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Human umbilical cord mesenchymal stem cell serum-free medium

A serum-free medium and stromal cell technology, which is applied to the formulation field of serum-free medium for human umbilical cord mesenchymal stem cells, can solve the problems of insufficient cell proliferation rate, cumbersome operation, poor cell adherence, etc., and achieves high cell proliferation rate. , Simple operation, good effect of stem cell characteristics

Pending Publication Date: 2019-03-01
青岛麦迪赛斯生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the commercial MSCs serum-free medium currently on the market, whether it is imported from abroad or domestically developed and produced, is not only expensive, but also the effect of cell culture is not ideal
Compared with the serum culture system, the cell state is poor, the cell adhesion is poor, the operation is cumbersome, the cell proliferation rate is not enough, and the cell aging speed is fast, and the differentiation ability of MSCs is reduced.

Method used

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  • Human umbilical cord mesenchymal stem cell serum-free medium
  • Human umbilical cord mesenchymal stem cell serum-free medium
  • Human umbilical cord mesenchymal stem cell serum-free medium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Screening of Human Umbilical Cord Mesenchymal Stem Cell Serum-Free Medium Proportion

[0029] Preparation method: Add glutamine mother solution, HEPEs mother solution, putrescine, transferrin mother solution, vitamin C mother solution, insulin mother solution, progesterone mother solution, cortisol mother solution, human blood to the α-MEM basal medium according to the stated concentration Albumin mother liquor, basic fibroblast growth factor mother liquor, transforming growth factor mother liquor, instant proteoglycan mother liquor, stirred and mixed, and filtered through a 0.2 μm filter membrane to obtain the product.

[0030] Glutamine mother solution: take 0.5g, dissolve in triple distilled water, make 200mM mother solution, and store at -20°C. Preparation of recombinant transferrin mother solution: take 50 mg, dissolve in α-MEM basal medium, prepare 1 mM mother solution, and store at -20°C. Preparation of vitamin C mother solution: take 1 g, dissolve it ...

Embodiment 2

[0036] Example 2 Isolation of human umbilical cord mesenchymal stem cells

[0037] Take the fresh umbilical cord of the newborn, put it into 100ml of DMEM-LG medium containing 100U / ml penicillin-streptomycin mixture, and transport it at 4°C to 12°C; process the umbilical cord in a 100-level biological safety cabinet, and wash it with 75% medical alcohol Soak the umbilical cord for 30s-60s, remove both ends of the umbilical cord, cut the middle section into 1-2cm long sections, rinse the small section of umbilical cord repeatedly with normal saline, cut it open, remove the arteries and veins in it, and rinse repeatedly with normal saline to remove blood stains , chopped to 2mm 3 . Put the shredded tissue blocks evenly in a 150mm cell culture dish (one solid tissue block per 1-1.5 square centimeters), let stand in a safety cabinet for 10 minutes (air-dry the liquid between the culture dish and the tissue block), and then Place the Petri dish upside down at 37°C, 5% CO2 , in a ...

Embodiment 3

[0038] Example 3 Detection of expression levels of cell surface markers by flow cytometry

[0039] Take the P3 human umbilical cord mesenchymal stem cells cultured with three kinds of media (A, B, C) in Example 2, and when the cells are fused to 90%, digest and collect the cells, wash them twice with PBS, add Fluorescently labeled CD44, CD73, CD90, CD105, CD34, CD45, and HLA-DR surface antibodies were incubated for 30 min, then washed twice with PBS, and the expression levels of cell surface markers were detected by flow cytometry. The results are shown in Table 2.

[0040] Table 2 Expression levels of cell surface markers

[0041]

[0042]

[0043] It can be seen from Table 2 that the surface marker proteins of P3 generation human umbilical cord mesenchymal stem cells cultured in three media (A, B, C) all conform to the biological characteristics of MSCs.

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Abstract

The invention provides a human umbilical cord mesenchymal stem cell serum-free medium characterized by comprising an alpha-MEM basal medium and further comprising the following components by final concentration: 1-20 mM of glutamine, 1-20 mM of HEPEs, 10-100 muM of putrescine, 0.1-10 muM of transferrin, 10-400 muM of vitamin C, 1-10 muM of recombinant insulin, 1-20 nM of progesterone, 10-200 nM ofcortisol, 1-20 mg / mL of human serum albumin, 1-10 ng / mL of basic fibroblast growth factor, beta 11-10 ng / mL of transforming growth factor and 1-50 mg / mL of spirulina fast-soluble proteoglycan. The human umbilical cord mesenchymal stem cell serum-free medium can significantly improve the proliferation rate and the adherence performance of human umbilical cord mesenchymal stem cells, is beneficialto the proliferation of the human umbilical cord mesenchymal stem cells and the maintenance of stem cell characteristics, and has the potential of adipogenic osteogenic induced differentiation. The medium is simple, clear and stable, does not contain any serum components, overcomes the risk of heterologous protein contamination and pathogenic microorganisms, and has high safety.

Description

technical field [0001] The invention belongs to the technical field of stem cell culture, and in particular relates to a formula of a serum-free medium for human umbilical cord mesenchymal stem cells and an application thereof. Background technique [0002] Mesenchymal stem cells (Mesenchymal Stem Cells, MSCs) are adult stem cells derived from early mesoderm, which have high self-renewal ability and multi-directional differentiation potential. For example, bone, cartilage, muscle, ligament, tendon, fat and stromal cells proliferate and differentiate, and have weak immunogenicity, so they are the source of seed cells for tissue engineering projects. Human umbilical cord mesenchymal stem cells (human umbilical cord mesenchymalstem cells, hUC-MSCs) are derived from Wharton's jelly in the umbilical cord of newborns. In addition to all the characteristics of MSCs, they are also rich in content, pure in cells, and less immunogenic , original cells, stronger differentiation abilit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
CPCC12N5/0665C12N2501/115C12N2500/24C12N2500/30C12N2500/46C12N2500/34C12N2500/38C12N2500/32C12N2501/15C12N2501/998C12N2501/33
Inventor 王玉娟徐矫健葛淑娟刘燕丽
Owner 青岛麦迪赛斯生物科技有限公司
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