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Culturing method for maintaining self-renewal state of human embryonic stem cells

A technology of human embryonic stem cells and embryonic stem cells, applied in the field of embryonic stem cell culture conditions, can solve problems such as unfavorable exploration, unstable state of human embryonic stem cells, inconvenient passage operation, etc.

Active Publication Date: 2018-05-11
ANHUI UNIVERSITY
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  • Application Information

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Problems solved by technology

[0005] In order to solve the problems of unstable state of human embryonic stem cells, inconvenient subculture operation, high cost of existing culture conditions and unfavorable follow-up exploration in traditional culture conditions, the present invention provides a culture method for maintaining the self-renewal state of human embryonic stem cells

Method used

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  • Culturing method for maintaining self-renewal state of human embryonic stem cells
  • Culturing method for maintaining self-renewal state of human embryonic stem cells
  • Culturing method for maintaining self-renewal state of human embryonic stem cells

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Embodiment 1

[0070] (1) HES2 human embryonic stem cells (Human Embryonic Stem Cell, hESCs) were provided by Professor Ying Qilong of the University of Southern California;

[0071] (2) Passage and culture of HES2 human embryonic stem cells under the condition of Activin A+BFGF+IWR1:

[0072] a. Take a 6-well cell culture plate, coat each well with 2ml DMEM+10μl Matrigel, and place it at 37°C, 5% CO 2 In a cell incubator with a concentration of 4 hours;

[0073] b. Take human embryonic stem cells grown to a density of 70-80%, discard the culture medium, and wash the cells once with PBS to remove the residual culture medium on the cell surface;

[0074] c. Add 1ml CTK to digest the embryonic stem cells, about 7min, the edge of the cells floats up, discard the CTK, add 2ml of conventional cell culture medium containing 10% FBS, pipette with a pipette to blow the cells off the culture plate, and then transfer into a 15ml sterile centrifuge tube;

[0075] d. After centrifugation at 1200 rpm ...

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Abstract

The invention discloses a culturing method for maintaining a self-renewal state of human embryonic stem cells. The method comprises passage and culturing of the human embryonic stem cells under the condition of using the culturing method and observation and detection of the self-renewal state of the human embryonic stem cells cultured under the condition of using the culturing method. According tothe method, the problems of easily differentiated state, complicated passage operation, high culturing condition cost and inconvenient subsequent research on mechanisms of the human embryonic stem cells under the current traditional culturing condition are solved by adding an inhibitor of a Wnt / beta-catenin signal channel; the method has the advantages of being stable in effect, economic and high-efficient, simple to operate, convenient to research and the like, clues for improving and establishing culturing conditions for multipotential stem cells of other kinds and species are provided, and the method has a positive promotion effect on smooth development of stem cell basis and application research in the future.

Description

technical field [0001] The present invention relates to the culture conditions of human embryonic stem cells, in particular to a culture method for maintaining the self-renewal state of human embryonic stem cells. Background technique [0002] Stem cells are self-renewing pluripotent cells that are isolated from early embryos and can survive in vitro culture with the ability to proliferate indefinitely (self-renewal), while maintaining differentiation into various types of cells in the body It has become one of the powerful tools for studying gene function, screening drugs and making animal models of diseases, and has great application prospects in the field of regenerative medicine. Mouse embryonic stem cells (mouse Embryonic StemCells, mESCs), which is the first stem cell line successfully established in vitro. Then in 1998, the "father of stem cells" Thomson first established a human ESC line. Although mouse and human ESCs share some common features, their biological pr...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0735
CPCC12N5/0606C12N2501/115C12N2501/16C12N2501/999
Inventor 叶守东尤宇
Owner ANHUI UNIVERSITY
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