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Culture method and serum-free medium for promoting proliferation and differentiation of mesenchymal stem cells

A serum-free medium and quality stem cell technology, applied in the field of stem cell culture, can solve problems such as risks, and achieve the effects of reducing risks, maintaining performance, and reducing culture costs

Active Publication Date: 2022-05-10
徐飞
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Platelet lysate still contains a small amount of protein, which makes its safety more reliable than serum, but it still has risks. At the same time, the current DMEM / F12 system containing platelet lysate needs to ensure that the content of platelet lysate is at least 5%. Better maintenance of the characteristics of mesenchymal stem cells in subculture expansion

Method used

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  • Culture method and serum-free medium for promoting proliferation and differentiation of mesenchymal stem cells
  • Culture method and serum-free medium for promoting proliferation and differentiation of mesenchymal stem cells
  • Culture method and serum-free medium for promoting proliferation and differentiation of mesenchymal stem cells

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Effect test

Embodiment 1

[0045] Example 1, Table 1 Serum-free medium L1-L7

[0046]

[0047] Preparation of serum-free medium:

[0048] According to the formula in Table 1 above, add the ingredients in the table to DMEM / F12 medium in order to obtain serum-free medium L1-L7.

Embodiment 2

[0049] Embodiment 2, umbilical cord mesenchymal stem cell culture method

[0050] 2.1 Primary isolation and culture of umbilical cord mesenchymal stem cells: Under sterile conditions, the umbilical cord was washed twice with normal saline solution containing a mixture of penicillin and streptomycin until there was no residual blood; into 1cm 3 Tissue blocks of left and right size; the tissue blocks were inoculated at a density of 1×10 4 / cm 2 Inoculate evenly in mesenchymal stem cell medium L1-L7, place at 37°C, 5% CO 2 Cultured in a constant temperature box, the liquid was replenished on the 2nd day after inoculation, and the liquid was changed every 3 days thereafter.

[0051] 2.2 Subculture and expansion culture of umbilical cord stem cells: when the adherent cells are fused to 80-90%, aspirate the medium respectively, wash with PBS buffer twice, and add an appropriate amount of 0.2% trypsin for digestion; aspirate the trypsin, add DMEM / F12 for culture After the cells w...

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Abstract

The invention belongs to the technical field of stem cell culture. More specifically, it relates to a culture method and serum-free medium for promoting the proliferation and differentiation of mesenchymal stem cells. It includes the following components: 0.1-3.5% of human platelet lysate, 1-3 mmol / L of L-glutamine, 5-30 μg / mL of vitamin C derivatives, and the basal medium is supplemented to 1 L. The serum-free medium of the present invention adds lentinan and 2-O-ether ascorbic acid, realizes that only a relatively low concentration of platelet lysate can meet the needs of continuous expansion and culture of mesenchymal stem cells in vitro, and can be well The ability to maintain the proliferation and high differentiation of mesenchymal stem cells greatly reduces the cost of culture and the risk of clinical application.

Description

technical field [0001] The invention belongs to the technical field of stem cell culture. More specifically, it relates to a culture method and serum-free medium for promoting the proliferation and differentiation of mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells can grow on a solid surface, express specific antigens on the cell surface (positive expression of CD29, CD73, CD90, CD105, and negative for CD34, CD45, etc.), and have the ability to induce differentiation into adipocytes in vitro, Potential of osteoblasts and chondrocytes. Therefore, mesenchymal stem cells can play an important regulatory role in promoting hematopoiesis, immune regulation, and promoting angiogenesis. [0003] The clinical application of mesenchymal stem cells must undergo in vitro culture and expansion of cells. In the in vitro culture system, conditions similar to body fluid environment and necessary nutrients for cell growth and proliferation need to be provided, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0775
CPCC12N5/0668C12N2500/90C12N2500/84C12N2500/38C12N2501/90
Inventor 黄炎明
Owner 徐飞
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