CHO (Chinese hamster ovary) cell serum-free protein-free culture medium and preparation method thereof

A protein-free medium, serum-free medium technology, applied in biochemical equipment and methods, culture process, tissue culture, etc., can solve problems such as unfavorable optimization and adjustment of medium, the maximum viable cell density of less than 3.5 million, and complex components.

Inactive Publication Date: 2016-12-07
BEIJING SL PHARMA +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Chinese patent application number 200710307076.9 discloses a protein-free cell culture medium containing transferrin and insulin substitutes, but the highest viable cell density is less than 3.5 million / ml
[0007] At present, most serum-free media developed in China can support cell growth, but most of them add various nutrients, insulin, transferrin, protein hydrolyzate, etc. on the basis of existing media such as DMEM, DMEM / F12, etc. The composition is complex, which puts the quality and safety of the product to the test, and also brings difficulties to the purification
Although the serum-free medium of foreign companies such as Life and Thermo does not contain protein, it is expensive and its ingredients are kept secret. The product needs to optimize and adjust the medium, which greatly reduces the subsequent optimization space

Method used

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  • CHO (Chinese hamster ovary) cell serum-free protein-free culture medium and preparation method thereof
  • CHO (Chinese hamster ovary) cell serum-free protein-free culture medium and preparation method thereof
  • CHO (Chinese hamster ovary) cell serum-free protein-free culture medium and preparation method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] 1) Preparation of serum-free and protein-free medium for CHO cells:

[0082] CHO cell serum-free protein-free medium dry powder components and dosage of the present invention are as follows:

[0083]

[0084]

[0085]

[0086] Add 1L of dry powder into 900ml of ultrapure water at a temperature of about 30°C. After stirring for half an hour, add a certain amount of sodium hydroxide to aid dissolution, then add sodium bicarbonate, and adjust the pH to 6.9-7.0 with hydrochloric acid. Filter through a 0.22 μm sterile membrane and store at 4°C.

[0087] 2) Cell culture:

[0088] CHO-S cells in 1×10 6 The density of cells / ml was inoculated into a 125ml Erlenmeyer shake flask containing 20ml medium, and the Erlenmeyer shaker flask was placed in 5% CO 2 Cultivate in a shaker at 37°C with a rotation speed of 180rpm; when the cell density is about 3×10 6 When cells / ml, take 1×10 6 The density of cells / ml was subcultured until the cells were passaged more than 10 tim...

Embodiment 2

[0091] 1) Preparation of CHO cell serum-free protein-free medium

[0092] CHO cell serum-free protein-free medium dry powder components and dosage of the present invention are as follows:

[0093]

[0094]

[0095]

[0096] Add 1L of dry powder into 900ml of ultrapure water at a temperature of about 30°C. After stirring for half an hour, add a certain amount of sodium hydroxide to aid dissolution, then add sodium bicarbonate, and adjust the pH to 6.9-7.0 with hydrochloric acid. Filter through a 0.22 μm sterile membrane and store at 4°C.

[0097] 2) Cell culture:

[0098] CHO-S cells in 1×10 6 The density of cells / ml was inoculated into a 250ml shake flask containing 50ml of culture medium, placed in a 5% CO2 incubator at 37°C, with a rotation speed of 180rpm, sampling and counting every 24 hours, and staining with trypan blue to calculate cell viability Rate. When the cells were cultured to the 4th day, the viable cell density reached the highest, and the peak cel...

Embodiment 3

[0100] 1) Preparation of CHO cell serum-free protein-free medium

[0101] CHO cell serum-free protein-free medium dry powder components and dosage of the present invention are as follows:

[0102]

[0103]

[0104]

[0105] Add 1L of dry powder into 900ml of ultrapure water at a temperature of about 30°C. After stirring for half an hour, add a certain amount of sodium hydroxide to aid dissolution, then add sodium bicarbonate, and adjust the pH to 6.9-7.0 with hydrochloric acid. Filter through a 0.22 μm sterile membrane and store at 4°C.

[0106] 2) Cell culture:

[0107] CHO-S cells in 1×10 6 The density of cells / ml was inoculated into a 250ml shake flask containing 50ml of culture medium, placed in a 5% CO2 incubator at 37°C, with a rotation speed of 180rpm, sampling and counting every 24 hours, and staining with trypan blue to calculate cell viability Rate. On the 4th day of cell culture, the density of living cells reached the highest, and the peak cell number ...

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PUM

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Abstract

The invention relates to preparation of a cell culture medium, and particularly provides preparation of a novel serum-free protein-free defined-chemical-component cell culture medium. The culture medium contains multiple amino acids, vitamins, inorganic salts, minor elements, carbohydrates and other supplementary factors. The culture medium is free of any extract or hydrolysate. The culture medium is free of any animal-derived component. The culture medium can well support in-vitro suspension culture of CHO (Chinese hamster ovary) cells, and satisfies the demands for CHO cell culture. The culture medium contains defined chemical components, and is low in cost.

Description

technical field [0001] The invention belongs to the field of cell culture, and in particular relates to a serum-free and protein-free medium suitable for large-scale suspension culture of CHO cells and a preparation method thereof. Background technique [0002] Chinese hamster ovary (CHO) cells are widely used in the production of recombinant protein drugs and antibody drugs, which can provide stable and accurate glycosylation modifications, making the expression products closest to natural protein molecules, with high safety characteristics . Traditionally, the cultivation of CHO cells is accomplished by adding 5-10% fetal calf serum to DMEM / F12 basal medium. In addition to supplying nutrients for cells, serum also provides growth factors necessary for cell proliferation in vitro. However, the use of serum has many disadvantages such as large batch-to-batch variance, easy contamination by mycoplasma and viruses, high cost, unfavorable separation and purification of product...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/0682C12N2500/10C12N2500/12C12N2500/14C12N2500/16C12N2500/20C12N2500/22C12N2500/24C12N2500/30C12N2500/32C12N2500/34C12N2500/35C12N2500/36C12N2500/38C12N2500/46C12N2500/50C12N2500/95
Inventor 于红阳吴彦卓张苗甘一迪贾东晨徐明波
Owner BEIJING SL PHARMA
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