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A special serum-free medium for differentiation of human embryonic stem cells

A technology of human embryonic stem cells and differentiation medium, applied in the field of stem cell medium, can solve the problems of slow growth, affecting cell expansion, and prone to aging, so as to control the risk of infection, keep the biological characteristics unchanged, and improve the differentiation efficiency and a high survival rate

Active Publication Date: 2018-11-06
集钧(上海)医疗科技发展有限公司
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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 special serum-free medium for differentiation of human embryonic stem cells
  • A special serum-free medium for differentiation of human embryonic stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1: recovery, cultivation and subculture of the first method human embryonic stem cells;

[0021] (1) A commercially purchased human embryonic stem cell line (H9) was purchased from Jiangsu Stanford Biotechnology Co., Ltd. Melt rapidly in a water bath (within 20s), and add to 20mL human embryonic stem cell medium (consisting of the following final concentrations of components: L-glutamine 2mmol / L, L-sodium pyruvate 0.2mmol / L, transfection Ferritin 6.5mg / L, sodium selenite 30mg / L and human epidermal growth factor: 0.15mg / L, fibronectin: 0.3mg / L, cell activity promoting peptide (sequence as shown in SEQ ID NO: 1) The final concentration is 50ppm, use DMEM / F12 (1:1) medium for constant volume) in a 50mL centrifuge tube, naturally precipitate for 30min, suck off the supernatant, gently resuspend the precipitate with 3mL medium, and then add the resuspension solution to Already layer MEFs on a 0.1% gelatin-treated 6 cm Petri dish. When the clones of human embryon...

Embodiment 2

[0023] Embodiment 2: second method recovery, cultivation and subculture of human embryonic stem cells;

[0024] (1) A commercially purchased human embryonic stem cell line (H9) was purchased from Jiangsu Stanford Biotechnology Co., Ltd. Melt rapidly in a water bath (within 20s), add to 20mL human embryonic stem cell culture medium (composed of the following components: L-glutamine 2mmol / L, L-sodium pyruvate 0.2mmol / L, transferrin 6.5mg / L, sodium selenite 30mg / L and human epidermal growth factor: 0.15mg / L, fibronectin: 0.3mg / L, the final concentration of cell activity promoting peptide (sequence shown in SEQ ID NO: 2) is 50ppm , use DMEM / F12 (1:1) medium to make constant volume) in a 50mL centrifuge tube, naturally precipitate for 30min, suck off the supernatant, gently resuspend the pellet with 3mL medium, and then add the resuspension to the well-plated MEF 0.1% gelatin-treated 6 cm Petri dishes. When the clones of human embryonic stem cells were overgrown, they were treat...

Embodiment 3

[0027] Example 3 Analysis of Differentiation Transformation Efficiency

[0028] From the 5th day of differentiation, observe and record the beating situation of clones every day under the microscope, as well as the frequency of beating, and finally statistical drawing such as ( figure 1 ) shown. It can be seen that the culture medium shown in Example 1 and Example 2 can make the beating cells achieve more than 50% transformation of stem cells into cardiomyocytes in a short period of time, while the control method has no cardiomyocytes within 5 days The conversion of 9.9% was not achieved until the 13th day, while Examples 1 and 2 achieved a conversion of 96.4%, a very significantly high conversion. Through statistics, it is found that the frequency distribution of most beating clones of cardiomyocytes obtained in Examples 1 and 2 is between 55-75 times / min, and the average beating frequency of beating clones is (67.86) times / min, which has better cell activity .

[0029] Ta...

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Abstract

The invention provides a commercial human embryonic stem cell serum-free differentiation medium, which consists of the following components: L-glutamine 2mmol / L, L sodium pyruvate 0.2mmol / L, transferrin 6.5mg / L, sub Sodium selenate 30mg / L and human epidermal growth factor: 0.15mg / L, fibronectin: 0.3mg / L, cell activity promoting peptide as shown in SEQ ID NO: 1 or 2, configure with DMEM / F12 medium . Since the differentiation medium does not contain animal-derived serum, the risk of infection can be controlled; a small peptide that specifically promotes the differentiation activity of stem cells is added to the differentiation medium, which significantly increases the cell growth and differentiation speed; the prepared cardiomyocytes The biological characteristics of cardiomyocytes remain unchanged; the biological characteristics of cardiomyocytes that have been verified by experiments do not change for a long period of time. Moreover, the differentiation efficiency and survival rate of cells are high.

Description

technical field [0001] The invention relates to the technical field of stem cell culture medium, in particular to a serum-free culture medium for embryonic stem cells. 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 life-saving method. Charitable behavior is the performance of ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/077C12N5/0735
CPCC12N5/0657C12N2500/12C12N2500/24C12N2500/30C12N2500/32C12N2500/90C12N2501/11C12N2501/998C12N2506/02
Inventor 朱猛汪雁归
Owner 集钧(上海)医疗科技发展有限公司
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