MDCK cell suitable for serum-free suspension culture obtained by a one-step acclimation method, but this method usually has a large chance, and has high requirements for the medium used for acclimation and acclimatization culture conditions, and reasonable design of culturing Only by using culture medium and optimized culture conditions can it support rapid domestication, but the obtained cell density cannot meet the requirements of large-scale production.

Method used

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  • Suspension culture domestication method of MDCK cell line
  • Suspension culture domestication method of MDCK cell line
  • Suspension culture domestication method of MDCK cell line

Examples

Experimental program
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Effect test

Embodiment 1

[0062] The domestication method steps of the MDCK suspension cell line that is suitable for the production of human influenza vaccine in the chemically defined culture medium in full suspension culture:

[0063] 1) When the MDCK adherent cells cultured in commonly used serum-containing medium are cultured to a confluence of 80-90%, discard the serum-containing medium in the square bottle, rinse twice with PBS; add trypsin solution Make it just cover the MDCK adherent cell layer, and digest for 5-10 minutes; after the cells become round, gently tap the bottom of the square bottle to dissociate the MDCK cells, and add serum-containing medium to terminate the digestion;

[0064] 2) Collect the cell suspension in step 1) and transfer it to a shake flask, add an appropriate amount of serum-containing medium and adjust the cell density to 0.8-1.5×10 6 cells / mL, in a shaker with a rotation speed of 100rpm, at a temperature of 37°C and 5% CO 2 cultured under the conditions;

[0065]...

Embodiment 2

[0091] The domestication method steps of the MDCK suspension cell line that is suitable for the production of human influenza vaccine in the chemically defined culture medium in full suspension culture:

[0092]1) When the MDCK adherent cells cultured in commonly used serum-containing medium are cultured to a confluence of 80-90%, discard the serum-containing medium in the square bottle, rinse twice with PBS; add trypsin solution Make it just cover the MDCK adherent cell layer, and digest for 5-10 minutes; after the cells become round, gently tap the bottom of the square bottle to dissociate the MDCK cells, and add serum-containing medium to terminate the digestion;

[0093] 2) Collect the cell suspension in step 1) and transfer it to a shake flask, add an appropriate amount of serum-containing medium and adjust the cell density to 0.8-1.5×10 6 cells / mL, in a shaker with a rotation speed of 100rpm, at a temperature of 37°C and 5% CO 2 cultured under the conditions;

[0094] ...

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Abstract

The invention provides a suspension culture domestication method of an MDCK cell line. The suspension culture domestication method comprises the following steps of: carrying out adherent culture on MDCK cells by using a serum-containing culture medium; after digestion, putting the obtained cell suspension into a shake flask for suspension culture; after carrying out passage for 3-4 times, centrifuging the culture solution, re-suspending cell clusters by using a chemically defined culture medium, continuing suspension culture; and carrying out passage until the MDCK cells completely grow in a single suspension manner. Compared with a gradual serum reduction method, the domestication method provided by the invention is faster and more efficient, and the domestication time only needs about 10generations and is much shorter than that of the gradual serum reduction method. Compared with a one-step domestication method, the suspension culture domestication method provided by the invention is milder, is not limited by the quality of a serum-free culture medium used for domestication, has a higher universal application value, and also spends a short domestication time as the one-step domestication method.

Description

technical field [0001] The present invention relates to a method for producing biological products, in particular to a method for acclimating cells so that they are suitable for suspension culture. Background technique [0002] In recent years, the global market demand for seasonal influenza vaccines has been steadily increasing year by year, and the fact that vaccination rates in many developing countries are low reflects the huge potential market development prospects of seasonal influenza vaccines. In addition, there has been no outbreak of pandemic influenza since 2009, and influenza virus still poses a threat to human society. Compared with the traditional chicken embryo production method, the use of animal cells to produce influenza vaccines is gradually becoming the mainstream production method. In the past ten years, great progress has been made in the use of adherent MDCK cells to amplify influenza virus. However, there are still many disadvantages in this culture ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N1/36
CPCC12N5/0686C12N1/36C12N2501/90C12N2760/16151Y02A50/30
Inventor 朱绍荣张成林
Owner SHANGHAI RONGSHENG BIOLOGICAL PHARM CO LTD
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