Serum-free culture medium used for cultivating human mesenchymal stem cells

A serum-free medium and stem cell technology, applied in the field of biochemistry, can solve the problems of difficult large-scale industrial application, foreign protein contamination of cell products, virus contamination, etc.

Inactive Publication Date: 2015-10-07
龚伟
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This culture method will affect cell proliferation efficiency and biological performance, and there is a risk of virus contamination caused by poor animal serum quality, resulting in foreign protein contamination of cell products
[0004] In view of the above problems, a solution proposed in the field is to replace the medium containing fetal bovine serum with serum-free medium when human mesenchymal stem cells are cultured to the last 1-2 passages, but residual foreign protein contamination always exists , cannot be completely removed; another solution is to use serum-free medium for the whole culture, which requires sufficient nutrients in the serum-free medium to maintain the good growth of cells, resulting in high-quality serum-free culture of human mesenchymal stem cells The cost of the base is extremely high, and at the same time, if the nutrient composition ratio design is unreasonable, the stem cell culture will be out of control, making it difficult for large-scale industrial application, and it is also difficult to control the quality of human mesenchymal stem cell products

Method used

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  • Serum-free culture medium used for cultivating human mesenchymal stem cells
  • Serum-free culture medium used for cultivating human mesenchymal stem cells
  • Serum-free culture medium used for cultivating human mesenchymal stem cells

Examples

Experimental program
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Effect test

Embodiment 1

[0030] In this implementation, several specific implementations of the serum-free medium of the present invention are provided, and human bone marrow mesenchymal stem cells BM-MSC are used to initially study the growth and proliferation of stem cells under various combination conditions.

[0031] Composition 1: 50 ng of growth hormone, 20 ng of cortisol, 12 μg of insulin, 8 μg of glycine, 9 μg of arginine, 12 μg of threonine, 30 μg of serine, 25 μg of leucine, 18 μg of isoleucine, L-lysine 9μg, methionine 30μg, L-tyrosine 28μg, endothelial cell growth factor 100ng, fibroblast growth factor 100ng, hepatocyte growth factor 40ng, folic acid 30ng, d-calcium pantothenate 17ng, choline chloride 3ng , vitamin B12 3ng, inositol 1ng, recombinant human transferrin 15ng, putrescine 20ng, recombinant human serum albumin 500ng, glutathione 95μg, vitamin C 10μg, vitamin E 10μg, calcium chloride 35μg, potassium chloride 70μg , sodium chloride 10mg, ferric citrate 0.8μg.

[0032] Composition...

Embodiment 2

[0041] Taking the serum-free medium of the above-mentioned composition 2 as an example, adipose-derived mesenchymal stem cells and bone marrow mesenchymal stem cells were respectively treated with cell culture medium (containing DMEM, 10v / v% fetal bovine serum, 2mML-glutamine). Adhesive culture, and then subculture with the serum-free medium of the present invention, observe the cell morphology obtained in different culture systems, and detect their activity.

[0042] Such as figure 1 As shown, the morphological changes of each generation of adipose-derived mesenchymal stem cells a and bone marrow mesenchymal stem cells b in the subculture process are shown respectively, which shows that the subculture quality of the serum-free medium of the present invention is good and the distribution is relatively uniform.

[0043] refer to figure 2 As shown, the area fraction of the rough structure area (that is, the disordered proliferation and aggregation of passaged stem cells) in th...

Embodiment 3

[0047] A serum-free suspension medium for cultivating human mesenchymal stem cells, the composition of which is as follows: 200 ng of growth hormone, 25 ng of cortisol, 14 μg of insulin, 12 μg of glycine, 12 μg of arginine, and threonine are contained in each 1 ml of DMEM medium. 12μg, Serine 35μg, Leucine 27μg, Isoleucine 20μg, L-Lysine 10μg, Methionine 35μg, L-Tyrosine 30μg, Endothelial Growth Factor 200ng, Fibroblast Growth Factor 120ng, Hepatocyte Growth Factor 60ng, folic acid 40ng, d-calcium pantothenate 20ng, choline chloride 5ng, vitamin B12 5ng, inositol 3ng, recombinant human transferrin 30ng, putrescine 20ng, recombinant human serum albumin 700ng, glutathione 100μg, vitamin C 10 μg, vitamin E 12 μg, calcium chloride 36 μg, potassium chloride 200 μg, sodium chloride 20 mg, ferric citrate 1 μg, polyoxyethylene-polyoxypropylene copolymer 1 mg.

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Abstract

The invention discloses a serum-free culture medium used for cultivating human mesenchymal stem cells. DMEM culture solution is taken as a basic culture medium, full nutritional components, namely amino acid, vitamin, protein nutrients and inorganic salt, and regulatory components, namely a glycoregulatory hormone, a growth factor and an antioxidant, are added into the DMEM culture solution. The serum-free culture medium used for cultivating the human mesenchymal stem cells can be used for cultivating the human mesenchymal stem cells with low cost, comprehensive nutrition is provided for the human mesenchymal stem cells, and growth of the human mesenchymal stem cells is effectively regulated and controlled, so that the human mesenchymal stem cells have good activity in a cultivation process, a mescenchymal stem cell cultivation process is improved, requirement on animal serum in the mesenchymal stem cell cultivation process is met, and animal protein residue and animal-sourced virus infection probability are reduced.

Description

technical field [0001] The invention relates to a serum-free culture medium, which belongs to the field of biochemistry. Background technique [0002] Due to their good self-proliferation ability and multi-directional differentiation potential, mesenchymal stem cells have the ability to differentiate into osteoblasts, chondrocytes, adipocytes, endothelial cells, nerve cells, muscle cells, liver cells, etc. under specific conditions in vivo and in vitro. The ability of adult cells to differentiate. Existing studies have shown that mesenchymal stem cells have immune regulation and hematopoietic support functions, and are easy to introduce and express foreign genes. Therefore, they can be used as seed cells in the construction of tissue engineered bone, cartilage and cardiac muscle, and important carrier cells in gene therapy. It also has broad application prospects in hematopoietic stem cell transplantation and organ transplantation. [0003] Due to the above-mentioned needs...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
Inventor 龚伟
Owner 龚伟
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