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Surface treatment method of cell separation equipment, related equipment, rapid and efficient separation method of peripheral blood rare cells or circulating tumor cells

A separation method and surface treatment technology, which is applied to the separation of rare cells in peripheral blood or circulating tumor cells, and in the field of cell separation. It can solve the problems of ineffective improvement, loss of target cells, and short action distance, so as to reduce non-specific adsorption and facilitate operation. , the effect of simple structure

Active Publication Date: 2021-07-09
上海透景诊断科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, using this method often fails to obtain the expected separation effect. The reasons include: 1. The surface of the utensil in contact with the cells in the process of handling the cells will non-specifically adsorb the cells, resulting in a loss of 10%-60% of the target cells. For example, after treatment with a certain concentration of serum, it cannot be effectively improved; 2. When applying a magnetic field for separation, due to the short distance of the magnetic field, the modification of the cells by the magnetic ball will generally be carried out in a small reaction system. , generally less than 1-5ml, which causes the density of cells in the sample to be too high, reaching 10 million to 70 million per ml. In the process of magnetic field separation, the cells modified with magnetic spheres will carry a large number of non-magnetic sphere modified cells. The cells are separated together, resulting in a large loss of target cells

Method used

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  • Surface treatment method of cell separation equipment, related equipment, rapid and efficient separation method of peripheral blood rare cells or circulating tumor cells
  • Surface treatment method of cell separation equipment, related equipment, rapid and efficient separation method of peripheral blood rare cells or circulating tumor cells
  • Surface treatment method of cell separation equipment, related equipment, rapid and efficient separation method of peripheral blood rare cells or circulating tumor cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0101] Embodiment 1 The sealing effect of fibrinogen on the tube wall

[0102] Use the following three methods to seal the surface of 1.5ml and 15ml centrifuge tubes (made of PP) and a silicone hose (inner diameter 5mm, outer diameter 9mm) with a length of 30 cm. Add a liquid containing a certain number of lymphocytes and MCF cells, and count the cells in the liquid after 30 minutes of incubation to calculate the cell yield. The three methods are respectively 1, 10mMPBS (pH7.0) (SIGMA, Cat#P5493) prepared 5% bovine fibrinogen (Shanghai Yeyuan, article number: 9001-32-5) solution, soaking the surface for 30 minutes (hereinafter referred to as Blocking method one); 2. 5% BSA solution prepared by 10mMPBS (pH7.0) (hereinafter referred to as blocking method two); 3. 30% bovine serum (Bioteck, Cat#P30-3302) prepared by 10mMPBS (pH7.0) ) (hereinafter referred to as blocking method three); 4, the control group adopts 10mPBS (pH7.0) solution (hereinafter referred to as blocking method...

Embodiment 2

[0107] Example 2 Enrichment and isolation of circulating tumor cells in 7.5ml whole blood samples (forward selection)

[0108] 1. Sample processing

[0109] Take 10 anticoagulated whole blood samples of 7.5ml healthy individuals, add an average of 100 rare cells to each sample, such as MCF-7 tracer cells (Cell Bank, Institute of Cells, Chinese Academy of Sciences, article number: TCHu 74), mix well, add 8ml of Red blood cell lysate (Tiangen Biotechnology Co., Ltd. product number: RT122-02), let stand at room temperature for 15 minutes, centrifuge at 300g for 20 minutes to collect white blood cells, and repeat the above lysis steps;

[0110] 2. Closed treatment

[0111] Use 5% bovine fibrinogen solution (preparation method is the same as in Example 1) to carry out 15-60 minute sealing to the contact cell surface of all cell separation instruments in the following steps, including centrifuge tube, liquid flowing through pipeline and pipette tip etc. deal with;

[0112] 3. Sep...

Embodiment 37

[0121] Example 3 Enrichment and separation of rare cells in 7.5ml whole blood samples (negative selection)

[0122] 1. Sample processing

[0123] Take 10 anticoagulated whole blood samples of 7.5ml healthy individuals, add an average of 100 SK-Br-3 tracer cells (Cell Bank, Institute of Cell Research, Chinese Academy of Sciences, catalog number: TCHu225) to each sample, mix well, and add 8ml red blood cell lysate (Cat. No. of Tiangen Biotechnology Co., Ltd.: RT122-02), let stand at room temperature for 15 minutes, centrifuge at 300g for 20 minutes to collect white blood cells, and repeat the above lysis steps;

[0124] 2. Closed treatment

[0125]Use 5% human fibrinogen solution (preparation method is the same as in Example 1) to carry out 15-60 minutes of blocking treatment on the contact cell surface of all cell separation instruments in the following steps, including centrifuge tubes, liquid flow through pipelines and pipette tips, etc. .

[0126] 3. Separation and recove...

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Abstract

The invention provides a method for rapidly and efficiently separating rare cells from peripheral blood, comprising: (1) separating leukocytes in peripheral blood, resuspending the leukocytes with a cell diluent to obtain a leukocyte solution; Mix and incubate with the white blood cell solution to obtain an incubation solution; (3) place a part of the ring pipeline closed with fibrinogen on the inner wall in a magnetic field, dilute the incubation solution with a cell diluent to obtain an incubation dilution, and make the incubation dilution in the ring pipeline Circulating flow in the medium, incubating the magnetic balls in the diluent through magnetic field adsorption, and collecting the liquid components in the circular pipeline. It also provides a rapid and efficient separation method for circulating tumor cells in peripheral blood, a surface treatment method for cell separation equipment and related equipment. The method for isolating rare peripheral blood cells or circulating tumor cells of the present invention can quickly and efficiently separate peripheral blood rare cells or circulating tumor cells, is ingenious in design, easy to operate, and is suitable for large-scale popularization and application.

Description

technical field [0001] The invention relates to the technical field of cell separation, in particular to the technical field of separation of rare peripheral blood cells or circulating tumor cells, and specifically refers to a method for surface treatment of cell separation equipment, related equipment, and a rapid and efficient separation method for rare peripheral blood cells or circulating tumor cells. Background technique [0002] As an important content of tumor liquid biopsy, the detection of circulating tumor cells in peripheral blood has great clinical diagnostic value. The main difficulties in the detection of circulating tumor cells in peripheral blood include: 1. The number of circulating tumor cells is very small, only a few to dozens in a 7.5ml blood sample; 2. The proportion in the sample is extremely low, several or several Tens of cells are distributed in 50-70 million normal cells; 3. There is a lack of clear cell surface markers of circulating tumor cells. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/078C12N5/09C12M1/42
CPCC12M23/20C12M47/04C12N5/0634C12N5/0693C12N2509/00C12N2533/56
Inventor 白艳军陶晟王珊
Owner 上海透景诊断科技有限公司