Application method of small molecules capable of promoting self-status-renewal of embryonic stem cells

An embryonic stem cell and application method technology, applied in the application field of small molecules, can solve the problems of easy differentiation and inconvenient passage operation, and achieve the effects of optimizing the culture system, good cell growth, and simplifying the operation steps and processes.

Active Publication Date: 2019-05-07
ANHUI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve the problems of easy differentiation and inconvenient subculture operation in the embryonic stem cell culture process, the present invention provides an application method of a small molecule that promotes the self-renewal state of embryonic stem cells

Method used

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  • Application method of small molecules capable of promoting self-status-renewal of embryonic stem cells
  • Application method of small molecules capable of promoting self-status-renewal of embryonic stem cells
  • Application method of small molecules capable of promoting self-status-renewal of embryonic stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Passage and culture of human embryonic stem cells under the condition of CID755673

[0044] (1) Take a 6-well cell culture plate, wrap each well with 2 ml DMEM+10 µl Matrigel, and place at 37°C, 5% CO 2 Concentration in the cell culture box, coated for 4h;

[0045] (2) Take human embryonic stem cells grown to 70-80% density, discard the culture medium, and wash the cells once with PBS to remove the residual culture medium on the cell surface;

[0046] (3) Add 1 ml CTK containing (1% Collagenase IV+0.25% Trypsin +20% KSR+1mM CaCl 2 PBS) to digest embryonic stem cells, for about 7 minutes, the edge of the cells floated, discarded CTK, added 2 ml of regular cell culture medium containing 10% FBS, blown with a pipette, the cells were blown off the culture plate, and then transferred to 15 ml sterile centrifuge tube;

[0047] (4) After centrifugation at 1200 rpm for 4 min, aspirate the supernatant;

[0048] (5) Add 2 ml N2B27 containing 10% KSR (5 ml N2B27 contains 1.875 m...

Embodiment 2

[0055] Passage and culture of mouse embryonic stem cells under the condition of CID755673

[0056] (1) Take a 6-well cell culture plate, wrap each well with 2 ml Gelatin, and place at 37°C, 5% CO 2 Concentration of cells incubator, coated for 1 h;

[0057] (2) Take a stable cell line with a growth density of about 60%, discard the culture medium, carefully wash once with PBS buffer, and discard the remaining washing solution;

[0058] (3) Immediately add 1 ml Trypsin-EDTA to digest the cells. After a few minutes, the edge of the cell colony floats, blow off carefully with a micropipette and transfer to a 15 ml centrifuge containing 2 ml DMEM serum culture medium. In the tube, continue to carefully pipette and mix to terminate the digestion;

[0059] (4) Centrifuge the mixed suspension in the centrifuge tube at 1000 rpm for about 3 minutes, discard the supernatant, add 1 ml PBS buffer to wash and centrifuge again;

[0060] (5) Discard the supernatant and add an appropriate a...

Embodiment 3

[0065] Detection of self-renewal indicators after using the small molecule CID755673

[0066] (1) Morphological observation

[0067] Using a Leica DMi8 inverted microscope to observe the embryonic stem cells of mice without adding any small molecules (as a control group), only adding CID755673 small molecules, and for different days of culture, it was found that compared with the control group, the cells showed a tightly packed monolayer Morphology, colony boundaries are clear, in line with the morphological characteristics of self-renewal, but it was found that only adding CID755673 can only maintain the short-term self-renewal of mouse embryonic stem cells, so it was found that adding two small molecules, CID755673 and PD0325901, can maintain mouse embryonic stem cells for a long time self-updating state.

[0068] Using a Leica DMi8 inverted microscope to observe the human embryonic stem cells without adding any small molecules (as a control group), only adding CID755673 sm...

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Abstract

The invention discloses an application method of small molecules capable of promoting self-status-renewal of embryonic stem cells. The application method of the small molecules capable of promoting self-status-renewal of the embryonic stem cells comprises the following steps: passaging and culturing human and mouse embryonic stem cells under small molecular state of the method; and then, observingand detecting self-renewal state of the cells according to the method. Being added with CID755673 small molecules, the application method of the small molecules capable of promoting self-status-renewal of embryonic stem cells can solve the problems of liable state differentiation, complex passages operation, high culture-condition cost, unfavorable follow-up mechanism research and so on of humanand mouse embryonic stem cells under existing traditional cultivation conditions; moreover, the application method of the small molecules capable of promoting self-status-renewal of embryonic stem cells has the advantages of being stable in effects, economical, highly efficient, easy to operate, convenient to study and so on. In addition, clues are further provided for improving and establishing culture conditions of pluripotent stem cells of other species and genera; and thus, the application method of the small molecules capable of promoting self-status-renewal of embryonic stem cells playsan active role in promoting smooth development of basic and applied research of stem cells in the future.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an application method of small molecules for promoting the self-renewal state of embryonic stem cells. Background technique [0002] Stem cells are a type of cells that come from fetuses, embryos or adults and have unlimited self-renewal, proliferation and differentiation capabilities under certain conditions. Under certain conditions, embryonic stem cells can be induced to differentiate into various tissues. This characteristic makes them have attractive application prospects in basic research, transplantation therapy and gene therapy. Unlimited proliferation, which provides an inexhaustible source of cells for the research and application of embryonic stem cells. These two main characteristics of embryonic stem cells have now become one of the powerful tools for studying gene function, screening drugs and making animal models of diseases, and have great application pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0735
Inventor 叶守东朱振华
Owner ANHUI UNIVERSITY
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