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Cartilage progenitor cell culture medium and preparation method and application thereof

A culture medium and progenitor cell technology, applied in the direction of cell culture active agents, bone/connective tissue cells, biochemical equipment and methods, etc., to achieve the effect of avoiding pollution, simple steps, and excellent self-renewal ability

Active Publication Date: 2020-04-24
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Third, the successful transplantation of CPCs in vivo has not been reported yet.

Method used

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  • Cartilage progenitor cell culture medium and preparation method and application thereof
  • Cartilage progenitor cell culture medium and preparation method and application thereof
  • Cartilage progenitor cell culture medium and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] 1) Mix DMEM medium, F12 medium, proline, sodium pyruvate, vitamin C, insulin growth factor, basic fibroblast growth factor 2 (FGF2), transferrin, ROCK inhibitor at 25°C , P38 inhibitor and transforming growth factor β signaling pathway inhibitor are mixed to prepare mixture M1; wherein the volume ratio of DMEM medium to F12 medium is 1:1, the concentration of vitamin C is 100 μg / mL, and the concentration of insulin growth factor The concentration is 100ng / mL, the concentration of FGF2 is 100ng / mL, the concentration of the GSK-3α / β inhibitor is 2uM, the concentration of the transferrin is 50μg / mL, the concentration of the ROCK inhibitor is 10uM, The concentration of the p38 inhibitor is 2.5uM, the concentration of the proline is 0.35mM, and the concentration of the sodium pyruvate is 1mM;

[0042] 2) After adding sodium hydroxide to the above mixture M1 to adjust the pH to 7.4, then adding sodium chloride to adjust the osmotic pressure to 340 mOsm / kg to obtain a mixture M2;...

Embodiment 2

[0045] 1) Mix DMEM medium, F12 medium, proline, sodium pyruvate, vitamin C, insulin growth factor, basic fibroblast growth factor 2 (FGF2), transferrin, ROCK inhibitor at 25°C , P38 inhibitor and transforming growth factor β signal pathway inhibitor are mixed to prepare mixture M1; wherein the volume ratio of DMEM medium and F12 medium is 1:2, the concentration of vitamin C is 200 μg / mL, and the concentration of insulin growth factor The concentration is 150ng / mL, the concentration of FGF2 is 20ng / mL, the concentration of the GSK-3α / β inhibitor is 30uM, the concentration of the transferrin is 10μg / mL, the concentration of the ROCK inhibitor is 50uM, The concentration of the p38 inhibitor is 20uM, the concentration of the proline is 10mM, and the concentration of the sodium pyruvate is 15mM;

[0046] 2) After adding sodium hydroxide to the above mixture M1 to adjust the pH to 7.4, then adding sodium chloride to adjust the osmotic pressure to 360 mOsm / kg to obtain a mixture M2;

[0...

Embodiment 3

[0049] 1) Mix DMEM medium, F12 medium, proline, sodium pyruvate, vitamin C, insulin growth factor, basic fibroblast growth factor 2 (FGF2), transferrin, ROCK inhibitor at 25°C , P38 inhibitor and transforming growth factor beta signal pathway inhibitor are mixed to prepare mixture M1; wherein the volume ratio of DMEM medium to F12 medium is 1:1.5, the concentration of vitamin C is 150μg / mL, and the concentration of insulin growth factor The concentration is 5ng / mL, the concentration of FGF2 is 10ng / mL, the concentration of the GSK-3α / β inhibitor is 20uM, the concentration of the transferrin is 100μg / mL, the concentration of the ROCK inhibitor is 30uM, The concentration of the p38 inhibitor is 10uM, the concentration of the proline is 0mM, and the concentration of the sodium pyruvate is 4mM;

[0050] 2) After adding sodium hydroxide to the above mixture M1 to adjust the pH to 7.4, then adding sodium chloride to adjust the osmotic pressure to 340 mOsm / kg to obtain a mixture M2;

[0...

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Abstract

The invention provides a cartilage progenitor cell culture medium which is composed of DMEM culture medium, F12 culture medium, proline, sodium pyruvate, vitamin C, insulin growth factor, FGF2, transferrin, ROCK inhibitor, p38 inhibitor and GSK-3 alpha / beta inhibitor. The preparation method comprises the following steps: mixing the components, adjusting the pH and osmotic pressure of an obtained mixture, and sterilizing. According to the invention, enough nutrient substances and a stable living environment are provided for culturing cells, so that the cells have excellent self-renewal capability. As no animal-derived component is used, pollution caused by animal-derived components is effectively avoided, and immune response is completely prevented from being induced. The preparation methodof the culture medium has the advantages that steps are simple, raw materials are easily available, and no serum is contained, the undifferentiated and pluripotent state of CPCs can be maintained, and the cartilage progenitor cell culture medium is specially used for culturing and amplifying cartilage progenitor cells. The invention disclsoes a long-lasting, continuous and efficient CPCs culturesystem, so that in vitro stable propagation of functional mice and human cartilage progenitor cells is possible to realize.

Description

Technical field [0001] The invention belongs to the field of culture media, and specifically relates to a culture medium for cartilage progenitor cells and a preparation method and application thereof. Background technique [0002] As an important part of synovial joints, hyaline articular cartilage has the function of maintaining the flexibility of bones and the smoothness of joints. After adulthood, articular chondrocytes, as a subgroup of chondrocytes, slowly maintain the stability of cartilage tissue. Hypertrophic chondrocytes are different from articular chondrocytes. They exist in the growth plate and their function is to produce new soft bone during bone formation. In order to treat degenerative joint diseases, replacing articular cartilage cells or articular cartilage with cells or tissues produced in vitro provides a new option for the current main treatment option-total joint replacement. For different cell therapies, ensuring a good source of stem cells or progenitor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077
CPCC12N5/0655C12N2500/24C12N2500/32C12N2500/38C12N2501/105C12N2501/115C12N2501/999
Inventor 梁成振夏楷顺李中伟李荣辉
Owner ZHEJIANG UNIV
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