Nucleus pulposus progenitor cell culture medium and preparation method and application thereof

A culture medium and progenitor cell technology, applied in the field of nucleus pulposus progenitor cell culture medium and its preparation, can solve the problems of insufficient simulation of microenvironment and low purity of NPPCs, achieve excellent self-renewal ability, avoid inducing immune response, raw materials easy-to-get effect

Active Publication Date: 2020-04-17
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, Tie2 is considered to be a specific marker gene of NPPCs, but the purity of NPPCs obtained by the above method is very low, and it can only be amplified for 3 generations and cultured for 7 days
Furthermore, the culture conditions of NPPCs in the above studies failed to fully simulate the low-pH microenvironment of intervertebral discs in vivo.

Method used

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  • Nucleus pulposus progenitor cell culture medium and preparation method and application thereof
  • Nucleus pulposus progenitor cell culture medium and preparation method and application thereof
  • Nucleus pulposus progenitor cell culture medium and preparation method and application thereof

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Embodiment 1

[0032] The invention provides a nucleus pulposus progenitor cell culture medium, which consists of DMEM medium, F12 medium, proline, sodium pyruvate, vitamin C, insulin growth factor, basic fibroblast growth factor 2 (FGF2), transgenic Composed of ferritin, ROCK inhibitors, p38 inhibitors and transforming growth factor beta signaling pathway inhibitors. Wherein, the volume ratio of DMEM medium and F12 medium is 1:1, the concentration of vitamin C is 100 μg / mL, the concentration of insulin growth factor is 100 ng / mL, the concentration of FGF2 is 100 ng / mL, and the transforming growth factor β The concentration of the signaling pathway 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, and the concentration of the proline is 0.35mM, the concentration of the sodium pyruvate is 1mM. Specifically prepared by the following methods:

[0033] 1) At 25°C, add DMEM medium,...

Embodiment 2

[0037] A nucleus pulposus progenitor cell medium, consisting of DMEM medium, F12 medium, proline, sodium pyruvate, vitamin C, insulin growth factor, basic fibroblast growth factor 2 (FGF2), transferrin, ROCK Inhibitors, p38 inhibitors and transforming growth factor beta signaling pathway inhibitors. Wherein, the volume ratio of DMEM medium to F12 medium is 1:2, the concentration of vitamin C is 200 μg / mL, the concentration of insulin growth factor is 150 ng / mL, the concentration of FGF2 is 10 ng / mL, and the transforming growth factor β The concentration of the signaling pathway inhibitor is 30uM, the concentration of the transferrin is 100μg / mL, the concentration of the ROCK inhibitor is 50uM, the concentration of the p38 inhibitor is 20uM, and the concentration of the proline is 10mM , the concentration of the sodium pyruvate is 15mM. Specifically prepared by the following methods:

[0038] 1) At 25°C, add DMEM medium, F12 medium, proline, sodium pyruvate, vitamin C, insuli...

Embodiment 3

[0042] A nucleus pulposus progenitor cell medium, consisting of DMEM medium, F12 medium, proline, sodium pyruvate, vitamin C, insulin growth factor, basic fibroblast growth factor 2 (FGF2), transferrin, ROCK Inhibitors, p38 inhibitors and transforming growth factor beta signaling pathway inhibitors. Wherein, the volume ratio of DMEM medium to F12 medium is 1:0.5, the concentration of vitamin C is 80 μg / mL, the concentration of insulin growth factor is 80 ng / mL, the concentration of FGF2 is 50 ng / mL, and the transforming growth factor β The concentration of the signaling pathway inhibitor is 20uM, the concentration of the transferrin is 80μg / mL, the concentration of the ROCK inhibitor is 30uM, the concentration of the p38 inhibitor is 1uM, and the concentration of the proline is 4mM , the concentration of the sodium pyruvate is 4mM. Specifically prepared by the following methods:

[0043] 1) At 25°C, add DMEM medium, F12 medium, proline, sodium pyruvate, vitamin C, insulin gr...

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Abstract

The invention provides a nucleus pulposus progenitor cell culture medium as well as a preparation method and an application thereof. The nucleus pulposus progenitor cell culture medium consists of a DMEM culture medium, an F12 culture medium, proline, sodium pyruvate, vitamin C, an insulin growth factor, FGF2, transferrin, a ROCK inhibitor, a p38 inhibitor and a transforming growth factor beta signal channel inhibitor. The preparation method comprises the following steps: mixing the components, adjusting the pH and osmotic pressure of the mixture, and performing sterilizing. The culture mediumdisclosed by the invention does not use animal-derived components; a undifferentiated and pluripotent state of the NPPCs can be maintained; the removal of animal serum in the culture medium greatly avoids the interference and pollution of animal components to nucleus pulposus progenitor cell culture, can completely avoid the induction of immune response in the cell treatment process, simplifies the culture process, increases the credibility of experimental results, and basically solves the key problem of durable culture of NPPCs. The preparation method is simple, raw materials are easy to obtain, and the culture medium is suitable for culture and amplification of nucleus pulposus progenitor cells under feeder-free cell conditions.

Description

technical field [0001] The present invention relates to the field of culture medium, in particular to the culture medium of nucleus pulposus progenitor cells and its preparation method and application. Background technique [0002] In embryonic development, nucleus pulposus cells originate from the notochord during embryonic development. The part of the notochord surrounded by the vertebral bodies will completely degenerate and disappear, but the part between the vertebral bodies will remain to form the nucleus pulposus, so the purity of the nucleus pulposus progenitor cells (NPPCs) derived from embryos will be significantly higher than that derived from adults NPPCs. However, because NPPCs can exist stably for a very short time, they will continue to differentiate into adult cells soon, and it is impossible to obtain a stable progenitor cell line. Therefore, it is necessary to provide the same or similar microenvironment as that of embryonic progenitor cells while obtaini...

Claims

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Application Information

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IPC IPC(8): C12N5/0797
CPCC12N5/0623C12N2500/24C12N2500/30C12N2500/32C12N2500/38C12N2501/105C12N2501/115C12N2501/15C12N2501/405
Inventor 梁成振夏楷顺章裕昂李方财陈其昕
Owner ZHEJIANG UNIV
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