Clinical-grade serum-free culture medium for stem cells

A serum-free medium and stem cell technology, which is applied in the field of clinical-grade stem cell serum-free medium and medium for cell culture, can solve problems such as batch variation, failure to maintain stem cell stemness, and imperfection

Active Publication Date: 2021-05-28
武汉济源高科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the traditional stem cell culture process, a certain amount of fetal bovine serum is usually added, but fetal bovine serum has several disadvantages: high cost, batch variation, limited availability, serum is susceptible to contamination by viruses, mycoplasma and other pathogens, and heteroimmunity The possibility of reaction, at the same time, serum contains a large number of amino acids, nucleosides, proteins, hormones, lipids and other trace components, the content and specific role of these components are still not fully determined, the lipids contained in serum can promote cell differentiation
In order to avoid the above defects in the use of fetal bovine serum, a medium that can replace animal serum has been developed. The most co

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  • Clinical-grade serum-free culture medium for stem cells
  • Clinical-grade serum-free culture medium for stem cells
  • Clinical-grade serum-free culture medium for stem cells

Examples

Experimental program
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Effect test

Embodiment 1

[0010] A serum-free medium for clinical-grade stem cells in this embodiment uses DMEM-F12 medium as the base medium, and removes lipid components and synthetic lipid components in DMEM-F12 medium that affect cell differentiation: including Inositol and linoleic acid also contain additives, the additives and their content (final concentration) are as follows: polyvinyl alcohol 5ng / ml, BMP-410ug / ml, vitamin C 40ug / ml, retinoic acid 1uM, nicotinamide 5ug / ml, azithromycin 20ug / ml.

[0011] The specific configuration method is that when configuring the DMEM-F12 medium, no inositol and linoleic acid components are added, or the DMEM-F12 medium is degreased with activated carbon. Add the above-mentioned components to the DMEM-F12 medium that does not contain lipid components and synthetic lipid components, so that the final concentration is the above concentration, thereby obtaining a clinical-grade stem cell serum-free medium.

Embodiment 2

[0013] A serum-free medium for clinical-grade stem cells in this embodiment uses E8 medium as the basal medium, and removes lipid components and synthetic lipid components in the E8 medium that affect cell differentiation: including inositol, sub- Oleic acid also contains additives, the additives and their content (final concentration) are as follows: polyvinyl alcohol 10ng / ml, BMP-4 20ug / ml, vitamin C 15ug / ml, retinoic acid 1uM, nicotinamide 1ug / ml, azithromycin 30ug / ml.

[0014] The specific configuration method is that when configuring the E8 medium, the components of inositol and linoleic acid are not added, or the E8 medium is degreased with activated carbon. The above-mentioned components are added to the E8 medium that does not contain lipid components and synthetic lipid components, so that the final concentration is the above concentration, thereby obtaining a clinical-grade stem cell serum-free medium.

Embodiment 3

[0016] Umbilical cord mesenchymal stem cells were cultured in vitro with a clinical-grade stem cell serum-free medium obtained in Example 1, and the steps were as follows:

[0017] (1) Collection and separation of umbilical cord tissue: One umbilical cord was taken from a healthy puerpera with full-term delivery. Wash and disinfect the umbilical cord tissue in a sterile biosafety cabinet in a GMP laboratory, remove the arterial and venous vessels and the epithelial membrane tissue of the amniotic membrane, and finally obtain the perivascular tissue and Wharton's jelly tissue, which are fully shredded into 1mm3 tissue blocks. Using the tissue block attachment method, add a clinical-grade stem cell serum-free medium prepared in Example 1, inoculate it into a culture dish or a culture bottle at an appropriate density, and culture it statically in a CO2 incubator at 37°C.

[0018] (2) Subculture, observation and detection of umbilical cord MSCs: when a large number of cells crawl ...

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Abstract

The invention discloses a clinical-grade serum-free culture medium for stem cells. According to the culture medium, an L-DMEM culture medium, an IMDM culture medium, a DMEM-F12 or alpha-MEM culture medium, an mTeSRTM1 culture medium, an E8 culture medium, a KSR conditioned culture medium and the like are used as basic culture media, and lipid substances such as inositol and linoleic acid and synthetic lipid substance components are removed; and meanwhile the culture medium contains the following added components: polyvinyl alcohol, bone morphogenetic protein BMP-4, vitamin C, retinoic acid, nicotinamide and azithromycin. The culture medium is clear in composition, controllable in quality and free of animal serum components, can be used for culturing various stem cells including pluripotent stem cells and multipotent stem cells, and can maintain the capability of stemness of the stem cells without differentiation after multiple passages.

Description

technical field [0001] The invention relates to a medium for cell culture, in particular to a serum-free medium for clinical-grade stem cells, which belongs to the field of biomedicine. Background technique [0002] The current basal medium cannot support the long-term culture of stem cells, and there is still room for optimization. Now, more research is looking at the effects of supplementation on stem cells. A certain amount of fetal bovine serum is usually added to the traditional stem cell culture process, but fetal bovine serum has several disadvantages: high cost, batch variation, limited availability, serum is susceptible to contamination by viruses, mycoplasma and other pathogens, and heteroimmunity The possibility of reaction, at the same time, serum contains a large number of amino acids, nucleosides, proteins, hormones, lipids and other trace components, the content and specific role of these components are still not fully determined, the lipids contained in seru...

Claims

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Application Information

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IPC IPC(8): C12N5/074C12N5/0775
CPCC12N5/0665C12N5/0696C12N2500/90C12N2500/50C12N2500/38C12N2501/155
Inventor 孔德照陈杰方松刚
Owner 武汉济源高科技有限公司
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