A clinical grade stem cell serum-free medium

A serum-free medium and stem cell technology, which is applied in the field of cell culture medium and clinical-grade stem cell serum-free medium, which can solve the problems of limited availability, high cost, and inability to maintain stem cell stemness well.

Active Publication Date: 2022-07-15
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 commonly used ones are human AB serum, human platelet lysate and serum-free chemically defined medium, but each has its own shortcomings. AB serum Both human platelet lysate and human platelet lysate have the possibility of virus contamination, and there are obvious differences between different batches; various growth factors released by human platelet lysate may have substances that inhibit cell growth, and change the expression of cell surface markers and Cell function, etc. Although the current chemically defined medium does not contain serum and can avoid potential risks brought by serum products, it is not perfect. It can promote stem cell differentiation and improve immunogenicity, but cannot maintain the stemness of stem cells well.

Method used

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  • A clinical grade stem cell serum-free medium
  • A clinical grade stem cell serum-free medium
  • A clinical grade stem cell serum-free medium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0010] The serum-free medium for clinical-grade stem cells of this embodiment uses DMEM-F12 medium as the basal medium, and removes lipid substances that affect cell differentiation and substances that synthesize lipids in the DMEM-F12 medium: including Inositol and linoleic acid also contain added ingredients. The added ingredients and their content (final concentration) are as follows: polyvinyl alcohol 5ng / ml, BMP-4 10ug / ml, vitamin C 40ug / ml, retinoic acid 1uM, niacinamide 5ug / ml, azithromycin 20ug / ml.

[0011] The specific configuration method is to not add inositol and linoleic acid components when configuring DMEM-F12 medium, or to delipidize DMEM-F12 medium with activated carbon. The above components were added to the DMEM-F12 medium containing no lipid components and synthetic lipid components to achieve the above concentrations, thereby obtaining a serum-free medium for clinical-grade stem cells.

Embodiment 2

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

[0014] The specific configuration method is, when configuring E8 medium, do not add inositol and linoleic acid components, or E8 medium is degreased with activated carbon. The above-mentioned components are added to the E8 medium containing no lipid components and synthetic lipid components, so that the final concentration is the above concentration, thereby obtaining a serum-free medium for clinical-grade stem cells.

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 from a healthy full-term puerpera was taken. The acquisition process was legal, and the detection items for hepatitis B, hepatitis C, HIV, syphilis, ALT, and mycoplasma were all negative. In the sterile biological safety cabinet of the GMP laboratory, the umbilical cord tissue was washed, disinfected, and the arteriovenous blood vessels and the fetal amniotic membrane tissue were removed. Finally, the perivascular tissue and Wharton's jelly tissue were obtained, which were fully cut into 1mm3 tissue pieces. A clinical-grade stem cell serum-free medium prepared in Example 1 was added by the tissue block adherence method, and seeded into a petri dish or flask at an appropriate density, and cultured at 37°C in a CO2 incubator.

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Abstract

The invention discloses a serum-free culture medium for clinical grade stem cells. The culture medium of the present invention is composed of L-DMEM, IMDM, DMEM-F12 or α-MEM, mTeSR TM 1 Medium, E8 medium and KSR conditioned medium are basal medium, remove lipids such as inositol and linoleic acid and synthetic lipids, and also contain the following additives: polyvinyl alcohol, bone morphogenetic protein BMP‑4, Vitamin C, Retinoic Acid, Niacinamide, Azithromycin. The medium has clear components, controllable quality, and does not contain animal serum components. It can cultivate various stem cells including pluripotent stem cells and multipotent stem cells, and can maintain the "stemness" of stem cells without the ability to differentiate after multiple passages.

Description

technical field [0001] The invention relates to a culture medium for cells, in particular to a serum-free culture 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 variability, limited availability, serum susceptibility to virus, mycoplasma and other pathogens contamination, and xenoimmunity The possibility of reaction is a problem. At the same time, serum contains a large number of amino acids, nucleosides, proteins, hormones, lipids and other trace components. The content and specific functions of these components are still not fully determined. The li...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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