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New method of screening and enriching solid tumor stem cell

A technology of tumor stem cells and a new method, applied in the field of cell culture and manufacturing, can solve the problems of low purity of stem cells, restrictions on the research of tumor stem cells and the development of new tumor treatment technologies, and high cost of separation

Inactive Publication Date: 2012-04-11
殷勤伟 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The purity of tumor stem cells obtained by flow cytometry is high, up to 95%, but the sorting process will cause great damage to the cells. Although the conditions of the magnetic bead sorting method are relatively mild, the purity of the sorted stem cells Lower, reaching 80% is a better separation result
A common problem faced by the above two methods is that the isolation of stem cells requires the use of high-quality monoclonal antibodies, and the cost of isolation is very high
In addition, how to expand the isolated tumor stem cells without losing or changing their stem cell characteristics is still a technical bottleneck that has not been solved (3), and it is a technical obstacle that restricts the research of tumor stem cells and the development of new tumor treatment technologies

Method used

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  • New method of screening and enriching solid tumor stem cell
  • New method of screening and enriching solid tumor stem cell
  • New method of screening and enriching solid tumor stem cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1. Cloning culture and expansion of human breast cancer MCF-7 tumor stem cells

[0018] Human breast cancer MCF-7 was purchased from the American cell bank ATCC, and the cells were cultured using DMEM medium (Beijing Niuyinhuaxin Technology Co., Ltd., DM10140021) containing 10% fetal bovine serum (PAA, FCS500), and T-25 produced by Coring Company Cultured in cell culture flasks at 37°C, 5% CO 2 . Cells were passaged every two days. When culturing MCF-7 stem cell clones, first trypsinize the cells cultured to a confluence of 90% in a cell culture dish, wash with normal saline, replace the stem cell medium (see Recipe 1) and resuspend the MCF -7 cells were inoculated into a culture well of a 24-well plate with 50,000 to 100,000 per well, and the cells continued to be cultured. After 1 day of culture, a small amount of cell proliferation was found, and some cells grew in an incomplete spherical structure. Suspension growth spheres with a similar growth pattern. ...

Embodiment 2

[0021] Example 2. Clonal culture and expansion of human prostate cancer Du-145 tumor stem cells.

[0022] Human prostate tumor Du-145 was purchased from the American cell bank ATCC, and the cell culture was the same as that of MCF-7. Collect the cells 1×10 6 , using a flow cytometer to sort out Hoechst 33342 staining negative SP cell side population, using the stem cell medium prepared by formula 2 to cultivate the SP side population cells, and gradually form cell spheres after 20 days of cultivation (see figure 2 ), cells that cannot form spheroids gradually die during the culture process. The cell sphere has the characteristics of tumor stem cells and is a tumor stem cell subtype of Du-145.

[0023] Formula 2: Medium formula for Du-145 tumor stem cell selection and expansion:

[0024] Element

Embodiment 3

[0025] Example 3. Expression of stem cell characteristics of MCF-7 stem cell clones

[0026] The alkaline phosphatase staining method is a commonly used feature to identify embryonic stem cells. During the development of embryonic stem cells, alkaline phosphatase is highly expressed in embryonic stem cells, and cells / tissues can be stained by alkaline phosphatase staining kit, but once embryonic stem cells differentiate, the expression of alkaline phosphatase decreases, and cells / tissues cannot be stained dyeing. One of the sources of cancer stem cells is the generation of embryonic stem cells with gene or / and epigenetic mutations, so positive alkaline phosphatase staining is one of the effective markers for identifying certain cancer stem cells. The alkaline phosphatase staining experiment was carried out using the alkaline phosphatase kit (Catalog No. SCR004) of Millpore Company and the operation method provided in the manual. image 3 The results of alkaline phosphatase s...

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Abstract

The invention provides a new rapid screening / culture amplification method and culture medium formulation of a novel solid tumor stem cell. By utilizing a novel solid tumor stem cell culture medium containing a GSK (GlaxoSmithKline) signal path inhibitor and a tyrosine kinase inhibitor, through SP (side population) screening or directly through a tumor stem cell screen / amplification culture medium, a solid tumor cell can selectively propagate a cell clone with tumor stem cell characteristics, accompanied by the high expression of an embryo stem cell characteristic gene Oct4, Nanog and a tumor stem cell surface characteristic antigen gene. The tumor stem cell prepared by the method provided by the invention has potential application prospects in the aspects of new anti-tumor medicament screening and efficient tumor treatment.

Description

technical field [0001] The invention belongs to the field of cell culture and manufacturing, and more specifically relates to a technology for screening, enriching, cultivating and amplifying corresponding tumor stem cell subgroups from solid tumor cells, which is used as a method for solid tumor therapeutic drug screening, biotherapy and gene therapy. Screening, in vitro evaluation of model methods and experimental materials. Background technique [0002] Solid tumors are the main types of tumor diseases and an important disease that threatens human health. Two different models exist regarding the origin of solid tumors. One model considers cancer to be a disease in which multiple genes malfunction. During the process of cell / tissue lesions, with the increase of oncogenes and epigenetic gene mutations, cells / tissues gradually form precancerous lesions, cancerous cells / tissues and malignant cancerous cells / tissues. Another type of model believes that tumors are derived fr...

Claims

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Application Information

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IPC IPC(8): C12N5/095C12Q1/02
Inventor 殷勤伟张洪杰黄兵张颖娱
Owner 殷勤伟