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Reagent and kit for enriching breast cancer stem cells from breast cancer cell strains

A breast cancer stem cell and breast cancer cell technology, applied in the field of stem cells, can solve the problems of high cost and long cycle, and achieve the effects of improving efficiency, high content and short cell sphere formation cycle

Inactive Publication Date: 2017-05-10
南京千年健干细胞基因工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this method has high enrichment efficiency for breast cancer stem cells, the method of in vivo passage in mouse models is costly and takes a long time

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Enrichment of breast cancer stem cells from human breast cancer MCF-7 cell line

[0025] 1. Experimental materials

[0026] Conventional serum-containing medium: 10% fetal bovine serum FBS and double antibody were added to DMEM / F12 culture medium, and 10 ml of penicillin-streptomycin solution (100× double antibody, Beijing Legen Biotechnology Co., Ltd.) was added to 1L medium.

[0027] Conventional serum-free medium: add double antibody (concentration as above), recombinant human epidermal growth factor EGF 20g / L, bovine serum albumin BSA4g / L, insulin 5mg / L, B272% (1L medium plus 20ml 50X B27).

[0028] Improved serum-containing medium: 10% fetal bovine serum FBS, double antibody (same concentration as above), 150nmol / L of galantamine and lycorine were added to DMEM / F12 culture medium, and the molar ratio of the two was 1:1.

[0029] Improved serum-free medium: add double antibody (concentration as above), recombinant human epidermal growth factor EGF 20g / ...

Embodiment 2

[0059] Example 2: Enrichment of breast cancer stem cells from human breast cancer MCF-7 cell line

[0060] 1. Experimental materials

[0061] Improved serum-containing medium: a total of 150nmol / L of galantamine and lycorine, the molar ratio of the two is 2:1, and the others are the same as in Example 1.

[0062]Improved serum-free medium: a total of 150nmol / L of galantamine and lycorine, the molar ratio of the two is 2:1, and the others are the same as in Example 1.

[0063] 2. Experimental method

[0064] With embodiment 1.

[0065] 3. Experimental results

[0066] (1) Formation of MCF-7 microspheres

[0067] Typical MCF-7 suspension cell spheres appeared on the 3rd day, and the cell sphere formation efficiency was (3.1±0.9)%.

[0068] (2) CD44 + CD24 - / low cell ratio

[0069] FCM showed that CD44 in microsphere cells on day 3 of the improved culture method + CD24 - / low The cell ratio was (59.7±5.4)%.

[0070] (3) SP cell ratio

[0071] The proportion of SP cel...

Embodiment 3

[0072] Example 3: Enrichment of breast cancer stem cells from human breast cancer MCF-7 cell line

[0073] 1. Experimental materials

[0074] Improved serum-containing medium: a total of 150nmol / L of galantamine and lycorine, the molar ratio of the two is 1:2, and the others are the same as in Example 1.

[0075] Improved serum-free medium: a total of 150nmol / L of galantamine and lycorine, the molar ratio of the two is 1:2, and the others are the same as in Example 1.

[0076] 2. Experimental method

[0077] With embodiment 1.

[0078] 3. Experimental results

[0079] (1) Formation of MCF-7 microspheres

[0080] Typical MCF-7 suspended cell spheres appeared on the 3rd day, and the cell sphere formation efficiency was (3.3±0.5)%.

[0081] (2) CD44 + CD24 - / low cell ratio

[0082] FCM showed that CD44 in microsphere cells on day 3 of the improved culture method + CD24 - / low The cell ratio was (56.5±5.8)%.

[0083] (3) SP cell ratio

[0084] The proportion of SP cel...

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Abstract

The invention discloses a reagent and kit for enriching breast cancer stem cells from breast cancer cell strains. The reagent includes galantamine and lycorine as a screening agent. The breast cancer cell strains are MCF-7 breast cancer cell strains. The kit comprises a double-antibody, galantamine and lycorine as a screening agent, a screening-agent-dissolving auxiliary agent, sodium sulfite as an anti-oxidant, a recombinant human epidermal growth factor (EFG) which is a conventional growth factor, bovine serum albumin (BSA), insulin and B27. The provided reagent can enrich breast cancer stem cells from human breast cancer MCF-7 cell strains in a convenient, fast and efficient way, the cell spheroids are short in forming period, and the content of cancer cells and SP cells marked by CD44<+>CD24<- / low> molecules in microsphere cells is high. The developed kit can improve the efficiency for enriching cancer stem cells by science researchers.

Description

technical field [0001] The invention belongs to the field of stem cells, and relates to a reagent and a kit for enriching breast cancer stem cells from breast cancer cell lines. Background technique [0002] Cancer stem cell research is a new field emerging from stem cell research. Although it started late, it is developing rapidly and showing good prospects. The existence of cancer stem cells and their role as a basis for tumor formation, growth and metastasis has been widely recognized. The basic theory is: there is a small part of tumor cells that can produce new tumor cells and self-replicate cells, called tumor stem cells. Different from ordinary tumor cells, cancer stem cells are resistant to all chemotherapy drugs, and after the end of chemotherapy, they will differentiate into new tumor cells with characteristics similar to stem cells. Therefore, cancer stem cells are the root cause of tumor recurrence [Cancer stem cells in solid tumors. Curr Opin Biotechnol, 2007;...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/095
CPCC12N5/0037C12N5/0695C12N2500/10C12N2500/90C12N2501/11C12N2501/33C12N2501/50C12N2501/999
Inventor 杨超高云雷其他发明人请求不公开姓名
Owner 南京千年健干细胞基因工程有限公司
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