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Enriching method and device for circulating tumor cells

A tumor cell enrichment technology, applied in the field of molecular biology, can solve the problems of difficult capture efficiency, balance between purity and cell viability, low purity, etc., and achieve the effect of simplified process and high capture efficiency

Pending Publication Date: 2020-08-25
SUZHOU INST OF BIOMEDICAL ENG & TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the above methods are still technically deficient in that it is often difficult to make a trade-off between capture efficiency, purity, and cell viability, which are important criteria for basic and clinical research
For example, the main problem with cell size-based filtration techniques is that if the pore size is small enough to achieve high tumor cell capture efficiency, it usually has high non-tumor cell retention, i.e., the purity is not high

Method used

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  • Enriching method and device for circulating tumor cells
  • Enriching method and device for circulating tumor cells
  • Enriching method and device for circulating tumor cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] This embodiment provides a device for enriching circulating tumor cells, such as figure 1 shown, including:

[0062] The sample collector 1 has a sample inlet 11 at the top and a sample outlet 12 at the bottom;

[0063] The separation column 2, located below the sample collector 1, includes a top opening 21 and a bottom opening 22, and the top opening 21 can be sealingly connected with the sample outlet 12;

[0064] The filter 3 is located below the separation column 2; the filter 3 has a cavity 31, and the top of the cavity 31 is provided with an inlet 32 ​​which can be sealed with the bottom opening 22 of the separation column 2, and the cavity An outlet 33 is provided at the bottom of the chamber; a filter membrane 34 is arranged horizontally in the cavity 31 , and the edge of the filter membrane 34 is closely connected with the inner wall of the cavity 31 .

[0065] In the above-mentioned enrichment device for circulating tumor cells, such as figure 2 As shown, ...

Embodiment 2

[0074] This embodiment provides a method for enriching circulating tumor cells, including the following steps:

[0075] (1) Mix 5ml of fresh peripheral blood sample with 500μl of CD45-labeled immunomagnetic bead solution, and incubate at 4°C for 0.5h;

[0076] (2) Add 15 ml of one-step lysis and fixation solution to the peripheral blood sample incubated in step (1) and incubate for 15 minutes;

[0077] (3) Add PBS buffer (pH7.2) to the peripheral blood sample incubated in step (2) to dilute, dilute to 5 times the original volume (5ml) of the peripheral blood sample, and then load it into the sample of Example 1. The sample collector 1 in the enrichment device for circulating tumor cells, and then turn on the peristaltic pump for suction filtration, and the sample is sequentially subjected to magnetic separation and membrane filtration at a flow rate of 0.25ml / min;

[0078](4) When the sample liquid level in the device reaches the top of the separation column 2, add PBS buffer...

Embodiment 3

[0084] This embodiment provides a method for enriching circulating tumor cells, including the following steps:

[0085] (1) Mix 5ml of fresh peripheral blood sample with 500μl of CD45-labeled immunomagnetic bead solution, and incubate at 4°C for 0.5h;

[0086] (2) Add 15 ml of one-step lysis and fixation solution to the peripheral blood sample incubated in step (1) and incubate for 15 minutes;

[0087] (3) Add PBS buffer (pH7.2) to the peripheral blood sample incubated in step (2) to dilute, dilute to 10 times the original volume (5ml) of the peripheral blood sample, and then load it into the sample of Example 1. The sample collector 1 in the enrichment device for circulating tumor cells, and then turn on the peristaltic pump for suction filtration, and the sample is sequentially subjected to magnetic separation and membrane filtration at a flow rate of 1 / 12ml / min;

[0088] (4) When the sample liquid level in the device reaches the top of the separation column 2, add PBS buff...

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Abstract

The invention relates to the field of molecular biology, in particular to an enriching method and device for circulating tumor cells. The enriching method comprises the steps of performing mixed incubation on samples and immunomagnetic beads which can be combined with leucocyte; adding erythrocyte lysate to the samples, and performing incubation; and diluting the incubated samples, and then sequentially performing magnetic separation and membrane filtration. According to the method, a negative enrichment technique and a membrane filtration technique are combined, so that the advantages of negative enrichment and the advantages of membrane filtration are synthesized, high recovery rate of CTCs is guaranteed, besides, purity and vitality of CTC can also be guaranteed, and reduced recovery rate of the CTCs, cell breakage and reduced vitality caused by steps of centrifuging, liquid transfer and the like are avoided; besides, membrane filtration is used for trapping the circulating tumor cells and removing other impurities and cells, so that the circulating tumor cells are reserved as possible; and in addition, a technological process is simplified, separation of 5ml of whole blood canbe completed within 2h, and capturing of 90% or above of CTC and removing of 95% or above of leukopheresis can be realized.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a method for enriching circulating tumor cells and a device thereof. Background technique [0002] Thomas Ashworth first discovered circulating tumor cells in 1869. With the development of repeatable detection technology, circulating tumor cells have received more and more attention. Circulating tumor cells (CTCs) are a general term for various types of tumor cells in peripheral blood. They are shed from primary tumor tissues and released into the blood, and develop into new tumors in distant places to achieve metastasis. Therefore, CTCs are considered to be a necessary prerequisite for tumor metastasis, and can then be used as the core research object for tumor disease diagnosis, prognosis assessment, and personalized monoclonal antibody drug screening, and become a "liquid biopsy" for metastatic tumors. However, due to the extreme rarity and high heterogeneity of CTCs, there a...

Claims

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Application Information

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IPC IPC(8): C12N5/09G01N1/30C12M1/00C12M1/42C12M1/12
CPCC12N5/0694G01N1/30C12M29/04C12M35/06C12N2509/00C12N2509/10G01N2001/302
Inventor 张威李豪李金泽周连群张芷齐李超李传宇姚佳郭振
Owner SUZHOU INST OF BIOMEDICAL ENG & TECH CHINESE ACADEMY OF SCI
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