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In-vitro culture solution of mesenchymal stem cells and culture and amplification method

A technology of bone marrow mesenchyme and in vitro culture, which is applied in the direction of cell culture active agent, bone/connective tissue cells, culture process, etc., can solve the problems of complicated operation, high cost, and long culture period, and achieve the purpose of improving amplification efficiency, Significant progress and the effect of highlighting substantive features

Inactive Publication Date: 2016-12-07
袁肇斌
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the classic Dexter-type longterm cultures (Dexter-LTC) is mostly used for in vitro expansion and culture of HBMSCs, which is high in cost, complicated in operation, prone to aging of cultured cells, and long in culture period, which is far from meeting the needs of clinical and scientific research.

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  • In-vitro culture solution of mesenchymal stem cells and culture and amplification method
  • In-vitro culture solution of mesenchymal stem cells and culture and amplification method
  • In-vitro culture solution of mesenchymal stem cells and culture and amplification method

Examples

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Effect test

Embodiment 1

[0024] Example 1: Preparation of in vitro culture medium and in vitro culture and expansion method

[0025] In vitro culture medium composition:

[0026] Prepare 950ml of DMEM culture medium as the basic medium, and add 20mg of compound (I), 300mg of L-glutamine, 75mg of vitamin C, 15mU of insulin, 30U of erythropoietin and 70μg of vascular endothelial growth factor in sequence, and then adjust it with sodium hydroxide. When the pH value reaches 7.0-7.2, 50ml of fetal bovine serum is finally added and sterilized by filtration to obtain an in vitro culture medium of bone marrow mesenchymal stem cells, which is stored at 4°C for later use.

[0027] Culture expansion method:

[0028] Step S1, preparing the above-mentioned bone marrow mesenchymal stem cell in vitro culture medium;

[0029] Step S2, collecting primary human bone marrow mesenchymal stem cells, digesting with 0.25% trypsin to make a cell suspension, counting the number of cells, adjusting the cell density to 2×10 ...

Embodiment 2

[0033] Embodiment 2: Preparation of in vitro culture medium and in vitro culture expansion method

[0034] In vitro culture medium composition:

[0035] Prepare 950ml of DMEM culture medium as the basic medium, and add 15mg of compound (I), 200mg of L-glutamine, 60mg of vitamin C, 10mU of insulin, 20U of erythropoietin and 20μg of vascular endothelial growth factor in sequence, and adjust with sodium hydroxide When the pH value reaches 7.0-7.2, 50ml of fetal bovine serum is finally added and sterilized by filtration to obtain an in vitro culture medium of bone marrow mesenchymal stem cells, which is stored at 4°C for later use.

[0036] Culture expansion method:

[0037] Step S1, preparing the above-mentioned bone marrow mesenchymal stem cell in vitro culture medium;

[0038] Step S2, collecting primary human bone marrow mesenchymal stem cells, digesting with 0.25% trypsin to make a cell suspension, counting the number of cells, adjusting the cell density to 2×10 5 Each / m...

Embodiment 3

[0042] Embodiment 3: Preparation of in vitro culture medium and in vitro culture expansion method

[0043] In vitro culture medium composition:

[0044] Prepare 950ml of DMEM culture medium as the basic medium, and add 25mg of compound (I), 400mg of L-glutamine, 90mg of vitamin C, 20mU of insulin, 40U of erythropoietin and 120μg of vascular endothelial growth factor in sequence, and then adjust it with sodium hydroxide. When the pH value reaches 7.0-7.2, 50ml of fetal bovine serum is finally added and sterilized by filtration to obtain an in vitro culture medium of bone marrow mesenchymal stem cells, which is stored at 4°C for later use.

[0045] Culture expansion method:

[0046] Step S1, preparing the above-mentioned bone marrow mesenchymal stem cell in vitro culture medium;

[0047] Step S2, collecting primary human bone marrow mesenchymal stem cells, digesting with 0.25% trypsin to make a cell suspension, counting the number of cells, adjusting the cell density to 2×10...

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Abstract

The invention discloses an in-vitro culture solution of mesenchymal stem cells and a culture and amplification method. According to the in-vitro culture solution, DMEM culture solution is taken as a basic culture medium, compound (I), L-glutamine, vitamin C, insulin, erythrogenin and vascular endothelial growth factors are sequentially added, then sodium hydroxide is adopted for adjusting the pH, finally, fetal calf serum is added, filtration sterilization is carried out, and thus the in-vitro culture solution of the mesenchymal stem cells is obtained, wherein the concentration of the compound (I) is 15-25 mg / L, the concentration of L-glutamine is 200-400 mg / L, the concentration of vitamin C is 60-90 mg / L, the concentration of insulin is 10-20 mU / L, the concentration of the erythrogenin is 20-40 U / L, and the concentration of the vascular endothelial growth factors is 20-120 [mu] g / L. With the adoption of the in-vitro culture solution of mesenchymal stem cells and the culture and amplification method, the amplification efficiency of the human mesenchymal stem cells can be improved, and compared with the prior art, the in-vitro culture solution and the culture and amplification method have the outstanding substantive features and prominent progress.

Description

technical field [0001] The invention relates to the cultivation of mesenchymal stem cells, in particular to an in vitro culture solution and a method for culturing and expanding bone marrow mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells (MSCs) are a group of stem cells with the ability to differentiate into osteoblasts, chondrocytes and adipocytes, which exist in adult bone marrow, bone, fat and muscle tissues. The in vitro adherence of MSC makes it easy to isolate and purify, and the MSC cultured in vitro has strong proliferation ability, so it is a kind of seed cell with important application value. [0003] Human bone marrow mesenchymal stem cells (HBMSCs) are pluripotent stem cells with self-renewal and multi-lineage differentiation potential, which can differentiate into bone, fat, cartilage, nerve, liver Cardiomyocytes and other cell tissues have great application potential in clinical treatment of radiation sickness, blood disease, and t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
CPCC12N5/0663C12N2500/30C12N2500/32C12N2500/38C12N2501/14C12N2501/165C12N2501/33
Inventor 袁肇斌
Owner 袁肇斌
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