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Method for enriching and screening tumor stem cells

A technology for cancer stem cells and screening methods, which is applied in the fields of tumors/cancer cells, biochemical equipment and methods, animal cells, etc. Effect

Active Publication Date: 2017-01-04
湖南艾佳生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] The purpose of the present invention is to provide a new compound tumor stem cell screening method to solve the problem that it is difficult to effectively sort and enrich tumor stem cells

Method used

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  • Method for enriching and screening tumor stem cells
  • Method for enriching and screening tumor stem cells
  • Method for enriching and screening tumor stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Combined sorting of renal cancer cell line ACHN tumor stem cells (CD133 / CD24 positive double selection)

[0054] 1. Multi-generation sphere culture

[0055] 1) Cultivate ACHN tumor cells until the bottle is 80-90% full, digest and collect the cells, and the single cell suspension is 5×10 4 cells / ml were inoculated into 6-well ultra-low adsorption cell culture plates, and each well was cultured with 2 mL of SFMD medium;

[0056] 2) 37°C, 5% CO 2 Cultivate for 96 hours, add 1mL culture medium every other day;

[0057] 3) After 96 hours, at 37°C, 5% CO 2 Digest with 0.05% trypsin in the cell culture incubator for 5 minutes, the tumor spheres become loose, add 2 mL of SFMD medium to stop digestion, pipette evenly, take 10 μL of uniform cell suspension for cell counting, continue to press 5×10 4 Cells per ml were planted in a new 6-well ultra-low adsorption cell culture plate to start P2 generation sphere culture;

[0058] 4) Repeat the above steps 1) to 3) to c...

Embodiment 2

[0130] Example 2 Joint sorting of bladder cancer cell line 5637 tumor stem cells (CD133 / CD44 positive double selection)

[0131] 1. Multi-generation sphere culture

[0132] 1) Cultivate 5637 tumor cells until the bottle is 80-90% full, digest and collect the cells, and press 5×10 single-cell suspension 4 cells / ml were inoculated into 6-well ultra-low adsorption cell culture plates, and each well was cultured with 2 mL of SFMD medium;

[0133] 2) 37°C, 5% CO 2 Cultivate for 96 hours, add 1mL culture medium every other day;

[0134] 3) After 96 hours, at 37°C, 5% CO 2 Digest with 0.05% trypsin in the cell culture incubator for 5 minutes, the tumor spheres become loose, add 2 mL of SFMD medium to stop digestion, pipette evenly, take 10 μL of uniform cell suspension for cell counting, continue to press 5×10 4 Cells per ml were planted in a new 6-well ultra-low adsorption cell culture plate to start P2 generation sphere culture;

[0135] 4) Repeat the above steps 1) to 3) to c...

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Abstract

The invention provides an enriching and screening method for tumor stem cell. The combination of ball culture and immunomagnetic beads separation method is used for enriching and screening the tumor stem cells, the details are as follows: continuously culturing the tumor cells in a no-serum ball culture medium, using the QPCR method to simultaneously examine the relative levels of tumor stem cell markers, when the expression levels of specific marker is relatively the highest, supplement with immunomagnetic and positive, single or double screening tumor stem cells. The combination of the two screening methods can significantly improve such practical problems as low separation rates, low purity levels and the inconveniences of after application for tumor stem cells.

Description

technical field [0001] The invention relates to the field of cell biology, in particular to a method for enriching and screening tumor stem cells. Background technique [0002] The traditional concept holds that tumors are formed by somatic cell mutations, each tumor cell can grow indefinitely, and the occurrence and development of tumors is the result of the joint proliferation of all tumor cells. But this cannot explain the phenomenon that not all tumor cells can grow unrestricted. The characteristics of tumor cell growth, metastasis and recurrence are very similar to the basic characteristics of stem cells. Therefore, some scholars put forward the theory of cancer stem cells (CSC). This theory provides a new direction and perspective for people to re-understand the origin and nature of tumors, as well as clinical tumor treatment. [0003] Recent studies have confirmed that there are a small number of stem cell-like cell populations in tumor tissues. These cells have sel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/095
CPCC12N5/0695C12N2500/32C12N2501/11C12N2501/115C12N2501/33C12N2501/998
Inventor 艾立平魏薇胡清云
Owner 湖南艾佳生物科技股份有限公司
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