Three-dimensional culture method for screening breast cancer stem cells

A technology of breast cancer stem cells and human breast cancer cells, which is applied in the field of three-dimensional culture of screening breast cancer stem cells, can solve the problems of cumbersome process, high cost, poor cell viability, etc., and achieve the effect of simple operation

Active Publication Date: 2013-11-20
GUANGZHOU SAGENE BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Existing technologies mainly use flow cytometry sorting or immunomagnetic bead sorting to separate breast cancer tumor stem c

Method used

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  • Three-dimensional culture method for screening breast cancer stem cells
  • Three-dimensional culture method for screening breast cancer stem cells
  • Three-dimensional culture method for screening breast cancer stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] 1) Digest the MCF-7 cell line with trypsin to obtain MCF-7 cells in a single-cell state, add complete medium (addition of high Sugar DMEM medium) to make breast cancer cell suspension, and use complete medium (high sugar DMEM medium supplemented with 10v / v% fetal bovine serum, 80U / ml penicillin and 80mg / ml streptomycin) to inoculate the cells Adjust the density to 10000 cell / mL;

[0030] 2) Mix 10 μL of salmon thrombin (0.1U / μL) and 250 μL of cell suspension evenly to obtain mixture A, and keep it on ice for later use;

[0031] 3) Dilute salmon fibrinogen to 2mg / mL with complete medium to obtain mixed solution B, which is kept on ice for later use;

[0032] 4) After mixing equal volumes of the mixture A and the mixture B, immediately inoculate into a 24-well plate with a volume of 500 μL per well; incubate at 37°C for 15 minutes until it solidifies into a soft gel;

[0033] 5) Add 1mL of complete medium on the top of the soft gel and place it in a cell culture incubat...

Embodiment 2

[0036] 1) Digest the MCF-7 cell line with trypsin to obtain MCF-7 cells in a single-cell state, add complete medium (high concentration of 10v / v% fetal calf serum, 150U / ml penicillin and 150mg / ml streptomycin Suspension of breast cancer cells was made in DMEM medium with sugar, and the cells were inoculated with complete medium (high-glucose DMEM medium supplemented with 15v / v% fetal bovine serum, 150U / ml penicillin and 150mg / ml streptomycin) Density adjusted to 1×10 3 cell / mL;

[0037] 2) Mix 5 μL of salmon thrombin (0.1U / μL) and 250 μL of cell suspension evenly to obtain mixture A, and keep it on ice for later use;

[0038] 3) Dilute salmon fibrinogen to 1.5mg / mL with complete medium to obtain mixed solution B, which is kept on ice for later use;

[0039] 4) After mixing equal volumes of the mixture A and the mixture B, immediately inoculate into a 24-well plate with a volume of 500 μL per well; incubate at 36°C for 18 minutes until it solidifies into a soft gel;

[0040...

Embodiment 3

[0043] 1) Digest the MCF-7 cell line with trypsin to obtain MCF-7 cells in a single-cell state, add complete medium (addition of high Sugar DMEM medium) to make breast cancer cell suspension, and use complete medium (high sugar DMEM medium supplemented with 12v / v% fetal bovine serum, 100U / ml penicillin and 100mg / ml streptomycin) to inoculate the cells Density adjusted to 1×10 5 cell / mL;

[0044] 2) Mix 20 μL of salmon thrombin (0.1U / μL) and 500 μL of cell suspension evenly to obtain mixture A, and keep it on ice for later use;

[0045] 3) Dilute salmon fibrinogen to 3mg / mL with complete medium to obtain mixed solution B, which is kept on ice for later use;

[0046] 4) After mixing equal volumes of the mixture A and the mixture B, immediately inoculate into a 24-well plate with a volume of 500 μL per well; incubate at 38°C for 15 minutes until it solidifies into a soft gel;

[0047] 5) Add 1mL of complete medium on the top of the soft gel, place it in a cell culture incubat...

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Abstract

The invention discloses a three-dimensional culture method for screening breast cancer stem cells. According to the three-dimensional culture method, hydrogel is adopted for conducting three-dimensional culture on the human breast cancer cells to directly obtain breast cancer stem cell microspheres which are in ESA+CD44+/CD24-/low phenotypes and obviously have the characteristics of tumor stem cells. The method is a reliable breast cancer stem cell screening method. The method is simple to operate and has the advantages that the costs of reagent consumption, instrument usage, human resources and the like are greatly lowered compared with the prior art such as a flow cytometer screening method.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a three-dimensional culture method for screening breast cancer stem cells. Background technique [0002] Breast cancer is one of the most common malignant tumors that seriously affects women's physical and mental health and even threatens their lives. In my country, the incidence of breast cancer is on the rise, and it has become the most common malignant tumor in women. Some scholars believe that the existence of cancer stem cells is the root of malignant tumors. Years of treatment studies have shown that the drug resistance and drug exclusion ability of breast cancer stem cells in radiotherapy, radiation resistance during chemotherapy, and postoperative recurrence and metastasis are still thorny issues in its treatment. Therefore, it is urgent to develop a suitable screening method to effectively screen out breast cancer stem cells, so as to be used in the research of breast canc...

Claims

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Application Information

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IPC IPC(8): C12N5/095
Inventor 曾宏彬戴果鲜康涛郭玲玲陈丽丹
Owner GUANGZHOU SAGENE BIOTECH
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