Vascular endothelial cell culture method

A technology of vascular endothelium and culture method, which is applied in the field of cell culture, can solve the problems of increasing error, inducing immune response, animal-derived contamination, etc., achieve excellent self-renewal ability, and avoid the effect of inducing immune response

Active Publication Date: 2015-09-23
宁波医诺生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • 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 di...

Method used

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  • Vascular endothelial cell culture method
  • Vascular endothelial cell culture method
  • Vascular endothelial cell culture method

Examples

Experimental program
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Effect test

preparation example 1

[0046] 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.

[0047] 2) adding sodium hydroxide to the above mixture W1 to adjust the pH to 7.8 to prepare mixture W2; ...

preparation example 2

[0050] 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

[0052] 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.

[0053] 2) adding sodium ...

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Abstract

The invention discloses a vascular endothelial cell culture method. The method comprises the following steps: 1) differentiating pluripotent stem cells into mesendoderm precursor cells in a culture medium A; 2) differentiating the mesendoderm precursor cells into progenitor cells of vascular endothelial cells in a culture medium B; 3) differentiating the progenitor cells of the vascular endothelial cells into the vascular endothelial cells in a culture medium C. The culture medium A contains a DMEM, an F12 culture medium, sodium selenate, sodium bicarbonate, vitamin C, insulin, an activin A , bone morphogenetic protein-4 and a glycogen synthase kinase-3 inhibitor; the culture medium B contains a DMEM, an F12 culture medium, sodium selenate, sodium bicarbonate, vitamin C, insulin, a vascular endothelial growth factor and a transforming growth factor beta signaling pathway inhibitor; the culture medium C contains a DMEM, an F12 culture medium, sodium selenate, sodium bicarbonate, vitamin C, insulin, a vascular endothelial growth factor, an epidermal growth factor and a fibroblast growth factor. According to the method, the pluripotent stem cells can be differentiated into the vascular endothelial cells.

Description

technical field [0001] The present invention relates to cell culture, in particular to a method for culturing vascular endothelial 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) according to 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 research hotspot for scientists from all over the world. In recent year...

Claims

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Application Information

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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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