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Method for optimizing culture-medium of high-flux mammal

A culture medium optimization, culture medium technology, applied in the field of high-throughput mammalian cell microculture and live cell concentration detection, cell culture medium composition optimization

Inactive Publication Date: 2007-12-19
上海中科伍佰豪生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no high-throughput cell culture condition screening method that combines cell culture and adds resazurin after culture for activity detection without affecting cell survival

Method used

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  • Method for optimizing culture-medium of high-flux mammal
  • Method for optimizing culture-medium of high-flux mammal
  • Method for optimizing culture-medium of high-flux mammal

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1: the relation of resazurin concentration and 605nm light absorption

[0047] (1) Add 100 μl of serum-free medium (prepared in our laboratory) to the 96-well cell culture plate;

[0048] (2) Add 5, 10, 20, 30, 40 and 50 μl of resazurin solution (Sigma, 1.2 mM, pH7.5 PBS) to each well, repeat 4 wells for each concentration, and mix well;

[0049] (3) Serum-free medium was used as a blank control, and the optical density at 605 nm was read with a microplate reader (Microplate Reader 550, Bio-Rad).

[0050] Experimental result shows (see Fig. 1), resazurin concentration is in a certain range and OD 605 The relationship is linear; it can be seen from the signal size that the proportion of resazurin solution is moderate when it is about 20-30 μl.

Embodiment 2

[0051] Embodiment 2: The influence of solution pH on resazurin from 605nm light absorption

[0052] (1) Prepare pH5.0, 5.5, 6.5, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0 buffers respectively (pH5.5-6.0 is citric acid buffer, pH6.5-8.0 is phosphate buffer, pH8 .5-9.0 is Tris-HCl buffer solution);

[0053] (2) Add 100 μl of buffer solution to a 96-well cell culture plate (Corning), and repeat 4 wells for each pH;

[0054] (3) Add 20 μl of resazurin solution to each well and mix well;

[0055] (4) Using the corresponding buffer as a blank control, read the optical density at 605 nm with a microplate reader.

[0056] The experimental results show (see Fig. 2) that the pH has a great influence on the light absorption of resazurin at 605 nm in the range of 5.0-6.0, while the pH has little influence in the range of 6.5-9.0.

Embodiment 3

[0057] Embodiment 3: the influence of temperature on resazurin OD605nm light absorption

[0058] (1) Add 100 μl of serum-free medium to a 96-well cell culture plate;

[0059] (2) Add 5, 10, 20, 30, 40 and 50 μl of resazurin solution to each well, repeat 4 wells for each concentration, and mix well;

[0060] (3) Place the 96-well cell culture plate at 4°C for 20 minutes and read the optical density at 605 nm with a microplate reader;

[0061] (4) Place the 96-well cell culture plate at room temperature for 20 minutes and read the optical density at 605 nm with a microplate reader;

[0062] (5) Place the 96-well cell culture plate at 37° C. for 20 minutes and read the optical density at 605 nm with a microplate reader.

[0063] The experimental results showed (see Fig. 3 ) that the temperature within the range of 4-37° C. had basically no effect on the optical density of resazurin at 605 nm.

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Abstract

The invention opened an optimization method of the cell medium. The process is: a. inoculating the cell into the different component medium which is at least two kinds in the porous sheet; b. culturing the cells in the same conditions; c. detecting the cells number; d. selecting the medium of the most cell numbers as the optimization medium.

Description

technical field [0001] The invention relates to the fields of cell culture and biopharmaceuticals, more specifically to a high-throughput mammalian cell microculture and a living cell concentration detection method for optimizing cell culture medium components. Background technique [0002] The development of recombinant DNA technology has made it possible to produce protein drugs on a large scale and replace the extraction from biological materials; there is no doubt that with the development of human genomics and proteomics research, more gene functions will be identified, and with them Correspondingly, more protein drugs will appear and be used in clinical trials and disease treatment. [0003] Several pathways are available for the biosynthesis of protein drugs, including mammalian cell culture, microbial fermentation, transgenic animals, and transgenic plants. At present, microbial fermentation is mainly used for the production of protein drugs that do not require modi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/06C12N5/071
Inventor 沈秉谦贡向辉杨胜利
Owner 上海中科伍佰豪生物工程有限公司
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