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Rare cell enrichment device and method

A technology of rare cells and cells, applied in the field of devices for screening and collecting rare cells with high recovery rate, can solve the problem of low recovery rate of negative selection of rare cells

Active Publication Date: 2018-03-06
HANGZHOU JUNHUI BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] What the present invention aims to solve is the problem of low recovery rate of rare cell negative selection

Method used

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  • Rare cell enrichment device and method
  • Rare cell enrichment device and method
  • Rare cell enrichment device and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Enrichment of Circulating Epithelial Cells from Human Peripheral Blood

[0036] (1) Pretreatment of blood samples

[0037] 2 ml of whole blood was subjected to density gradient centrifugation to extract the mononuclear cell layer, and the density gradient centrifugation medium used was Lymphoprep from Stemcell Company TM (#07801), the density gradient centrifuge tube is SepMate from Stemcell TM -15 (#15415), the extracted mononuclear cell layer was diluted with diluent (Hank's balanced salt solution + 0.1% bovine serum albumin + 5mM anticoagulant EDTA), and 100 cells were added according to each white blood cell obtained Ratio of immunomagnetic beads Add CD45 antibody-labeled immunomagnetic beads (about 1 micron in size), mix and incubate for 1 hour.

[0038] (2) Remove the remaining white blood cells in the sample

[0039] Add the diluent to the gradient magnet in the circulating epithelial cell enrichment device, then further dilute the cell suspension incubated ...

Embodiment 2

[0048] Sensitivity and Stability Experiments

[0049] 5, 10, 50, and 100 colon cancer tumor cells HCT116 were stained with the dye Vybrant DiI (Life Tehnologies) and mixed into 1 ml of healthy human blood, and then the tumor cells were captured by the method of Example 1, and each experiment was repeated 3 times . After the capture, the HCT116 cells in the chip were directly counted with a fluorescence microscope to calculate the capture efficiency and experimental deviation. The results show that the capture efficiencies corresponding to the incorporation of 5, 10, 50, and 100 tumor cells and the experimental deviations are 73±12%, 77±12%, 79±6%, and 78±4%, which can reach clinical sample detection required sensitivity and stability.

Embodiment 3

[0051] Single cell recovery and sequencing experiments

[0052] 100 non-small cell lung cancer cells H1975 were stained with the dye Vybrant DiI (Life Tehnologies) and mixed into 1 ml of healthy human blood, and then the tumor cells were captured by the method of Example 1, wherein the microfiltration membrane used to recover the cells was as follows: image 3 The shown microfiltration membrane has a double-layer structure, and its bottom is a silicon nitride film with a thickness of only 1 micron. After the capture, the position of the H1975 cells on the filter membrane was directly counted with a fluorescence microscope, and the mechanically fragile silicon nitride membrane at the bottom of the microwell where the H1975 cells were located was impacted by a fine needle, so that the entire silicon nitride film was broken and the cells fell into the membrane where the cells were located. In the wells of a 384-well plate directly below it for cell recovery, 4 μl of phosphate b...

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Abstract

An enrichment device for rare cells comprises a cell screening part and a cell collecting part. The cell screening part comprises two cell screening channels formed between the two opposite surfaces, at least one of the two opposite surfaces is a magnetic adsorption surface, the thickness of the cell screening channels is arranged in the mode that when cell suspension flows through the cell screening channels, a thin liquid layer enabling cells to be distributed in a single-layer mode basically is formed, and therefore when most cells combined with immunity magnetic spheres are adsorbed to the magnetic adsorption surface in the cell screening channels, the rare cells are prevented from being wrapped. The cell collecting part comprises a filtering unit, and the filtering unit is detachably arranged on the downstream portion of the cell screening part to intercept the rare cells which are not magnetically adsorbed. The two key problems of adsorption wrapping and centrifugal loss causing rare cell loss easily in the flotation enrichment process of the rare cells are solved, and therefore the recovery rate of the rare cells can be high. The invention further discloses a method for recovering the rare cells with the enrichment device.

Description

technical field [0001] The invention relates to the field of cell separation and detection, in particular to a device and method for screening and collecting rare cells from cell suspension with high recovery rate. Background technique [0002] For the purpose of medical diagnosis and treatment, it is necessary to screen and separate specific target cells from human or animal blood, tissue fluid or lymph fluid, especially blood, mainly diseased cells or cells exfoliated from diseased tissues and organs . Frequently, the target cells are present in very rare quantities in the aforementioned body fluids. [0003] For example, circulating epithelial cells are a kind of rare cells present in the blood, which are exfoliated from diseased epithelial tissues, among which those exfoliated from benign lesion tissues are benign epithelial cells, and those exfoliated from malignant lesion tissues (cancer) are tumor cells. Extremely rare, every 10ml of blood may only contain a few to ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12M1/00C12M1/12C12N5/078
CPCC12M45/00C12N5/0634C12N2509/00
Inventor 阎灼辉
Owner HANGZHOU JUNHUI BIOTECHNOLOGY CO LTD
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