Serum-free medium suitable to culturing ovary cells of Chinese hamster

An ovarian cell, Chinese hamster technology, applied in tissue culture, microorganisms, biochemical equipment and methods, etc., can solve the problems of lack of CHO cell culture, affecting product separation and purification, and high cost

Inactive Publication Date: 2009-09-09
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] To sum up, although scientists from various countries have developed many serum-free media, each media is only suitable for one or a type of cell growth. At present, most serum-free media contain macromolecular protein additives such as albumin , which seriously affects the separation and purification of the product, and the cost is also high
In particular, there is a lack of medium suitable for CHO cell culture, and the culture effect is equivalent to or better than that of medium with serum

Method used

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  • Serum-free medium suitable to culturing ovary cells of Chinese hamster

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0154] Orthogonal method to determine the best medium for CHO cell culture

[0155] On the basis of the initial medium, the concentrations of vitamin C, putrescine, and lipids were changed (see Table 1 below), and different mediums were prepared. The same number of the same CHO cells (2.0×10 5 Cells / mL) were inoculated in a 125ml square bottle filled with 10ml culture medium, at 37°C, CO 2 Culture in the incubator for about 8 days, and then measure the number of CHO cells.

[0156] The initial medium is a mixture of DMEM / 12 medium (purchased from Sigma Company) and trace elements, etc., and the following components are added:

[0157] Element Content (mg / L) Transferrin 5 insulin 5 ethanolamine 2.5 Sodium Selenite 0.01

[0158] Table 1 Orthogonal test header

[0159] A B C Vitamin C(mg / L) Putrescine (mg / L) lipid mixture * (v / v%) 1 5 2 0.1 2 10 5 0.2 3 30 10 0.5

[0160] *Note: The lip...

Embodiment 2

[0166] Preparation of media and CHO cell culture

[0167] (a) Medium formula:

[0168] DMEM / F12 medium (purchased from Sigma company) and in it simultaneously add the following supplements:

[0169] Part I: Amino Acids

[0170] L-alanine 9.0mg / L L-Aspartic Acid 13mg / L L-Asparagine 85mg / L L-Arginine 170mg / L L-glutamic acid 75mg / L L-Glycine 25mg / L L-histidine 62mg / L L-isoleucine 100mg / L L-leucine 150mg / L L-Lysine 110mg / L

[0171] L-Methionine 50mg / L L-phenylalanine 86mg / L L-proline 34mg / L L-serine 76mg / L L-threonine 43mg / L L-tryptophan 47mg / L L-valine 88mg / L L-tyrosine 76mg / L

[0172] Part II Trace Elements

[0173] AgNO 3 0.0194mg / L AlCl 3 ·6H 2 o 1.00mg / L Ba(C 2 h 3 o 2 ) 2 0.0186mg / L CaCl 2 77.7mg / L 3CdSO 4 ·8H 2 o 0.003mg / L CoCl 2 ·6H 2 o 0.101mg / L CuSO 4 ·5H ...

Embodiment 3

[0185] Wild-type CHO cell culture

[0186] The experiments of Example 2 and Comparative Example 1 were repeated, except that the wild-type CHO cells were used to replace the CHO cells with glutamine synthetase expression system, and aminosulfoxide-methionine was not added to the medium.

[0187] The highest viable cell density was 1.6 × 10 in serum- and serum-free media, respectively. 6 cells / mL and 1.8×10 6 cells / mL indicates that the serum-free medium of the present invention can completely replace the medium with serum for the cultivation of CHO cells.

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Abstract

The invention provides a serum-free medium for CHO cell culture. Said culture medium is based on DMEM / F12 with addition of transferrin, insulin, ethanolamine and other substances. The serum-free medium of the invention can make CHO cells grow vigorously, and the cell density and cell activity are comparable to those of the serum-containing medium, and can completely replace the serum-containing medium for the cultivation of CHO cells.

Description

technical field [0001] The present invention relates to the field of cell culture. More specifically, the present invention relates to a medium for CHO cell culture and a corresponding culture method. Background technique [0002] Since mammalian cells have the function of post-transcriptional modification, exogenous proteins produced by mammalian cells have incomparable advantages over proteins produced by prokaryotes, and this technology has received more and more attention. Chinese hamster ovary cells—CHO cells (Chinese hamster ovary cells) are currently the most widely used cells and have been widely used to produce various genetically engineered protein products, such as human erythropoietin (rEPO), human thrombopoietin (rTPO), tissue plasminogen activator (tPA), various antibodies, etc. [0003] Foreign proteins can usually be expressed in two different ways in CHO cells, namely DHFR-deficient (dihydrofolate reductase-deficient) and glutamine synthetase system. The ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/06
Inventor 张立张元兴张芳
Owner EAST CHINA UNIV OF SCI & TECH
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