A serum-free medium for mesenchymal stem cells and its preparation method and application

A serum-free medium and stem cell technology, which is applied in the formulation and preparation of a new type of mesenchymal stem cell serum-free medium, primary culture and large-scale subculture, and can solve the problem of standardization of stem cell production and stem cell toxicity. , the high price of serum, etc., to achieve the effect of being conducive to standardization and continuous production, reducing the chance of contamination, and reliable experimental results

Active Publication Date: 2020-02-14
湖南省生宝生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But there are also many problems: (1) Serum is a very complex mixture with various components. At present, its exact composition, content and mechanism of action are still unclear, and some may have inhibitory or toxic effects on the growth of stem cells; (2) ) Serum is produced in batches, and the components or contents of biologically active substances and factors are inconsistent between batches, resulting in poor reproducibility of experimental results or products, making it difficult to standardize stem cell production; (3) animal serum may contain exogenous The risks of infectious viruses and pathogenic factors, such as mycoplasma, viruses, etc., lead to contamination of stem cell production; (4) In large-scale stem cell culture, serum is expensive and is one of the main parts of production costs; (5) The clinical application of stem cells During application, residual serum may cause allergic reactions in patients

Method used

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  • A serum-free medium for mesenchymal stem cells and its preparation method and application
  • A serum-free medium for mesenchymal stem cells and its preparation method and application
  • A serum-free medium for mesenchymal stem cells and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Embodiment 1: the preferred version of the present invention is as follows:

[0072] The final concentration of each component in the basal medium after mixing the basal medium with concentrated supplements:

[0073] DMEM-LG medium powder 10g / L; Purmorphamine 2.5μM; Insulin 10.0mg / L; Transferrin 0.60mg / L; Ethanolamine 0.4mg / L; L-ascorbic acid-2-phosphate 15μmol / L; ×10 -5 mol / L; L-glutamine 150mg / L; L-alanyl-L-glutamine 50mg / L; sodium selenite 2.5mg / L; sodium pyruvate 150mg / L; ferrous sulfate 0.8mg / L L; zinc chloride 0.4mg / L; dexamethasone 1.0nmol / L; sodium bicarbonate 2000mg / L; 4-hydroxyethylpiperazineethanesulfonic acid 1000mg / L;

[0074] Concentrate Additive Components and Concentrations Prepared Before Mixing:

[0075] Human serum albumin 20g / L; fibronectin 10mg / L; α-antitrypsin 5mg / L; linoleic acid 5mg / L; linolenic acid 5mg / L; lecithin 2mg / L; BMP-7 5μg / L ; Wnt5a 125 μg / L; EGF 50 μg / L; bFGF 100 μg / L; PDGF 10 μg / L; TGF-β 2 μg / L.

Embodiment 2

[0076] Embodiment 2: The experiment is divided into three groups, which are respectively three kinds of culture media, as follows:

[0077] A culture medium: the serum-free medium for mesenchymal stem cells in the present invention is composed of basal medium and concentrated additives mixed in a volume ratio of 9:1;

[0078] B medium: commercial serum-free medium, StemPro MSC SFMCTS human mesenchymal stem cell serum-free medium from Life technologies;

[0079] C medium: commercial medium with serum, MEM+10% fetal bovine serum.

[0080] In this example, the cultivation of primary cells of human umbilical cord mesenchymal stem cells by three culture media was compared. Take the umbilical cord tissue, sterilize it with 75% alcohol, remove the arteries, veins and amniotic membrane under aseptic conditions, tear out Huatong glue, and cut it into pieces to about 1-3mm 3 Use a pipette to absorb the Huatong glue block, and divide it into three 75cm mediums respectively equipped wit...

Embodiment 3

[0084] Example 3: Primary umbilical cord mesenchymal stem cells were cultured using the serum-free A medium, B medium, and C medium of the present invention to detect their effects on cell phenotype. The primary umbilical cord mesenchymal stem cells cultured in three kinds of media were harvested, washed twice with normal saline, the supernatant was discarded, and the cell concentration was adjusted to 2×10 6 pieces / ml. Each sample was labeled with fluorescent antibodies. The labeled antibodies included CD13, CD14, CD19, CD29, CD31, CD34, CD44, CD73, CD90, CD105, HLA-ABC, and HLA-DR. Isotype antibodies were used as controls. Add 5 μl of the corresponding fluorescent antibody to each tube, and then add 100 μl of umbilical cord mesenchymal stem cells to each tube, shake well with a vortex shaker, and incubate at 4°C in the dark for 30 min. Add 3ml of normal saline to each tube, wash, centrifuge at 1200rpm for 5min and discard the supernatant; add 2ml of PBS to each tube, centri...

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Abstract

The invention discloses a serum-free medium for mesenchymal stem cells as well as a preparation method and applications of the serum-free medium. Cytokines in serum are replaced by adding EGF, PDGF, bFGF, beta-TGF, BMP-7 and wnt5a; lipids in serum are replaced by adding linoleic acid, linolenic acid and lecithin; through orthogonal design, the additives are compounded, and the good proliferation effect is obtained. In addition, the ingredients are definite, and the batch-to-batch differences are reduced; the potential risks of exogenous viruses and pathogenic factors are eliminated; the untoward effects of clinical research are reduced; and the application prospect is good.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a formula and a preparation method of a novel serum-free medium for mesenchymal stem cells, and its application in primary culture and large-scale subculture of human umbilical cord mesenchymal stem cells. Background technique [0002] With the rapid development of regenerative medicine, the research and clinical application of stem cells has become one of the most cutting-edge and hottest fields in life sciences. It not only brings hope for human beings to overcome many diseases, but also has huge market potential. The culture medium closely related to stem cell research and clinical application is an indispensable part. At present, a certain amount of animal-derived serum, such as newborn bovine serum or fetal bovine serum, is added to the culture of stem cells to meet the needs of stem cell growth and proliferation. But there are also many problems: (1) Serum is a ver...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0775
CPCC12N5/0665C12N2500/12C12N2500/22C12N2500/24C12N2500/30C12N2500/32C12N2500/38C12N2500/44C12N2500/46C12N2501/11C12N2501/115C12N2501/15C12N2501/33C12N2501/998
Inventor 唐升斌徐峰波卢开红肖笑
Owner 湖南省生宝生物科技有限公司
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