CHO cell culturing method

A cell culture and cell technology, applied in the field of cell engineering, can solve the problems of low mechanical strength, high production cost and high added value, and achieve the effects of uniform and stable quality, reducing labor intensity and improving utilization rate.

Inactive Publication Date: 2017-05-31
鼎正生物药业(天津)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Although CHO cells can be cultured on a large scale in artificially controlled bioreactors like microbial cells, their cell structure and culture characteristics are significantly different from those of microbial cells.
Animal cells are much larger than microbial cells, without cell walls, low mechanical strength, sensitive to shear force, and poor adaptability to the environment; long doubling time, slow growth, susceptible to microbial contamination, antibiotics must be used for culture; oxygen demand during culture During the culture process, the cells adhere to each other and exist in the form of clusters; the primary culture cells generally degenerate and die after 50 generations of reproduction; the metabolites are biologically active, and the production cost is high, but the added value is also high
[0003] In the prior art, experiments have confirmed that too many passages of cells have a significant impact on the growth of CHO cells in monolayer culture and the expression of adenovirus vectors, but for the growth of CHO cells in suspension culture and the expression of adenovirus vectors does not affect the expression level of CHO cells, but there are few reports on the suspension culture process of CHO cells in the prior art

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] A method for culturing CHO cells, comprising the steps of:

[0016] 1) Cultivate CHO cells with a stirred cell culture flask and make a cell suspension;

[0017] 2) moving the cell suspension in step 1) into a small-scale reactor for serum-free suspension culture;

[0018] 3) moving the cell suspension in step 2) into a large-scale reactor for serum-free suspension culture;

[0019] In the above preparation method, in step 1), the CHO cells are domesticated cell lines suitable for full suspension culture. Resuscitate CHO cells from the liquid nitrogen bank from the cell work, and now subculture and expand in T-shaped tissue culture flasks, culture them into stirred cell culture flasks, and then digest the spinner bottle cells and transfer them to small-scale reactors for culture, and then stir them Amplify the number of animal cells in cell culture flasks or small-scale reactors, and select healthy and well-growing cells as seed cells for the lower-level reactors.

...

Embodiment 2

[0023] A method for culturing CHO cells, comprising the steps of:

[0024] 1) Cultivate CHO cells with a stirred cell culture flask and make a cell suspension;

[0025] 2) moving the cell suspension in step 1) into a small-scale reactor for serum-free suspension culture;

[0026] 3) moving the cell suspension in step 2) into a large-scale reactor for serum-free suspension culture;

[0027] In the above preparation method, in step 1), the CHO cells are domesticated cell lines suitable for full suspension culture. Resuscitate CHO cells from the liquid nitrogen bank from the cell work, and now subculture and expand in T-shaped tissue culture flasks, culture them into stirred cell culture flasks, and then digest the spinner bottle cells and transfer them to small-scale reactors for culture, and then stir them Amplify the number of animal cells in cell culture flasks or small-scale reactors, and select healthy and well-growing cells as seed cells for the lower-level reactors.

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PUM

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Abstract

The invention provides a CHO cell culturing method. The CHO cell culturing method realizes maximization of cell culture density, reduces the volume of cell culture, lowers subsequent labor intensity and enables uniform and stable quality to be obtained; and production process is compact in link and easy to operate. According to the invention, liquid supplementation is carried out in the process of cell culture and virus propagation, so nutrients needed in cell proliferation are provided, the utilization rate of a nutrient solution is increased, and cost is saved. With a production process for an adenovirus vector vaccine in the invention, virus titer can reach 8 * 10<8> TCID50 / mL.

Description

technical field [0001] The invention relates to the technical field of cell engineering, in particular to a method for culturing CHO cells. Background technique [0002] Although CHO cells can be cultured on a large scale in artificially controlled bioreactors like microbial cells, their cell structure and culture characteristics are significantly different from those of microbial cells. Animal cells are much larger than microbial cells, without cell walls, low mechanical strength, sensitive to shear force, and poor adaptability to the environment; long doubling time, slow growth, susceptible to microbial contamination, antibiotics must be used for culture; oxygen demand during culture During the culture process, the cells adhere to each other and exist in the form of clusters; the primary culture cells generally degenerate and die after 50 generations of reproduction; the metabolites are biologically active, and the production cost is high, but the added value is also high....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
Inventor 刘鼎阔王立红张凤洪张俊霞
Owner 鼎正生物药业(天津)有限公司
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