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Culture method of vascular endothelial progenitor cells

A culture method and vascular endothelial technology, applied in animal cells, vertebrate cells, artificial cell constructs, etc., can solve the problems of inducing immune response, increasing errors, mycoplasma contamination, etc., to avoid inducing immune response and excellent self-renewal ability. Effect

Active Publication Date: 2015-11-18
宁波医诺生物技术有限公司
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  • Abstract
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  • Application Information

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Problems solved by technology

Although the above medium can guarantee the excellent self-renewal ability of stem cells and induced differentiation cells, due to the introduction of a large amount of animal-derived substances into the medium, it brings uncertainties to the self-renewal and differentiation process of stem cells and induced differentiation cells. factors, increasing the error; at the same time, it may introduce animal-derived contamination, such as mycoplasma contamination; in addition, it will induce immune response during the cell therapy

Method used

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  • Culture method of vascular endothelial progenitor cells
  • Culture method of vascular endothelial progenitor cells
  • Culture method of vascular endothelial progenitor cells

Examples

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preparation example 1

[0038] 1) DMEM medium, F12 medium, sodium selenate, vitamin C, sodium bicarbonate, insulin, activin A, bone morphogenic protein 4, Rho protein kinase inhibitor (brand Y27632), glycogen synthase kinase- 3 inhibitor (brand CHIR99021) and glycogen synthase kinase-3 inhibitor (brand BIO) were mixed to prepare mixture W1; wherein, the volume ratio of DMEM medium to F12 medium was 1:2, and the concentration of vitamin C The concentration of insulin is 50 μg / ml, the concentration of insulin is 50 μg / ml, the concentration of activin A is 55 ng / ml, the concentration of bone morphogenic protein 4 is 60 ng / ml, and the concentration of glycogen synthase kinase-3 inhibitor (brand name is CHIR99021) The concentration of glycogen synthase kinase-3 inhibitor (brand is BIO) is 25 μM, the concentration of sodium selenate is 20 μg / L, and the concentration of sodium bicarbonate is 500 mg / L.

[0039] 2) adding sodium hydroxide to the above mixture W1 to adjust the pH to 7.8 to prepare mixture W2; ...

preparation example 2

[0042] Cell culture medium A2 was prepared according to the method of Preparation Example 1, except that no Rho protein kinase inhibitor was used in step 1), and the concentration of Rho protein kinase inhibitor was 10 μM.

preparation example 3

[0044] 1) DMEM medium, F12 medium, sodium selenate, vitamin C, sodium bicarbonate, insulin, activin A, bone morphogenic protein 4, Rho protein kinase inhibitor (brand Y27632), glycogen synthase kinase- 3 inhibitor (brand CHIR99021) and glycogen synthase kinase-3 inhibitor (brand BIO) were mixed to prepare mixture W1; wherein, the volume ratio of DMEM medium to F12 medium was 1:1, and the concentration of vitamin C The concentration of insulin is 5 μg / ml, the concentration of insulin is 3 μg / ml, the concentration of activin A is 7 ng / ml, the concentration of bone morphogenic protein 4 is 3 ng / ml, and the concentration of glycogen synthase kinase-3 inhibitor (brand name is CHIR99021) It is 2 μ M, the concentration of glycogen synthase kinase-3 inhibitor (brand is BIO) is 1 μ M, the concentration of sodium selenate is 2 μ g / L, the concentration of sodium bicarbonate is 400 mg / L, Rho protein kinase inhibitor (brand is Y27632) at a concentration of 2 μM.

[0045] 2) adding sodium ...

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Abstract

The invention discloses a culture method of vascular endothelial progenitor cells, which comprises the following steps: 1) inducing differentiation of multipotent stem cells in a culture medium A to form mesendoderm precursor cells; and 2) inducing differentiation of the mesendoderm precursor cells in a culture medium B to form the vascular endothelial progenitor cells. The culture medium A contains a DMEM (dulbecco's modified eagle medium) culture medium, an F12 culture medium, sodium selenate, sodium bicarbonate, vitamin C, insulin, activin A, a bone formation protein 4 and a glycogen synthetase kinase-3 inhibitor. The culture medium B contains a DMEM culture medium, an F12 culture medium, vitamin C, insulin, a vascular endothelial growth factor and a transforming growth factor beta signal pathway inhibitor. The culture media used in the culture method do not contain any animal-derived component, and can provide sufficient nutrient substances and stable living environments for the cultured cells, thereby efficiently inducing the differentiation of the human multipotent stem cells to form the vascular-grid vascular endothelial progenitor cells which are highly similar to the human cord vessel endothelial cells.

Description

technical field [0001] The invention relates to a cell culture method, in particular to a culture method for vascular endothelial cell progenitor cells. Background technique [0002] Stem cells refer to a type of cell that is capable of self-renewal, division, and differentiation into other cell types. Stem cells can be divided into embryonic stem cells (embryonic stem cells, ES cells) and adult stem cells (adult stem cells) from the stage of stem cells in development. Embryonic stem cells are a kind of pluripotent stem cells (pluripotent stem cells), which can proliferate indefinitely and differentiate into more than 200 types of cells in the human body, forming tissues and organs of the body, that is, stem cells with the potential to differentiate into various cells and tissues. Since Evans and Kaufman isolated embryonic stem cells from the inner cell mass (ICM) of mice in 1981, ES cell research has become a hot spot for scientists from all over the world. In recent years...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
Inventor 张竞方秦小平
Owner 宁波医诺生物技术有限公司
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