Serum-free medium for mesenchymal stem cells as well as preparation method and applications of serum-free medium

A serum-free medium and mesenchymal stem cell technology, applied in primary culture and large-scale subculture, the formula and preparation of new mesenchymal stem cell serum-free medium, can solve the difficulties of standardization of stem cell production, stem cell toxicity, Serum is expensive and other problems, to achieve the effects of standardization and continuous production, reducing the chance of contamination, and reliable experimental results

Active Publication Date: 2017-05-31
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

Benefits of technology

This patented invented an improved method for culturing cells without any added substances that may harm them during their cultivation process. Instead, it uses natural ingredients instead of artificial ones like proteins found naturally on animal blood plasma. These methods help prevent contamination from other sources while still allowing us to study specific types of cells with greater reliability than existing techniques such as bacteria lysis.

Problems solved by technology

This patents discuss different methods used during culturing stem cells (SCs). However, these techniques can result in variations from unknown compositions that affect their quality and performance. Additionally, current methodologies involve adding animal blood into the media which could potentially introduce harmful agents like virus strains or other organisms involved in disease transmission. Therefore, this problem becomes more challenging when producing high purity SC samples containing specific compounds needed for medical applications.

Method used

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  • Serum-free medium for mesenchymal stem cells as well as preparation method and applications of serum-free medium
  • Serum-free medium for mesenchymal stem cells as well as preparation method and applications of serum-free medium
  • Serum-free medium for mesenchymal stem cells as well as preparation method and applications of serum-free medium

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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

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Claims

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Application Information

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Owner 湖南省生宝生物科技有限公司
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