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Novel three-dimensional culture method of glioma stem cells and application thereof

A technology of glioma stem cells and glioma cells, which is applied in the field of cell biology, can solve the problems of difficult observation of cell spheroids and poor repeatability of spheroid culture, so as to reduce the waste of patient resources and follow-up research funds, and achieve good biocompatibility sexual effect

Inactive Publication Date: 2014-07-02
THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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  • Application Information

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Problems solved by technology

Its advantages are: it can reflect the self-renewal and differentiation ability of a single cell from the cell level in a relatively simple and intuitive way; but the disadvantages are also obvious: in addition to the poor repeatability of sphere culture and the difficulty of observing the inside of the cell sphere, the more important thing is It is very different from the internal environment

Method used

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  • Novel three-dimensional culture method of glioma stem cells and application thereof
  • Novel three-dimensional culture method of glioma stem cells and application thereof
  • Novel three-dimensional culture method of glioma stem cells and application thereof

Examples

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Embodiment 1

[0026] A new three-dimensional culture method for glioma stem cells, using a three-dimensional collagen scaffold, and exploring a suitable low serum culture concentration, the culture method includes the following steps:

[0027] 1) Cut the three-dimensional collagen scaffold into 1*5*5mm cubes, sterilize with Co60 irradiation overnight before use, store in a dry environment at 4°C for later use, and soak in cell culture medium for 12 hours before inoculating cells;

[0028] 2) Digest and centrifuge the glioma cell lines to a single-cell suspension, wash them twice with PBS to remove serum components, and perform cell counting. 4 ~5×10 4 Density of 1 cell: Inoculate glioma cells on the three-dimensional collagen scaffold treated in step 1) and let it stand for 4 hours. After the cells are effectively adhered to the scaffold, place them in a cell culture plate for culture, and replace 1 gel every 2 days. The subculture medium was used to obtain a 3D glioma cell model.

[0029...

Embodiment 2

[0034] This example is an optimized solution based on Example 1, and provides a new three-dimensional culture method for glioma stem cells. For the parts in this embodiment that are the same as those in Embodiment 1, please refer to the disclosed content in Embodiment 1 for understanding. The disclosed content in Embodiment 1 should also be used as the content of this embodiment, and will not be repeated here.

[0035] The three-dimensional culture method comprises the following steps:

[0036] The conventional two-dimensionally cultured U87 cells were digested and centrifuged, prepared into a single cell suspension, and then seeded on a collagen scaffold with a pore size of about 50 μm, and cultured under the condition of 1% fetal bovine serum (FBS) + DMEM medium. Observe whether the cells can better adhere to the collagen matrix of the scaffold and grow, observe the proliferation of U87 cells transfected with green fluorescent protein (GFP) with a fluorescence microscope, ob...

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Abstract

The invention realizes enrichment culture of glioma stem cells under low serum concentration by utilizing a three-dimensional collagen scaffold and provides a novel stem cell culture method closer to an in-vivo real environment and application of the method for in-vitro glioma experimental study. According to the three-dimensional culture method, collagen serves as a material for manufacturing a three-dimensional (3D) scaffold, the collagen is the most important component of extracellular matrix can provide a good adhesion and migration environment for cells, and has high biocompatibility, high mechanical strength, high degradation coefficient and low immunogenicity. According to the invention, the influence of three-dimensional culture of a porous collagen scaffold on glioma cell dryness is researched, the mechanism of the three-dimensional culture of the porous collagen scaffold is discussed, a targeted GSCs drug trial method closer to the in-vivo tumor stem cell environment is groped, a drug sensitive test is performed, a glioma resistance mechanism is further researched, a foundation is laid for a new thought about the development of GSCs targeted drugs, and the achievement has a certain theoretical significance and application value.

Description

technical field [0001] The invention belongs to the field of cell biology, and in particular relates to a three-dimensional culture method and application of novel glioma stem cells. Background technique [0002] Malignant glioma is the malignant tumor with the highest incidence rate in the nervous system. In the past 10 years, even with the great progress of surgery, radiation and chemotherapy, the median survival period has only increased from 10 months to 14 months. months, and in multiple large-scale, multi-center new drug trials for glioma, all failed to achieve satisfactory results, most of the drugs failed in the second phase of clinical trials, failed to reflect the The conclusion is consistent with that of the in vitro test. This discrepancy between the in vitro test and the clinical results is common in anti-glioma treatment research, which indicates that there are still many gaps in the understanding of the pathogenesis, development characteristics and drug resis...

Claims

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Application Information

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IPC IPC(8): C12N5/095A61K35/12A61P35/00
Inventor 吕东来卞修武陈叶苗
Owner THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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