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Myocardial cells preparation method

A technique for cardiomyocytes and pluripotent stem cells, applied in the field of preparing cardiomyocytes from pluripotent stem cells, can solve the problems of limited application and uneven quality of cardiomyocyte products, and achieve the effect of good electrophysiological properties

Active Publication Date: 2019-03-22
HELP STEM CELL INNOVATIONS CO LTD
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  • Description
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AI Technical Summary

Problems solved by technology

[0002] With the development of induced pluripotent stem cell technology, cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSC-CMs) have shown great potential in drug screening, disease model construction and cell therapy, but at present, pluripotent The diversification of stem cell differentiation methods of cardiomyocytes, the quality of cardiomyocyte products is uneven
The physiological state of cardiomyocytes differentiated from pluripotent stem cells, such as body structure, action potential and calcium operation ability, is far from the physiological state of mature cardiomyocytes. These deficiencies in the quality of cardiomyocytes largely limit their clinical application. application in research
[0003] At this stage, the research on the method of differentiating pluripotent stem cells into cardiomyocytes mainly focuses on how to improve the differentiation efficiency of cardiomyocytes, and there is no technical plan for improving the quality of cardiomyocytes and promoting the maturation of cardiomyocytes.

Method used

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] The preparation method of monolayer binary colloidal crystal

[0021] Prepare silica colloidal suspension and PMMA colloidal suspension with ultrapure water, mix the two suspensions evenly and deposit them on the glass surface of a limited area coated with polystyrene, after the water evaporates, the formed BCCs surface Heating at 200°C for 1 min, part of the PMMA melted to bind the particles and stabilize the BCCs structure, and the BCCs were sterilized under ultraviolet light for 30 min.

[0022] In the embodiment of the present invention, rubber rings are used to define a region with a diameter of 3 cm on the glass surface coated with polystyrene, and regions of other sizes can also be defined according to actual needs, and the region is used to form single-layer BCCs.

[0023] In the embodiment of the present invention, single-layer BCCs are used as the cell culture substrate, and the number of silica colloid particles required to form a single-layer silica film in...

Embodiment 2

[0026] Detection of monolayer binary colloidal crystal structure parameters

[0027] In the examples of the present invention, the properties of the single-layer binary colloidal crystals prepared in were analyzed from the perspective of roughness and wettability.

[0028] Use an atomic force microscope (AFM) to analyze its roughness at a scan rate of 0.5 Hz, scan any 5 areas on the surface of BCCs, and calculate the average value;

[0029] The contact angle is analyzed by a fully automatic contact angle measuring instrument. The lower the contact angle, the higher the hydrophilicity and the easy attachment of the surface; the higher the contact angle, the stronger the hydrophobicity of the surface;

[0030] The performance parameters of the monolayer binary colloidal crystals are shown in Table 1:

[0031] Table 1. Roughness and wettability of monolayer binary colloidal crystals

[0032] Binary colloidal crystal performance parameters

Embodiment 3

[0033] cardiomyocyte preparation method

[0034] The monolayer binary colloidal crystals prepared in were used as the cell culture substrate for the preparation of cardiomyocytes, and glass slides were used as the cell culture substrate as the control group.

[0035] Matrigel was used to coat the single-layer binary colloidal crystals and slides of the control group, and the pluripotent stem cells were mixed with 0.2×10 6 cells / cm 2 seeded in pluripotent stem cell medium at 5% CO 2 , cultured at 37°C, in which the pluripotent stem cell medium is TeSR TM -E8 TM Medium.

[0036] When the density of pluripotent stem cells is higher than 40%, culture pluripotent stem cells in cardiomyocyte differentiation medium I containing CHIR99021 at a final concentration of 7.5 μM for 48 h, and then differentiate with cardiomyocytes containing IWR-1 at a final concentration of 2 μM Culture medium I continued to culture for 72 hours, and then cultured with cardiomyocyte differentiation ...

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Abstract

The invention provides a myocardial cells preparation method. Multipotential stem cells are differentiated into myocardial cells in a culture substrate on a monolayer binary colloid crystal, wherein the monolayer binary colloid is prepared from silicon dioxide colloid particles and PMMA colloid particles; and the surface of the culture substrate is coated with biogel. Through the preparation method of the myocardial cells provided by the invention, the maturity of the myocardial cells can be improved, and the myocardial cells can be endowed with relativley good electrophysiological characteristics.

Description

technical field [0001] The invention relates to the technical field of differentiation of pluripotent stem cells, in particular to a method for preparing cardiomyocytes from pluripotent stem cells. Background technique [0002] With the development of induced pluripotent stem cell technology, cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSC-CMs) have shown great potential in drug screening, disease model construction and cell therapy, but at present, pluripotent Due to the diversification of stem cell differentiation methods for cardiomyocytes, the quality of cardiomyocyte products is uneven. The physiological state of cardiomyocytes differentiated from pluripotent stem cells, such as body structure, action potential and calcium operation ability, is far from the physiological state of mature cardiomyocytes. These deficiencies in the quality of cardiomyocytes largely limit their clinical application. application in research. [0003] At this s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077
CPCC12N5/0657C12N2533/10C12N2533/30
Inventor 王嘉显崔畅徐轶冰王倩
Owner HELP STEM CELL INNOVATIONS CO LTD
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