Blood serum-free culture medium for supporting suspension culture of CHO cells with large scale and high density

A serum-free medium and suspension culture technology, applied in the field of cell engineering, can solve the problems such as the inability to meet the needs of high-density culture of CHO cells, the application status and development trend of difficult animal cell culture, and the limitation of serum-free medium for CHO cells, etc. To achieve the effect of clear ingredients, simple composition and easy preparation

Inactive Publication Date: 2010-06-09
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, although a variety of serum-free media have been developed for CHO cells in China, most of the cell culture methods are adherent culture, which is difficult to adapt to the current application status and development trend of animal cell culture.
At the same time, the maximum growth density of the cells it supports is often low, which cannot meet the needs of large-scale high-density culture of CHO cells
Hyclone, Invitrogen and other foreign media suppliers have developed a variety of commercial serum-free media for CHO cell suspension culture, including CHO serum-free media, CHO protein-free media and chemically defined CHO serum-free media Medium, but the composition of commercial serum-free medium has always been a secret core business secret. In addition, the high price and the narrow range of applicable cell lines also limit the serum-free culture of commercial CHO cells to a large extent. base application

Method used

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  • Blood serum-free culture medium for supporting suspension culture of CHO cells with large scale and high density
  • Blood serum-free culture medium for supporting suspension culture of CHO cells with large scale and high density
  • Blood serum-free culture medium for supporting suspension culture of CHO cells with large scale and high density

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1: Using the statistical method Plackett-Burman and response surface method to determine the supplementary components of CHO cell serum-free medium and their optimal dosage

[0066] CHO cells (CHO-K1, ATCC Cat.No.CCl-61) were pressed at 3-3.5×10 5 The cells / m were inoculated in a 100ml Erlenmeyer flask with a culture volume of 35ml. Serum-free medium containing 5mM glutamine, 0.1% (v / v) Pluronic F-68 and 25μg / ml dextran sulfate or containing DMEM / F12 (v / v, 1:1) medium with 5% (v / v) serum, the Erlenmeyer flask was placed in a 37°C incubator for culture on a shaker with a shaker speed of 90r / min.

[0067] The design of serum-free medium for CHO cell suspension culture was based on DMEM / F12 (v / v, 1:1), and the Plackett-Burman method was used to investigate the insulin (X 1 ), putrescine (X 2 ), transferrin (X 3 ), trace elements (X 4 , Table 1), ascorbic acid (X 5 ), lecithin (X 6 ), β-mercaptoethanol (X 7 ), glutathione (X 8 ), ethanolamine (X 9 ), β-cyc...

Embodiment 2

[0080] Embodiment 2: the preparation of culture medium and CHO cell culture

[0081] 1. Medium preparation: Add the following substances to the basal medium based on DMEM / F12 (v / v, 1:1):

[0082] Insulin 2mg / L

[0083] Putrescine 0.5mg / L

[0084] Transferrin 5mg / L

[0085] Glutathione 0.5mg / L

[0086] Ethanolamine 1mg / L

[0087] Lecithin 1mg / L

[0088] Hydroxypropyl-β-cyclodextrin 100mg / L

[0089] β-Mercaptoethanol 10μM

[0090] Ascorbic acid 5mg / L

[0091] Said supplement also contains the following trace elements:

[0092] Na 2 SeO 3 5nM

[0093] MnSO 4 ·H 2 O 0.5nM

[0094] Na 2 MoO 4 2H 2 O 5nM

[0095] NaVO 3 5nM

[0096] CuSO 4 ·5H 2 O 5nM

[0097] ZnSO 4 ·7H 2 O 1μM

[0098] FeC 6 h 5 o 7 3μM

[0099] The additive also contains the following substances

[0100] Dextran Sulfate 25mg / L

[0101] Glutamine 5mM

[0102] Pluronic F-68 1000mg / L

[0103] HEPES 15mM

[0104] The above me...

Embodiment 3

[0107] Embodiment 3: Compared with Hyclone's CHO cell serum-free medium SFM-CHO

[0108]0.1% (v / v) Pluronic F-68 and 25 μg / ml dextran sulfate were added to Hyclone's SFM-CHO medium, and the CHO cells (CHO-K1) were cultured in the same culture method as in Example 2. The whole cultivation process lasted 8 days. On the 4th day of culture, the living cell density of CHO cells in the serum-free medium of the present invention and the SFM-CHO medium of Hyclone Company reached 4.2×10 6 cells / ml and 3.3×10 6 cells / ml( image 3 ).

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Abstract

The invention discloses a blood serum-free culture medium suitable for the suspension culture of CHO cells with large scale and high density. The culture medium takes DMEM/F12 (v/v, 1:1) as a base culture medium, and adds the other substances such as insulin, putrescine, transferrin and microelement, etc. The blood serum-free culture medium has the advantages that: the culture medium can support the CHO cells to fast growth under the condition of suspension culture; the protein content is very low, and the chemical composition is basically clear; the cells are grown one by one by means of suspension, so that the living cell density and the cell living rate are higher than those of the serum culture and the similar commercialized serum-free culture; and the cost is lower.

Description

technical field [0001] The invention relates to a serum-free medium supporting mammalian cell culture, in particular to a serum-free medium supporting large-scale high-density suspension culture of CHO cells, belonging to the technical field of cell engineering. Background technique [0002] Compared with other animal cells, CHO (Chinese hamster ovary) cells have foreign proteins that are easily synthesized in the cells and secreted into the medium; they can correctly assemble multi-subunit proteins; folding and modification of recombinant proteins, physical and chemical properties, biological The properties are almost the same as the natural protein; it can be cultured in a large scale without serum and high density. Therefore, CHO cells have been widely used in biopharmaceuticals. More than 70% of genetically engineered drugs that have been marketed or are in different stages of clinical trial research are mammalian cell-expressed products, and CHO cell-expressed products ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/075
Inventor 刘兴茂陈昭烈刘红李世崇吴本传叶玲玲谢靖王启伟
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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