Extracellular matrix gel model used for researching development and differentiation of embryonic stem cells

A technology of embryonic stem cells and extracellular matrix, applied in a series of key technical fields, can solve problems such as ignoring the development process

Inactive Publication Date: 2010-05-26
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

However, most of the existing 3D model studies start from the embryoid bodies that have been differentiated from embryonic stem cells. The study of the important developmental process from the formation of embryoid bodies artificially separates the process of differentiation and development of embryonic stem cells

Method used

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  • Extracellular matrix gel model used for researching development and differentiation of embryonic stem cells
  • Extracellular matrix gel model used for researching development and differentiation of embryonic stem cells
  • Extracellular matrix gel model used for researching development and differentiation of embryonic stem cells

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Example 1. Construction of extracellular matrix gel three-dimensional scaffold in vitro

[0035] Mix type I liquid collagen and Matrigel glue evenly at a ratio of 4:1, then mix it with H-DMEM concentrated culture solution in equal volume, use an appropriate amount of 0.1M NaOH to adjust the pH of the mixture to 7.2-7.4, and add it to the prepared Inject 1ml into each well of a good 12-well plate mold. Place it at 37°C, 5% CO 2 Incubate at 5mim to 30mim in an incubator until the liquid complex is solidified to form a three-dimensional extracellular matrix gel model. This part of the operation is carried out on the ice box. exist figure 1 The lamellar-like three-dimensional differentiation and developmental model that can be seen in the formation.

Embodiment 2

[0036] Example 2. Embryonic stem cells were uniformly compounded into the three-dimensional model of extracellular matrix gel at different densities:

[0037] Mix type I liquid collagen and Matrigel gel evenly at a ratio of 4:1, then mix it with H-DMEM concentrated culture solution containing different concentrations of ESC in equal volume, and adjust the pH value of the mixture to 7.2~ with an appropriate amount of 0.1M NaOH 7.4, add to the prepared 12-well plate mold, inject 1ml into each well. (This part of the operation is carried out on the ice box). Place it at 37°C, 5% CO 2 When incubating in the incubator at 5mim to 30mim, after the liquid complex is solidified, add 1ml of ESC full culture medium without LIF to each well. Change the solution every day and observe. From figure 2 We can see that 10 4 The cell seeding density is too low, and the cells cannot survive; 10 7 The cell seeding density is too high, and there are a large number of cells; while at 10 5 -1...

Embodiment 3

[0038] Example 3. Research on induced differentiation and development of embryonic stem cells:

[0039] In the three-dimensional differentiation and development model of embryonic stem cells in vitro, different inducers were added to observe the ability of embryonic stem cells to differentiate and develop into different adult cells. Take the addition of V-C inducer as an example, add (0.1 mg / ml) V-C to the three-dimensional model that has been compounded with embryonic stem cells, and observe its effect on the formation of embryonic stem cells and their later differentiation and development. Figure 4 Embryonic stem cells were shown to form embryoid bodies and differentiate to form beating cardiac syncytium-like structures, and cTnT staining, a specific staining of the myocardium, revealed undifferentiated cardiomyocytes.

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Abstract

The invention relates to an extracellular matrix gel model used for researching development and differentiation of embryonic stem cells, belonging to the research field of tissue engineering and regenerative medicine, and mainly solving the problem that no ideal model exists for the research of differentiation and development of embryonic stem cells in vitro. We the extracellular matrix gel adopt as the in vitro three dimensional model of the differentiation and development of embryonic stem cells, inoculate the embryonic stem cells to the three dimensional model in different cell densities, and observe the phenomena of the forming of embryoid body by self-differentiation of the embryonic stem cells and the law of the embryoid body being differentiated into cells of different ancestries; and the microenvironment in which the embryonic stem cells grow is regulated, the extracellular matrix gel is taken as the main body to carry out the directional differentiation and development research of the embryonic stem cells, thus disclosing the inherent law and mechanism of the in vitro differentiation and development of the embryonic stem cells, and searching for new methods for directional differentiation of embryonic stem cells to the functional cells of important vital organs at the terminal differentiation period; and the invention will provide a new technical method for exploring the development mechanism of the embryonic stem cells.

Description

technical field [0001] The invention belongs to the research field of tissue engineering and regenerative medicine, and relates to a series of key technologies such as preparation of a three-dimensional differentiation and development model of embryonic stem cells in vitro, cultivation of embryonic stem cells and differentiation and development of embryonic stem cells in vitro. This method can construct a three-dimensional extracellular matrix gel scaffold in vitro, and combine embryonic stem cells with this scaffold to make it a research model for cell differentiation and development. Carry out research on the differentiation and development of embryonic stem cells in the model, and then reveal the internal rules and mechanisms of embryonic stem cell differentiation and development in vitro, and seek new methods for the directed differentiation of embryonic stem cells into functional cells that are in the terminal differentiation stage of important vital organs. Background o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61L27/38A61L27/24
Inventor 王常勇周瑾郝彤段翠密林秋霞
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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