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Three-step separation method for enriching stem cells

A separation method, a technology of liver cancer stem cells, applied in the field of three-step separation method for enriching stem cells, can solve the problems of high cost of sorting and low sorting accuracy, achieve low price, low cost of separation, and remove false positives Effect

Active Publication Date: 2015-03-04
中国人民解放军西部战区总医院
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Problems solved by technology

[0003] The purpose of the present invention is to provide a three-step separation method for enriching stem cells, which solves the problems of low sorting accuracy and high sorting cost in the prior art

Method used

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  • Three-step separation method for enriching stem cells
  • Three-step separation method for enriching stem cells
  • Three-step separation method for enriching stem cells

Examples

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Embodiment

[0026] This example is aimed at the isolation of rat liver cancer stem cells. First, the rats were allowed to drink water containing 0.05% DEN for 6 weeks, and then changed to normal drinking water to create a rat liver cancer model. At 18 weeks, the rats were anesthetized, and then the abdominal cavity of the rats was dissected to observe the tumor formation in the liver. Remove liver cancer nodules from rats, soak in serum-free medium containing 0.1% type IV collagenase and 0.005% trypsin, and incubate with shaking at 37°C for 20min, collect digested cell aggregates, pass through diameter Use a 100 μm nylon mesh to prepare a cell suspension and put it in a centrifuge for 8 minutes at 1000×g. The bottom cell aggregates were collected, resuspended in PBS to become a cell suspension, and formed a colony of liver cancer cells after adherent culture. The specific results of the isolation and culture of liver cancer cells are as follows: figure 1 shown. figure 1 Middle Ⅰ is the...

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Abstract

The invention discloses a three-step separation method for enriching stem cells, aiming at solving the problems that the prior art is not high in sorting precision and high in sorting cost. The method comprises the following steps of: (1) purifying cells: purifying an original cell group through a time difference adherence method and a time difference nitration method; (2) grouping the cells: grouping the purified cells by a Percoll continuous density gradient centrifugation method; and (3) enriching the cells: cultivating the grouped cells by culture solution, and selecting the cells which form the maximum cloning balls from the cells, i.e. the stem cells. After the method provided by the invention is used, the cells are sorted in combination with the adhesion intensity characteristic, the intensity size characteristic and the cloning formation characteristic of the stem cells, so that the stem cells are exactly enriched.

Description

technical field [0001] The invention relates to a three-step separation method for enriching stem cells. Background technique [0002] Currently, the use of surface markers to sort stem cells is a classic method, usually by flow cytometry for positive or negative sorting. Using this technology for sorting, the prerequisite is that the stem cells must have certain surface markers. However, there is a lack of relatively definite and recognized markers for some types of stem cells (such as liver stem cells and liver cancer stem cells). At the same time, this kind of separation technology itself has other shortcomings: (1) false positives of marker sorting are inevitable; (2) in order to ensure the accuracy of sorting, joint sorting of multiple markers is often required, so the price is relatively expensive. Contents of the invention [0003] The purpose of the present invention is to provide a three-step separation method for enriching stem cells, which solves the problems ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/095C12N5/07
Inventor 刘卫辉汤礼军闫洪涛任丽娜陈涛马利红崔剑锋刘立业王涛
Owner 中国人民解放军西部战区总医院
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