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

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

Active Publication Date: 2021-08-13
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 for promoting proliferation and differentiation of mesenchymal stem cells and serum-free culture medium
  • Culture method for promoting proliferation and differentiation of mesenchymal stem cells and serum-free culture medium
  • Culture method for promoting proliferation and differentiation of mesenchymal stem cells and serum-free culture medium

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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, and particularly relates to a culture method for promoting proliferation and differentiation of mesenchymal stem cells and a serum-free culture medium. The serum-free culture medium is prepared from the following components: 0.1%-3.5% of human platelet lysate, 1-3mmol / L of L-glutamine, 5-30[mu]g / mL of vitamin C derivative and the balance of a basic culture medium, wherein the total volume is 1L. According to the serum-free culture medium, lentinan and 2-O-diethyl ether ascorbic acid are added, so that the requirement of in-vitro continuous multiplication culture of the mesenchymal stem cells can be met by only adopting a platelet lysis solution with relatively low concentration, the proliferation and high differentiation performance of the mesenchymal stem cells can be well maintained, so that the culture cost is greatly reduced, and meanwhile, the clinical application risk is reduced.

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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IPC IPC(8): C12N5/0775
CPCC12N5/0668C12N2500/90C12N2500/84C12N2500/38C12N2501/90
Inventor 黄炎明
Owner 徐飞
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