Method for separating stem cell of breast cancer

A breast cancer stem cell and breast cancer cell technology, applied in the field of tumor research, can solve the problems of limited ability of tumor stem cell enrichment, rough method, impossible to achieve high sorting efficiency and purity, etc.

Inactive Publication Date: 2007-10-10
JIANGSU PROVINCE HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although the above method is relatively simple and can increase the content of tumor stem cells, this method is relatively rough, and the ability to enrich tumor stem cells is limited, and it is impossible to achieve high sorting efficiency and purity (such as more than 95%)

Method used

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  • Method for separating stem cell of breast cancer
  • Method for separating stem cell of breast cancer

Examples

Experimental program
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Effect test

Embodiment 1

[0026] In this example, the tumor stem cell-related subgroups in the breast cancer cell line MCF-7 were isolated according to the following steps:

[0027] 1) The breast cancer cell line MCF-7 was digested with trypsin-EDTA into a single cell suspension.

[0028] 2) Configure six Percoll separation fluids with different densities, the densities are 1.035g / ml, 1.040g / ml, 1.050g / ml, 1.060g / ml, 1.070g / ml, 1.080g / ml, 2ml for each density layer, Stacked in sequence according to density to make Percoll discontinuous density gradient separation liquid.

[0029] 3) The prepared single cell suspension was carefully superimposed on the top of the Percoll discontinuous density gradient separation medium, and centrifuged at 2000 rpm for 30 minutes.

[0030] 4) Carefully suck out the cells of each subpopulation layered in the Percoll separation medium after centrifugation, and wash twice with PBS for later use.

[0031] 5) For each subgroup of cells separated above, each subgroup is prov...

Embodiment 2

[0034] In this example, the tumor stem cell-related subgroups in the breast cancer cell line MDA-MB-231 were isolated according to the following steps:

[0035] 1) Digest breast cancer cell lines or primary breast cancer tissues into trypsin-EDTA single cell suspension.

[0036] 2) EGF, bFGF, insulin and other growth factors were added to DMEM / F12 serum-free culture medium to prepare serum-free culture medium.

[0037] 3) Place the digested single cell suspension in serum-free medium and culture for 3 weeks.

[0038] 4) The MDA-MB-231 enriched in tumor stem cells after 3 weeks of serum-free culture was digested with trypsin-EDTA into a single cell suspension.

[0039] 5) Divide the above cell suspension into experimental tubes and control tubes, the number of cells in each tube is 10 6 cells / ml, add CD to the test tube 44 -FITC, CD 24 -PE, not added to the control tube, stored in the dark for 20 minutes at room temperature, washed twice with PBS, resuspended in 400ul of PB...

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Abstract

This invention relates to a method for separating breast cancer stem cells. The method comprises: digesting breast cancer cells into single cell suspension, preparing Percoll discontinuous density gradient separating medium, mixing with single cell suspension, centrifuging to separate cell subsets, detecting by comparing the experimental tube and the control tube, adjusting the density of Percoll discontinuous density gradient separating medium until the content of the cell subsets related to the breast cancer stem cells reaches predetermined enrichment degree, and screening with a flow cytometry. The method utilizes Percoll screening or serum-free culture to enrich breast cancer stem cells, and then screens with a flow cytometry to ensure the screening efficiency. The ratio of the breast cancer stem cells after primary enrichment is higher than 25%, while that after low cytometry screening is higher than 99%.

Description

technical field [0001] The invention relates to a stem cell separation method, in particular to a breast cancer stem cell separation method, which belongs to the technical field of tumor research. Background technique [0002] In 2003, Al-Hajj et al. used the differences in surface antigens of breast cancer cells, used specific fluorescently labeled antibodies and flow cytometry, to sort breast cancer cells into separate groups, and then sorted cells with different phenotypes into separate groups. Injected into immunodeficient mice, it was found that only the surface antigen was marked as ESA + Lin - CD44 + CD24 - / low A small group of cells has continuous tumorigenic properties, and is the only one able to self-replicate, start the occurrence of tumors, and its proportion is very small, accounting for only 2% of tumor cells, this is breast cancer stem cells. In addition, the stem cell functional index SP phenotype can also be used to sort tumor stem cells. In 2004, Kond...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/08C12N5/095
Inventor 王水刘晓安许健凌立君
Owner JIANGSU PROVINCE HOSPITAL
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