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Separation method of breast cancer stem cells

A breast cancer stem cell and separation method technology, which is applied in the field of cell separation, can solve the problem of low purity of target cells, achieve the effects of shortening separation time, efficient separation, and reduction of separation cost

Inactive Publication Date: 2013-08-14
四川全组生命科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The purity of the target cells initially obtained by immunomagnetic bead separation is not high, so it is necessary to further enrich and purify the target cells

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] This example mainly uses pleural effusion collected by thoracentesis of breast cancer patients in Toronto General Hospital, Canada to separate breast cancer stem cells. The collected pleural effusion is processed within 2-4 hours. The specific steps are as follows:

[0025] (1) Preparation of pleural fluid single-cell suspension:

[0026] Centrifuge the pleural fluid collected by thoracentesis at 500 rpm and 4°C for 20 minutes to minimize damage to cells and fully collect target cells; use 2.0% heat-inactivated fetal bovine serum and 2 mmol / L After washing the cell fragments with Hank's balanced salt solution, centrifuge, add red blood cell lysate Tris-NH 4 Cl lysed red blood cells, after the red blood cells were lysed, washed with 2mmol / L Hank's balanced salt solution, centrifuged, and removed the supernatant;

[0027](2) Negative sorting by immunomagnetic beads, separation and removal of non-breast cancer cells, and breast cancer epithelial cell fluid:

[0028] Resu...

Embodiment 2

[0032] This example mainly uses pleural effusion collected by thoracentesis of breast cancer patients in Toronto General Hospital, Canada to separate breast cancer stem cells. The collected pleural effusion is processed within 2-4 hours. The specific steps are as follows:

[0033] (1) Preparation of pleural fluid single-cell suspension:

[0034] The pleural fluid collected by thoracentesis was centrifuged at 400rpm at 4°C for 25 minutes to minimize damage to the cells and fully collect the target cells; After washing the cell fragments with Hank's balanced salt solution, centrifuge, add red blood cell lysate Tris-NH 4 Cl lysed red blood cells, after the red blood cells were lysed, washed with 2mmol / L Hank's balanced salt solution, centrifuged, and removed the supernatant;

[0035] (2) Negative sorting by immunomagnetic beads, separation and removal of non-breast cancer cells, and breast cancer epithelial cell fluid:

[0036] Resuspend the cells with MACS buffer, pass through...

Embodiment 3

[0040] This example mainly uses pleural effusion collected by thoracentesis of breast cancer patients in Toronto General Hospital, Canada to separate breast cancer stem cells. The collected pleural effusion is processed within 2-4 hours. The specific steps are as follows:

[0041] (1) Prepare pleural effusion single-cell suspension: centrifuge the pleural effusion collected by thoracentesis at 600 rpm and 4°C for 20 minutes to minimize damage to the cells and fully collect the target cells; Inactivated fetal bovine serum and 2mmol / L Hank's balanced salt solution washed the cell fragments, centrifuged, and added red blood cell lysate Tris-NH 4 Cl lysed red blood cells, after the red blood cells were lysed, washed with 2mmol / L Hank's balanced salt solution, centrifuged, and removed the supernatant;

[0042] (2) Negative sorting of immunomagnetic beads, separation and removal of non-breast cancer cells: resuspend cells in MACS buffer, pass through a 100 μm filter to obtain suspen...

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PUM

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Abstract

The invention provides a separation method of breast cancer stem cells and belongs to the field of cell separation. The separation method comprises the following steps of: (1) preparing a hydrothorax single-cell suspension; (2) carrying out immunomagnetic bead negative sorting, and separating and removing non-breast-cancer cells, so as to obtain breast cancer epithelial cell sap; and (3) carrying out enrichment and purification on the breast cancer epithelial cell sap obtained in the step (2) by utilizing a non-adhesive culture method, so as to obtain breast cancer stem cell spheroids. Compared with the prior art, the separation method has the beneficial effects that the surfaces of the cells obtained by utilizing the separation method are not interfered by antibodies and magnetic beads; the separation efficiency of the breast cancer cells and the purity of the breast cancer cells after the breast cancer stem cells are enriched can be guaranteed; and not only can a small quantity of breast cancer epithelial cells be effectively separated, but also the separation time is shortened, and the production cost is lowered, so that the separation method is safe and effective.

Description

technical field [0001] The invention relates to the separation of cells, in particular to a method for the separation of breast cancer stem cells. Background technique [0002] Around the world, about 1.2 million women suffer from breast cancer every year, and 500,000 die from breast cancer. In developed countries such as Western Europe and North America, the incidence of breast cancer ranks first among female malignant tumors. Relevant data show that the peak incidence of breast cancer in Western women is 50-55 years old, and the incidence rate continues to increase with age. [0003] Compared with developed countries, although China is a low-incidence area of ​​breast cancer, it is one of the countries with the fastest-growing incidence of breast cancer. Statistics released by the Chinese Anti-Cancer Association show that the incidence of breast cancer in my country has increased by 3% per year in recent years. %, and tripled in the past five years, making it one of the f...

Claims

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Application Information

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IPC IPC(8): C12N5/095
Inventor 邓涛赵西娟魏薇魏丽婉高天权
Owner 四川全组生命科技有限公司
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