Serum-free medium for mammalian cell

A serum-free medium and mammalian technology, applied in animal cells, vertebrate cells, tissue culture, etc., can solve problems such as biological contamination, process instability, and increased difficulty in product purification

Inactive Publication Date: 2007-05-16
上海中科伍佰豪生物工程有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The use of animal serum has several disadvantages for protein expression, such as increased difficulty in product purification, source restrictio

Method used

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  • Serum-free medium for mammalian cell

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Experimental program
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Effect test

Embodiment 1

[0026] Embodiment 1: Serum-free medium configuration (1 liter)

[0027] The medium was formulated according to Table 1 below:

[0028] Table 1. Components of animal cell serum-free medium

[0029] amino acid

(mM)

Ala (alanine)

Arg (arginine)

Asn (asparagine)

Asp (aspartic acid)

Cys (cysteine)

Glu (glutamic acid)

Gly (glycine)

His (histidine)

Ile (isoleucine)

Leu (leucine)

Lys (Lysine)

Met (methionine)

Phe (phenylalanine)

Pro (proline)

Ser (serine)

Thr (threonine)

Trp (tryptophan)

Tyr (tyrosine)

Val (valine)

Gln (glutamine)

0.03

1.95

0.54

0.4

0.13

0.16

0.25

0.13

2.56

1.15

1.05

0.53

1.05

0.02

0.71

0.3

0.1

0.14

1.4

4

vitamins

(mM)

Biotin (Biotin)

Choline chloride (choline chloride)

D-Calcium pantothenate (D-calcium panto...

Embodiment 2

[0034] Embodiment 2: The domestication of 293 cells in serum-free medium

[0035] Resuscitate 293 cells in the original medium at 37°C 5% CO 2 Incubator agitation culture. After the cells were passaged three times, the cells were divided into 2 × 10 5 / mL density inoculated into this serum-free medium to start acclimatization. Samples were taken every 24 hours, and the cell density was counted by trypan blue method. After culturing for about 72 hours, subculture by centrifugation, and the seeding density was 2×10 5 / mL. The results showed that after a period of adaptation at the initial stage, the cells gradually adapted to the serum-free medium, and the growth ability of the cells gradually increased, and the cells were basically fully adapted after 7 passages. The results are shown in Figure 1.

Embodiment 3

[0036] Example 3: Growth of 293 cells in serum-free medium

[0037] The domesticated 293 cells were treated with 2×10 5 / mL density in this serum-free medium, placed in 37 ° C 5% CO 2 Incubator agitation culture. Samples were taken every 24 hours, and the cell density was counted by trypan blue method. The concentration of glucose and lactic acid in the medium was determined by YSI 2003 STAT plus, and the concentration of ammonium ion was determined by enzyme electrode method. The results showed that the viable cell density reached the highest after 6 days of culture, 2×10 6 / mL. In the stage of higher glucose concentration (the first 3 days), the lactate formation rate was faster, but the lactate concentration was basically maintained after the glucose concentration was reduced to 5mM. The results are shown in Figure 2.

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Abstract

The invention discloses a taming method of non-serum culture medium for mammal cell, which contains cross tumour cell, Chinese hamster oophoron cell (CHO) and 293-cell.

Description

technical field [0001] The present invention relates to the fields of cell culture and biopharmaceuticals, and more specifically relates to the composition of mammalian cell culture medium. 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 modification, while large-scale animal cell culture is mainly used for the p...

Claims

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Application Information

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IPC IPC(8): C12N5/06C12N5/071
Inventor 沈秉谦杨胜利方强毅
Owner 上海中科伍佰豪生物工程有限公司
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