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Mesenchymal stem cell serum-free medium and culture method

A technology of serum-free medium and mesenchymal stem cells, which is applied in the field of serum-free medium and culture of mesenchymal stem cells, and can solve problems such as difficult stem cell climbing out, difficult cell primary culture, and treatment failure

Active Publication Date: 2018-07-06
SHANGHAI HUAXIN HIGH BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, studies have shown that stem cells can phagocytize proteins in the medium during the culture process, which contains bovine serum albumin (7mg-30mg / 10 8 individual cells), which can cause the recipients to produce anti-bovine protein antibodies and cause an immune response, leading to treatment failure in patients, especially after repeated infusions of mesenchymal stem cells.
[0005] The existing serum-free medium is difficult to make stem cells climb out of the tissue, and it is difficult to carry out primary cell culture

Method used

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  • Mesenchymal stem cell serum-free medium and culture method
  • Mesenchymal stem cell serum-free medium and culture method
  • Mesenchymal stem cell serum-free medium and culture method

Examples

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Embodiment 1

[0062] The serum-free medium for mesenchymal stem cells in this embodiment includes a basal medium and additional components, and the basal medium is one of L-DMEM, DMEM-F12 or IMDM. Added ingredients and their content ranges are as follows: Force Growth Factor 20-50ng / ml, Hypoxia Inducible Factor-1 10-20ng / ml, Laminin LN521 1-4ug / mL, Chemically DefinedLipid Concentrate 1-5%, bFGF 20- 40ng / ml, EGF 20-30ng / ml, VEGF 20-40ng / ml, HGF20-40ng / ml, PDGF 10-20ng / ml, transferrin 0.2-2ng / ml, trypsin 10-30ug / ml, aprotinin Enzyme 0.1mg / ml, insulin 1-10U / ml, leukemia inhibitory factor 500-2000U / mL, choline dicitrate 20-30nM, phosphatidylcholine 1-10ng / ml, phosphatidic acid sodium salt 1-10ng / ml ml, soybean lecithin 1-10ng / ml, vitamin C 50ug / ml, vitamin E30ug / ml, vitamin B12 30ug / ml, estradiol 1-5ng / ml, testosterone 1-5ng / ml, progesterone 1-5ng / ml ml, ACTH 0.5-1ug / ml, dexamethasone 0.2-0.3nM, heparin 50-200μg / mL, ethanolamine 20-50nM, glutathione (reduced form) 2.5-5ug / ml, sodium selenite ...

Embodiment 2

[0064] This embodiment provides a serum-free medium for mesenchymal stem cells, adding the following additional components to the L-DMEM (or DMEM-12 / IMDM) medium, and making the final concentration the following concentration, thus To obtain serum-free medium for mesenchymal stem cells:

[0065] Force Growth Factor 20ng / ml, Hypoxia Inducible Factor-1 10ng / ml, Laminin LN521 2.5ug / mL, CDLC 2%, bFGF 20ng / ml, EGF 30ng / ml, VEGF 20ng / ml, HGF 20ng / ml, PDGF 20ng / ml, transferrin 0.2ng / ml, trypsin 10-30ug / ml, aprotinin 0.1mg / ml, insulin 5U / ml, leukemia inhibitory factor 500U / mL, choline dicitrate 20nM, phospholipids Acylcholine 1ng / ml, phosphatidic acid sodium salt 1ng / ml, soybean lecithin 1ng / ml, vitamin C 50ug / ml, vitamin E 30ug / ml, vitamin B12 30ug / ml, estradiol 1ng / mL, testosterone 2ng / ml , progesterone 2ng / mL, corticotropin 0.5ug / ml, dexamethasone 0.2nM, heparin 50μg / mL, ethanolamine 20nM, reduced glutathione 5ug / ml, sodium selenite 15nM, iron citrate 2.5 ng / mL, L-glutamine 5mM, ...

Embodiment 3

[0067] This example provides a serum-free medium for mesenchymal stem cells. The difference between this example and Example 1 is that the added ingredients also include vitamin B2 50ug / ml;

[0068] In another option, the added ingredients also include spirulina polysaccharide 15ug / ml;

[0069] More preferably, the added ingredients also include vitamin B2 50ug / ml and spirulina polysaccharide 15ug / ml.

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Abstract

The invention discloses a mesenchymal stem cell serum-free medium and a culture method, the serum-free medium comprises a basal medium and an additive component, the additive component comprises hypoxia-inducible factor-1 and a force growth factor, the concentration of the hypoxia-inducing factor -1 is 10-20 ng / ml; and the concentration of the force growth factor is 20-50 ng / ml. The serum-free medium is used for culturing mesenchymal stem cells under 5% hypoxic culture conditions. The serum-free medium is determined in component and controllable in quality, can be used for tissue adherent separation of the mesenchymal stem cells, can make growth of the mesenchymal stem cells normal; the serum-free medium is combined with the hypoxic culture conditions to play a synergistic effect on the growth of the mesenchymal stem cells, and the growth of the mesenchymal stem cells is greatly improved. At the same time, the '' stemness'' of the mesenchymal stem cells and differentiation capability of the mesenchymal stem cells into osteoblast, chondrocyte, adipocyte and other cells are remained after several passages.

Description

technical field [0001] The invention belongs to the field of cell culture, and in particular relates to a serum-free medium and a culture method for mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells (MSCs) are pluripotent stem cells with high self-renewal ability and multi-lineage differentiation potential, which are derived from the mesoderm and ectoderm in the early stages of development. Mesenchymal stem cells were originally found in bone marrow, and have attracted increasing attention because of their multi-directional differentiation potential, hematopoietic support, promotion of stem cell implantation, immune regulation, and self-replication; , umbilical cord blood, umbilical cord and other tissues. Mesenchymal stem cells can differentiate into fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelial and other tissue cells under specific induction conditions in vivo or in vitro. Potential for multidirection...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
CPCC12N5/0662C12N5/0668C12N2500/02C12N2500/25C12N2500/30C12N2500/32C12N2500/36C12N2500/44C12N2500/46C12N2500/90C12N2501/105C12N2501/11C12N2501/115C12N2501/12C12N2501/135C12N2501/165C12N2501/235C12N2501/30C12N2501/39C12N2501/392C12N2501/60C12N2501/734C12N2501/90C12N2501/91C12N2501/999C12N2533/52
Inventor 施坤宁刘惠
Owner SHANGHAI HUAXIN HIGH BIOTECH
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