Brain tumor stem cell separation method

Abstract

The invention provides a brain tumor stem cell separation method. The method includes the following steps: the obtainment of a primary brain tumor cell; the digestion and passage of the primary brain tumor cell: the brain tumor cell is inoculated into first serum-free stem cell culture medium, and is then added with MG-132 for culture, first serum-free stem cell culture medium is used for replacing the medium, docetaxel and adriamycin are then added for secondary culture, and thereby primary brain tumor sphere cells are obtained, wherein the content of MG-132 in the first serum-free stem cellculture medium is 10 to 25Mu mol / L; and the collection and passage of the primary brain tumor sphere cells: the primary brain tumor sphere cells are washed and collected, and are then inoculated intosecond serum-free stem cell culture medium and cultured for passage, thereby brain tumor stem cells are obtained, wherein the second serum-free stem cell culture medium contains ESGRO. By the method,the differentiation of the brain tumor stem cells can be inhibited and the multiplicative division of the brain tumor stem cells can be promoted, so that a large number of high-purity brain tumor stem cells can be obtained.

Description

Technical field [0001] The invention belongs to the technical field of stem cells, and specifically is a method for separating brain tumor stem cells. Background technique [0002] The primary problem of brain cancer stem cell (Brain cancer stem cells, BCSCs) research is how to isolate a very small amount of BCSCs from a heterogeneous brain tumor cell population. At present, there are mainly the following methods to isolate BCSCs. [0003] 1. Use the human leukocyte differentiation antigen 133 (CD133) marker molecule on the surface of BCSCs to screen: [0004] Commonly used screening methods are fluorescence activated cell sorting (FACS) and magnetic activated cell sorting (MACS). Among them, the FACS method is currently a widely used separation method. After fluorescently labeled CD133 specific antibodies are combined with CD133 molecules on the cell surface, the fluorescent BCSCs are sorted out. [0005] 2. Use biological characteristics for functional sorting (SP cell sorting): [...

AI Technical Summary

Problems solved by technology

However, the fluorescent dyes used in the SP sorting method may have certain toxic effects on BCSCs, which will change the activity and function of the isolated BCSCs and affect subsequent culture studies.

[0007] Both of the above 1 and 2 sorting methods still have the following disadvantages: the process is cumbersome, the time is lengthy, the cost is relatively expensive, and there is a certain degree of subjectivity, which can be different due to differences in cell preparation methods, gating, etc. There are some differences, and there are certain errors. The most important thing is that after a long period of staining and sorting, the viability of the cells will inevitably be affected, which in turn will affect the next step of culture or biological characteristics research.

However, BCSCs may differentiate into brain tumor non-stem cells in the process of in vitro suspension culture, and the purification degree of BCSCs obtained by existing methods is low (20%-30%), which cannot well meet the needs of a large number of later studies

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