Serum-free and protein-free all-chemical-component-definition culture medium for supporting CHO high-density suspension growth

A serum-free medium and chemical composition technology, applied in the field of cell engineering, can solve the problems of poor nutritional balance, complex composition, influence, etc., and achieve the effects of low cost and long maintenance time.

Inactive Publication Date: 2013-01-16
NINGBO PD ACRO BIO SYST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In recent years, a variety of serum-free media have been developed for CHO cells in China, but most of them are based on the existing basic media such as DMEM, DMEM / F12, IMDM, etc. by adding various vitamins, growth factors, transgenic Ferritin, protein hydrolyzate and trace elements have complex components, poor nutritional balance and basically contain protein or its hydrolyzate, which is difficult to quality control and affects downstream purification components

Method used

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  • Serum-free and protein-free all-chemical-component-definition culture medium for supporting CHO high-density suspension growth
  • Serum-free and protein-free all-chemical-component-definition culture medium for supporting CHO high-density suspension growth
  • Serum-free and protein-free all-chemical-component-definition culture medium for supporting CHO high-density suspension growth

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Medium preparation

[0043] The basic components of serum-free medium are divided into 10 groups of concentrated solutions:

[0044] S1 is an inorganic salt solution, a 50X concentrated solution

[0045]

[0046] S2 is a trace element, 5000X concentrate

[0047]

[0048] S3 is amino acid solution, 10X concentrate

[0049]

[0050]

[0051] S4 is L-tyrosine solution, 100X concentrate

[0052]

[0053] S5 is carbon source and other organic molecules

[0054]

[0055] S6 is L-glutamine solution, 40X concentrate

[0056]

[0057] S7 is vitamin concentrate, 3500X concentrate

[0058]

[0059] S8 is folic acid solution, 200X concentrate

[0060]

[0061] S9 is riboflavin solution, 1500X concentrate

[0062]

[0063] S10 is sodium bicarbonate solution, 100X concentrate

[0064]

[0065] Add the above-mentioned concentrated culture to a 2L container in turn, prepare 1X liquid medium and name it as T11 medium, adjust the osmotic pressure to between 270~275mosm with 5M NaCl and use 7.5% NaHCO 3 O...

Embodiment 2

[0067] Cell culture

[0068] Prepare the medium and place it in a 37°C water bath for 30 minutes, then take it out for use; take one each of CHO-K1, CHO-S, CHO-DG44 and CHO-DUKX-B11 to recover to DMEM containing 8% FBS / F12 medium, after expansion for two generations, spare;

[0069] Use 5.0X10 to recover the above various cells 5 The density of cells per milliliter was inoculated into a 125mL Erlenmeyer flask containing 25mLT11 medium, and the concentration of FBS (fetal bovine serum) in the medium was maintained at about 4%; the Erlenmeyer flask was placed in 5% CO 2 Culture in a shaker culture at 37 degrees, rotating speed 120rpm; count cells every day, when the cell density is greater than 1.5X10 6 When the density of cells per milliliter is 5.0X10 5 The cell density per milliliter was passed to a new 125 mL Erlenmeyer flask, and the FBS concentration in the medium was maintained at about 1%; this was repeated until all the serum was removed by centrifugation and passage after 3...

Embodiment 3

[0072] Cell culture comparison with other companies’ media

[0073] Acclimatize CHO-K1 cells to Life technologies / GIBCO CD-CHO medium and Thermofisher / Hyclone CDM4CHO medium according to the method in Example 2;

[0074] Take the respective domesticated CHO-K1 cells for parallel experiments; use CHO-K1 to 3.0X10 5 The density of cells per milliliter is inoculated into a 500mL Erlenmeyer flask containing 150mL; all Erlenmeyer flasks are placed in 5% CO 2 Cultivate at 37 degrees in a shaker culture, rotate at 120 rpm, sample and count cells every day, and use trypan blue staining to calculate cell viability. Maintain cell culture, when the viability rate drops and the density of viable cells is lower than 2.0X10 6 The cell density per milliliter terminates the experiment in the group. The final experimental results found that the CHO-K1 in the CD-CHO experimental group reached a peak of 4.24X10 on the 4th day after vaccination. 6 The density of cells per milliliter, the cell density ...

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Abstract

The invention discloses a serum-free and protein-free all-chemical-component-definition culture medium for supporting the CHO high-density suspension growth. The serum-free culture medium contains amino acids, inorganic salts, trace elements, vitamins, carbohydrates and other organic molecules. The serum-free culture medium has the advantages of no containment of any extracted or recombinant proteins, polypeptides or hydrolyzates, short cell growth time, high density, low metabolic refuse accumulation, good cell vitality maintenance, realization of the protein expression higher than that of products of same kind, and low cost because of all chemical component definition.

Description

Technical field [0001] The present invention relates to a serum-free medium that supports the culture of animal cells, in particular to a serum-free and protein-free fully chemically defined medium that supports the high-density suspension growth of CHO, and supports the high-density suspension culture of CHO cells while having chemical components The defined characteristics belong to the field of cell engineering. Background technique [0002] The expression of genetically engineered protein has risen abroad in the 1980s, and it has been nearly 30 years since it has been widely used in biological research and medical research industries. In particular, because the biomedical industry, especially biotechnology companies that use therapeutic monoclonal antibodies as their products, emerged after the 1990s, the industry has developed rapidly at a compound growth rate of more than 50% every year. Worldwide, more than 70-80% of the recombinant therapeutic proteins that have been app...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N5/02
Inventor 陈宜顶
Owner NINGBO PD ACRO BIO SYST
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