Serum-free protein-free culture medium supporting HEK293 cell suspension culture and preparation method and application thereof

A protein-free medium, cell suspension technology, applied in the field of cell engineering, can solve the problems of cell clumping, affecting cell activity, culture pollution, etc., to achieve the effect of promoting expression and promoting metabolism

Active Publication Date: 2020-06-19
XINXIANG MEDICAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, most synthetic cell culture media need to be supplemented with animal serum to support the growth and proliferation of cells in vitro, and the use of animal serum may cause the culture to be contaminated by animal pathogenic microorganisms and introduce foreign proteins, which will bring troubles to the separation and purification of recombinant protein cell products. come very difficult
In addition, HEK293 cells are very easy to clump during suspension culture, and cell clumps directly affect cell viability, resulting in the inability of high-density expansion and continuous passage of HEK293 cells
[0004] In the prior art, the imported brand HEK293 suspension culture serum-free medium formula is confidential and expensive, and there are still problems such as poor culture effect, low protein expression and cell clumping.
The Chinese invention patent with the publication number CN109294976A discloses a serum-free medium that supports the suspension culture of HEK293 cells. The medium contains a large amount of protein components, such as albumin, insulin, and transferrin, which on the one hand increases the cost of use, and on the other hand is not conducive to the purification and production of downstream proteins

Method used

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  • Serum-free protein-free culture medium supporting HEK293 cell suspension culture and preparation method and application thereof
  • Serum-free protein-free culture medium supporting HEK293 cell suspension culture and preparation method and application thereof
  • Serum-free protein-free culture medium supporting HEK293 cell suspension culture and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] In this embodiment, the serum-free and protein-free medium supporting the suspension culture of HEK293 cells is composed as follows (the unit of concentration of each substance is mg / L):

[0051] Amino acid composition:

[0052] L-histidine hydrochloride monohydrate 94.44;

[0053] L-isoleucine 163.41;

[0054] L-Leucine 177.15;

[0055] L-Lysine 273.75;

[0056] L-Methionine 50;

[0057] L-phenylalanine 106.44;

[0058] L-threonine 160.35;

[0059] L-tryptophan 51.1;

[0060] L-valine 158.55;

[0061] Glycine 56.25;

[0062] L-alanine 13.35;

[0063] L-Glutamine 1626;

[0064] L-arginine hydrochloride 442.5;

[0065] L-asparagine 200;

[0066] L-Aspartic Acid 39.95;

[0067] L-cysteine ​​hydrochloride monohydrate 52.68;

[0068] L-cystine hydrochloride 62.58;

[0069] L-glutamic acid 22.05;

[0070] L-proline 115;

[0071] L-serine 72;

[0072] L-tyrosine disodium salt dihydrate 167.37;

[0073] Trace element composition:

[0074] Ammonium metavanad...

Embodiment 2

[0135] In this example, the serum-free and protein-free medium that supports the suspension culture of HEK293 cells contains, in addition to the amino acids, trace elements, vitamins, lipids, inorganic salts, additives and anti-agglomeration substances in Example 1, it also contains autophagy Inhibitor 3-methyladenine. Before adding autophagy inhibitors, dissolve 500mM 3-methyladenine stock solution in 0.5M HCl, and add 3-methyladenine to a final concentration of 10mM one day after the cell concentration reaches its peak, and then use 7.5% w / vNaHCO 3 The solution adjusted the pH of the culture to 7.0.

[0136] The preparation method of the serum-free and protein-free medium in this example is the same as that in Example 1.

Embodiment 3

[0138] In this embodiment, the serum-free and protein-free medium supporting the suspension culture of HEK293 cells is composed as follows:

[0139] Amino acid components: L-histidine hydrochloride monohydrate 100mg / L, L-isoleucine 160mg / L, L-leucine 185mg / L, L-lysine 265mg / L, L-methionine 55mg / L, L-Phenylalanine 100mg / L, L-Threonine 165mg / L, L-Tryptophan 45mg / L, L-Valine 165mg / L, Glycine 50mg / L, L-Alanine Acid 18mg / L, L-glutamine 1600mg / L, L-arginine hydrochloride 450mg / L, L-asparagine 190mg / L, L-aspartic acid 45mg / L, L- Cysteine ​​hydrochloride monohydrate 45mg / L, L-cystine hydrochloride 70mg / L, L-glutamic acid 18mg / L, L-proline 120mg / L, L-serine 65mg / L , L-tyrosine disodium salt dihydrate 175mg / L;

[0140] Trace element components: ammonium metavanadate 0.0012mg / L, sodium selenite 0.05mg / L, manganous chloride tetrahydrate 0.3mg / L, copper sulfate pentahydrate 0.3mg / L, zinc chloride 0.75mg / L ;

[0141] Vitamin components: vitamin H (biotin) 0.15mg / L, folic acid 8mg / L, vit...

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Abstract

The invention discloses a serum-free protein-free culture medium supporting HEK293 cell suspension culture and a preparation method and application thereof, and belongs to the technical field of cellengineering. According to the serum-free protein-free culture medium disclosed by the invention, different nutrient substances are selected according to the nutritional requirements of in-vitro growthof cells so as to replace animal serum, and the proportion of the different nutrient substances is reasonably adjusted. By further adding an anti-shearing force substance and an anti-caking substance, high-density suspension culture of HEK293 cells can be maintained without supplementing serum, and the normal form of the cells is maintained, so that the cells are dispersed and not caked, meanwhile, the normal growth speed is maintained, and the transfection and expression of target genes are facilitated.

Description

technical field [0001] The invention relates to a serum-free and protein-free medium for sustaining HEK293 cell suspension culture, a preparation method and application of the medium, and belongs to the technical field of cell engineering. Background technique [0002] HEK293 cells are human-derived mammalian cell lines widely used in the production of proteins, vaccines, anti-cancer reagents and recombinant adenovirus coatings. The cell line was first constructed in 1976 by F.L.Graham and J.S.Miley of McMaster University in Canada using DNA transfection technology. HEK293 cells are human embryonic kidney subtriploid cells transformed with type 5 adenovirus 75 strain and containing Ads E1 region. HEK293 cells, as a complementary cell line with defects in the E1 region, can sustainably express the ElA and E1B genes. HEK293 cells can grow rapidly in the culture system, and after proper acclimation, high-density suspension culture can be achieved for large-scale expression. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/0686C12N2500/95C12N2500/32C12N2500/38C12N2500/05C12N2500/36C12N2501/90C12N2500/46C12N2500/40C12N2500/33C12N2500/34C12N2500/74C12N2500/76
Inventor 林艳王天云李照熙米春柳赵春澎樊振林王蒙段树燕
Owner XINXIANG MEDICAL UNIV
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