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Two breast cancer stem cell lines capable of being stably cultured and keeping original characteristics, separation method and uses thereof

A breast cancer stem cell, stable technology, applied in the field of medicine and biology, to achieve the effect of stable and repeatable passage

Inactive Publication Date: 2016-01-27
SUN YAT SEN UNIV CANCER CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, as far as we know, no laboratory has established a clinically derived breast cancer stem cell line that can be stably passaged. Therefore, there is an urgent need to establish a clinically derived breast cancer stem cell line that can be stably passaged and used in clinical and scientific research

Method used

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  • Two breast cancer stem cell lines capable of being stably cultured and keeping original characteristics, separation method and uses thereof
  • Two breast cancer stem cell lines capable of being stably cultured and keeping original characteristics, separation method and uses thereof
  • Two breast cancer stem cell lines capable of being stably cultured and keeping original characteristics, separation method and uses thereof

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Experimental program
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Effect test

Embodiment 1

[0032] 1. Isolation and culture of clinically derived breast cancer stem cell lines that can be stably passaged and maintain their original characteristics

[0033] (1) Take freshly resected breast cancer tumor specimens, add 250 units of collagenase III per milliliter of tissue fluid, place them in a constant temperature incubator at 37°C for 4 hours for digestion, and then use a 40mm filter to filter;

[0034] (2) CD44 was sorted by flow cytometry (CytomicsFC500) + , CD24 - and ALDH1 + Cells, regulation requires cell purity above 98% (CD44 and CD24 antibodies are from CellSignaling; ALDH1 antibody is from Technologies);

[0035](3), preparation of breast cancer stem cell culture medium: add 10ng / mL basic fibroblast growth factor (purchased from Invitrogen Company), 20ng / mL epidermal growth factor (purchased from Invitrogen Company) to serum-free DMEM-F12 (purchased from Invitrogen Company) Invitrogen), 2% B27 (purchased from Invitrogen Company) and 5ug / mL insulin (purchas...

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Abstract

The invention discloses two breast cancer stem cell lines capable of being stably cultured and keeping original characteristics, separation method and uses thereof. A breast cancer tumor specimen just cut off from the tumor is taken, then is subjected to collagenase digestion and is filtered; a flow cytometer sorts CD44<+>, CD24<-> and ALDH1<+> cells, the cells are adjusted to a purity of more than 98%; a stem cell culturing agent for breast cancer cell is prepared; the sorted cells are screened in a monoclonal manner and the obtained monoclone cells are inoculated in an ultralow adhesion culture dish containing breast cancer stem cell culturing agents; penicillin and streptomycin are added in the original culturing medium; culturing is performed in CO<2> cultivation box. In the continuous culturing process, the breast cancer stem cell lines capable of being stably cultured and keeping original characteristics keep ball formation ability, self-updating ability, CD44<+>, CD24<-> / low Lin- ALDH1<+> phenotype, pluripotency differentiation ability, in vitro soft agar tumorigenesis ability, in vivo tumorigenesis ability in nude mouse, and in vivo tumorigenesis pluripotency differentiation ability in nude mouse.

Description

Technical field: [0001] The invention belongs to the field of medicine and biology, and specifically relates to two breast cancer stem cell lines that can be stably passed down and maintain the original characteristics, their separation method and application. Background technique: [0002] Breast cancer is the most common malignant tumor in women, and its incidence rate is increasing year by year. According to statistics from the American Cancer Society (ACS) in 2012, there were 230,000 newly diagnosed cases in the United States, an increase of 29%, and 40,000 deaths, an increase of 14%. With the change of lifestyle in my country, the acceleration of urbanization, and the promotion of mammography, the number of newly detected cases increases year by year. The incidence of breast cancer in Shanghai, China, rose from 17 / 100,000 in 1972 to 2007. 69.22 / 100,000. At the same time, the incidence of breast cancer in China is younger than that in Western countries. The peak age of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/095A61K48/00A61P35/00
Inventor 谢小明林小提刘远芳唐海林刘晓萍杨露刘鹏叶锋谢新华
Owner SUN YAT SEN UNIV CANCER CENT
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