A kind of special culture medium and method for differentiating human embryonic stem cells into endothelial cells

A technology of human embryonic stem cells and differentiation medium, which is applied in the field of medium for differentiation of human embryonic stem cells into endothelial cells. The effect of high efficiency and survival rate, cell growth and differentiation speed improvement

Active Publication Date: 2018-05-08
GENECAST WUXI PRECISION MEDICAL DIGNOSTIC LAB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Based on the above method or the lack of medium composition, in the process of culturing cells in the medium, the growth rate is slow and aging characteristics are prone to appear, thus affecting the large-scale expansion of cells

Method used

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  • A kind of special culture medium and method for differentiating human embryonic stem cells into endothelial cells
  • A kind of special culture medium and method for differentiating human embryonic stem cells into endothelial cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment 1: recovery, cultivation and subculture of the first method human embryonic stem cells;

[0020] (1) A commercially purchased human embryonic stem cell line (H9) was purchased from Jiangsu Stanford Biotechnology Co., Ltd. Melt rapidly in a water bath (15s), add to 20mL human embryonic stem cell medium (L-glutamine 2mmol / L, retinoic acid concentration 3×10 -9 mol / L, transferrin 6mg / L, sodium selenite 30mg / L and human epidermal growth factor: 0.15mg / L, fibronectin: 0.5mg / L, TGF-β1 its concentration is 5ng / ml, cells Differentiation-promoting peptides (sequence shown in SEQ ID NO: 1) with a final concentration of 100ppm, prepared in DMEM / F12 (1:1) medium) in a 50mL centrifuge tube, naturally precipitated for 30min, sucked off the supernatant, and used 3mL Gently resuspend the pellet in the culture medium and add the resuspension to a 6 cm Petri dish treated with 0.1% gelatin on which MEFs have been laid. When the clones of human embryonic stem cells were overgr...

Embodiment 2

[0022] Embodiment 2: second method recovery, cultivation and subculture of human embryonic stem cells;

[0023] (1) A commercially purchased human embryonic stem cell line (H9) was purchased from Jiangsu Stanford Biotechnology Co., Ltd. Melt rapidly in a water bath (15s), add to 20mL human embryonic stem cell medium (L-glutamine 2mmol / L, retinoic acid concentration 3×10 -9 mol / L, transferrin 6mg / L, sodium selenite 30mg / L and human epidermal growth factor: 0.15mg / L, fibronectin: 0.5mg / L, TGF-β1 its concentration is 5ng / ml, cells Differentiation-promoting peptides (sequence shown in SEQ ID NO: 2) with a final concentration of 100ppm, prepared in DMEM / F12 (1:1) medium) in a 50mL centrifuge tube, naturally precipitated for 30min, sucked off the supernatant, and used 3mL Gently resuspend the pellet in the culture medium and add the resuspension to a 6 cm Petri dish treated with 0.1% gelatin on which MEFs have been laid. When the clones of human embryonic stem cells were overgrown...

Embodiment 3

[0026] Example 3 Analysis of Differentiation Transformation Efficiency

[0027] From the 5th day of differentiation, the expression of the surface markers of the differentiated cells was detected every day. By detecting the expression marker of endothelial cell-specific marker CD144, almost no expression of CD 144 can be detected on days 1-4 of differentiation, but starting at day 5, the up-regulation of CD144 expression rises sharply, reaching the expression of endothelial cells level, indicating successful differentiation of endothelial cells. By detecting the differentiation situation day by day, the conversion rate of endothelial cells on day 5-8 is as follows.

[0028] Table 1 Conversion rate table

[0029]

[0030]

[0031] As can be seen from the results in Table 1, the conversion efficiency of the highest value can be realized on the 6th day, and the final conversion efficiency reaches close to 97.6%, which is significantly improved compared with the prior art, a...

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Abstract

The invention provides a serum-free differentiation medium for commercial human embryonic stem cells, which is composed of the following final concentration components: L-glutamine 2mmol / L, retinoic acid concentration of 3×10-9mol / L, transferrin 6mg / L, sodium selenite 30mg / L and human epidermal growth factor: 0.15mg / L, fibronectin: 0.5mg / L, the concentration of TGF-β1 is 5ng / ml, the concentration of cell differentiation promoting peptide is 100ppm, The formulation was performed with DMEM / F12 (1:1) medium. Since the differentiation medium does not contain animal-derived serum, the risk of infection can be controlled; small peptides that specifically promote stem cell differentiation activity are added to the differentiation medium, which significantly improves cell growth and differentiation speed.

Description

technical field [0001] The invention relates to the technical field of stem cell culture medium, in particular to a special culture medium for differentiating human embryonic stem cells into endothelial cells and a method thereof. Background technique [0002] Embryonic stem cells (ESCs, referred to as ES, EK or ESC cells.) Embryonic stem cells are a type of cells isolated from early embryos (before the gastrula stage) or primitive gonads, which have the ability of unlimited proliferation and self-renewal in vitro and multi-directional differentiation characteristics. Whether in vitro or in vivo, ES cells can be induced to differentiate into almost all cell types in the body. Embryonic stem cell research has always been a controversial field in the United States. Supporters believe that this research can help to cure many intractable diseases, because embryonic stem cells can be differentiated into APSC pluripotent cells with multiple functions, which is considered to be a ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0735C12N5/071
CPCC12N5/0606C12N5/069C12N2500/24C12N2500/30C12N2500/32C12N2501/11C12N2501/15C12N2501/998C12N2506/02
Inventor 杜波王海波胡莹朱猛汪雁归
Owner GENECAST WUXI PRECISION MEDICAL DIGNOSTIC LAB
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