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Cell enriching, separating and extracting method and instrument and single cell analysis method

An extraction method and cell technology, applied in the field of cell separation and analysis, can solve the problems of limited selection space of fluorescence spectrum, limited space of target cell characteristics, etc.

Active Publication Date: 2012-09-26
GD TECH INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, the identification of blood circulating cancer cells is currently mostly performed by immunofluorescence staining, and the choice of fluorescence spectra used is limited, thus limiting the space for identifying the characteristics of target cells

Method used

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  • Cell enriching, separating and extracting method and instrument and single cell analysis method
  • Cell enriching, separating and extracting method and instrument and single cell analysis method
  • Cell enriching, separating and extracting method and instrument and single cell analysis method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Target cell capture, washing and release operations are completed in a standard, single, 6-well cell culture plate (suitable for purification of cultured cells):

[0084] DMEN (Invitrogen) cultured cells were harvested with TRIPLE (Life Tech). Pipette nearly 1000 MCF cells into a 15mL test tube containing 10ml of DMEM, add 10μl of EpCAM micro-magnetic beads (DYNAL), mix and rotate for 30 minutes to 1 hour at room temperature. Then, pour this mixed sample into the sample well of the 6-well cell culture plate, and in the other well, add 10 ml of PBS buffer (pH = 7.2) (it can be any other buffer suitable for the cell assay). With the Cytometer CI-101, capture capture, wash and release operations are performed. The above operations can be one-time or multiple-time. If it is a one-time operation, the entire operation will be completed within 10 minutes. This method is suitable for the separation and purification of target cells with fewer background cells, and to obtain r...

Embodiment 2

[0086] Capture, wash and release of target cells multiple times and in different combinations:

[0087] Take about 1000MCF7 cells into 10 ml of normal human blood, add 10 microliters of EpCAM micro-magnetic beads (DYNAL), and incubate at 4 degrees for 1 hour with rotation (the temperature can be adjusted as needed). Pour the mixed blood sample into one end of the first standard 6-well plate, and add 10 ml of PBS buffer to the other wells (including the first and second standard 6-well plates). Capture, wash and release of target cells is performed with Cytometer CI-101. The CI-101 machine automatically performs three operations of capturing, washing and releasing target cells, and releases the target cells and other products captured each time into the first release tank. At this point, the CI-101 robotic arm takes the empty magnet set from the country to the demagnetization box, replaces the used magnet set, puts on a new magnet set, and moves it to the first release slot. ...

Embodiment 3

[0089] Remove free micro-beads that are not bound to target cells from the capture of positive magnetic bead enrichment method: collect 10 ml of blood samples from breast cancer patients with EDTA blood collection tubes, and place them at room temperature for a short period of time. Add 10 microliters of EpCAM micromagnetic beads (DYNAL) to the blood sample, and incubate at room temperature for 1 hour with rotation. Pour this pooled blood sample into the sample well of the first standard 6-well plate. Add 10 ml of PBS buffer solution to other required tanks. Start the CI-101 and perform the ultra-clean cell isolation method. The CI-101 instrument will automatically perform three rounds of capturing, washing and releasing target cells, and release the captured objects into the first release tank. In order to avoid contamination of the magnet sleeve, CI-101 will automatically replace the magnet sleeve, return to the first release tank, and perform the last operation of capturi...

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Abstract

The invention discloses a method and an automatic instrument device for enriching and extracting target cells and separating single cells by using a positive magnetic bead method and performing immunity and molecular biology identification and analysis on single cells. By the method and the instrument device, the on / off of a capture magnet and a release magnet is controlled by various methods to complete target cell searching, capturing, cleaning and releasing operation once or for multiple times, so that the target cell detection sensitivity and stability are improved. When the capture magnet searches and captures the target cells, the search line is circular, square, comb-shaped, S-shaped or U-shaped. In addition, the captured substances are filtered, most free micro magnetic beads are removed, the purity of the product is further improved and the product can be used as a good experimental material. By combining a special filter and the adsorption of the capture magnet, more than 95 percent of free micro magnetic beads can be effectively removed. Meanwhile, the types of the single cells are identified and biological characteristics of the single cells are analyzed by an immunofluorescence staining method and a method in molecular biology, and effective biological indexes are provided for clinical diagnosis and treatment of cancers.

Description

technical field [0001] The invention belongs to the field of cell separation and analysis, and in particular relates to a cell enrichment, separation and extraction method, a cell enrichment, separation and extraction device and a single cell analysis method. Background technique [0002] The study of cytology provides effective methods and means for the analysis of the principles of many human diseases, clinical diagnosis and treatment. Some diseased cells (or target cells) can or must be identified and analyzed as specific indicators for clinical diagnosis, patient tracking and patient prognosis. Circulating tumor cells are an example. Circulating cancer cells were first reported more than a century ago by Thomas Ashworth [1]. However, for quite a long period of time, people have little research on blood circulating cancer cells, and their knowledge and understanding are quite limited. One of the reasons is that the technology for detecting and extracting blood circulat...

Claims

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Application Information

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IPC IPC(8): C12N5/09C12N5/095C12N5/078C12N5/073C12N15/10C07K1/14C12M1/42C12Q1/68G01N33/53
CPCC12M47/12C12M35/06C12M25/16C12M29/04C12M47/04
Inventor 邓亚光张杰田放
Owner GD TECH INC
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