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A kind of cartilage progenitor cell culture medium and its preparation method and application

A culture medium, 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: 2021-08-03
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

Method used

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  • A kind of cartilage progenitor cell culture medium and its preparation method and application
  • A kind of cartilage progenitor cell culture medium and its preparation method and application
  • A kind of cartilage progenitor cell culture medium and its preparation method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] 1) At 25°C, add DMEM medium, F12 medium, proline, sodium pyruvate, vitamin C, insulin growth factor, basic fibroblast growth factor 2 (FGF2), transferrin, ROCK inhibitor , p38 inhibitor and transforming growth factor β signaling pathway inhibitor were mixed to prepare mixture M1; wherein, the volume ratio of DMEM medium to F12 medium was 1:1, the concentration of vitamin C was 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) adding sodium hydroxide to the above mixture M1 to adjust the pH to 7.4, and then adding sodium chloride to adjust the osmotic pressure to 340mOsm / kg to obtain the mixt...

Embodiment 2

[0045] 1) At 25°C, add DMEM medium, F12 medium, proline, sodium pyruvate, vitamin C, insulin growth factor, basic fibroblast growth factor 2 (FGF2), transferrin, ROCK inhibitor , p38 inhibitor and transforming growth factor β signaling pathway inhibitor were mixed to prepare mixture M1; wherein, the volume ratio of DMEM medium to F12 medium was 1:2, the concentration of vitamin C was 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) adding sodium hydroxide to the above mixture M1 to adjust the pH to 7.4, and then adding sodium chloride to adjust the osmotic pressure to 360mOsm / kg to prepare the mixtu...

Embodiment 3

[0049] 1) At 25°C, add DMEM medium, F12 medium, proline, sodium pyruvate, vitamin C, insulin growth factor, basic fibroblast growth factor 2 (FGF2), transferrin, ROCK inhibitor , p38 inhibitor and transforming growth factor β signaling pathway inhibitor were mixed to prepare mixture M1; wherein, the volume ratio of DMEM medium to F12 medium was 1:1.5, the concentration of vitamin C was 150 μg / 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) adding sodium hydroxide to the above mixture M1 to adjust the pH to 7.4, and then adding sodium chloride to adjust the osmotic pressure to 340mOsm / kg to obtain the mixture...

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Abstract

The invention provides a chondrogenic progenitor cell culture medium, which consists of DMEM medium, F12 medium, proline, sodium pyruvate, vitamin C, insulin growth factor, FGF2, transferrin, ROCK inhibitor, p38 inhibitor and GSK ‑3α / β inhibitor composition. It is prepared by mixing the components, adjusting the pH and osmotic pressure of the mixture, and then sterilizing. The invention provides sufficient nutrients and a stable living environment for cultured cells, so that they have excellent self-renewal ability. Because no animal-derived ingredients are used, the occurrence of pollution caused by this is effectively avoided, and the occurrence of immune response is completely avoided. The medium preparation method has simple steps, readily available raw materials, does not contain serum, can maintain the undifferentiated and pluripotent state of CPCs, and is specially used for the cultivation and expansion of cartilage progenitor cells. The invention is a long-term, continuous and high-efficiency CPCs culture system, which makes it possible to stably propagate functional mouse and human cartilage progenitor cells in vitro.

Description

technical field [0001] The invention belongs to the field of culture medium, and in particular relates to a chondrocyte progenitor cell culture medium and its preparation method and application. Background technique [0002] As an important component of synovial joints, hyaline articular cartilage has the function of maintaining the flexibility of bones and the smoothness of joints. In adulthood, articular chondrocytes, as a subset of chondrocytes, slowly maintain the stability of cartilage tissue. Hypertrophic chondrocytes, unlike articular chondrocytes, are present in the growth plate and function to generate new cartilage during bone formation. For the treatment of degenerative joint diseases, the replacement of articular chondrocytes or articular cartilage with cells or tissues produced in vitro provides a new alternative to the current main treatment option, total joint replacement. For different cell therapies, ensuring a good source of stem cells or progenitor cells...

Claims

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Application Information

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Patent Type & Authority Patents(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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