CN112812945A - Malignant pleural effusion tumor cell separation device and separation method - Google Patents

Abstract

The application provides malignant pleural effusion tumor cell separation device and separation method, and separation device is including holding jar, vibration filter sieve, drainage funnel and filter cup, and vibration filter sieve, drainage funnel and filter cup all are located the holding jar, and vibration filter sieve is equipped with first microfiltration membrane, and the filter cup is equipped with second microfiltration membrane, and the filter cup sets up in vibration filter sieve for first microfiltration membrane is aimed at to second microfiltration membrane. The micropore diameter of the first microporous filter membrane and the second microporous filter membrane is set, so that when pleural effusion containing cells to be removed sequentially passes through the first microporous filter membrane and the second microporous filter membrane, thymus tumor cells can be remained on the second microporous filter membrane. The separation method comprises a use method of the separation device. The invention has the advantages of fast and high-efficiency separation and enrichment of tumor cells from pleural effusion, can provide high-purity and high-concentration samples for downstream detection and identification of the tumor cells in the pleural effusion, and improves the time and accuracy of pathogen detection.

Description

Malignant pleural effusion tumor cell separation device and separation method

Technical Field

The application relates to the technical field of advanced manufacturing and biomedical technology, in particular to a malignant pleural effusion tumor cell separation device and a separation method.

Background

Malignant Pleural Effusion (MPE) refers to pleural effusion caused by the metastasis of a malignant tumor that originates in the pleura or other sites to the pleura. The MPE epidemiology investigation and research data are not available at present in China. According to statistics, the number of MPE attacks is more than 150000 in the United states every year, corrected by population base, and about 1200000 new malignant pleural effusion is estimated to occur every year in China. MPE can appear in almost all malignant tumors, lung cancer is the most common cause, which accounts for 1/3 of MPE, secondary to breast cancer, and MPE appears in lymphoma, ovarian cancer and digestive tract tumors. 5% -10% of MPE can not find primary tumor focus. The appearance of MPE indicates that the tumor has spread or progressed to an advanced stage, and the life expectancy of the patient will be significantly shortened. MPE is calculated from the confirmed diagnosis, the median survival time is 3-12 months, and the life time of MPE caused by lung cancer is the shortest. Therefore, the diagnosis of the patients in time is very important.

The diagnostic criteria for malignant pleural effusion is the discovery of malignant tumor cells in the pleural fluid or the finding of malignant tumor tissue on the pleura. The traditional diagnosis idea is that the pleural effusion is identified as effusion and effusion; secondly, the possibility of tumor, tuberculosis, inflammation, trauma and the like is considered according to the condition of the patient, such as the need of combining the effusion with the condition of the patient. The current clinical examination methods commonly used include cytopathology of pleural effusion, blinding or pleural biopsy under B-ultrasound/CT guidance, and medical thoracoscopy or thoracosurgical thoracotomy biopsy is still not considered unequivocally. The positive rate of the pleural effusion cell pathology is 30-62%, the positive rate of pleural biopsy is about 30-40%, and more than 90% of pleural effusion can be diagnosed definitely by the medical thoracoscope. But the latter three are very traumatic and are more prone to pleural effusion cytology examination for the elderly and the patients with systemic failure. Although the etiology can be clearly diagnosed by the existing hydrops cytology examination, the diagnosis rate is only 40-87%, so how to utilize the pleural effusion to the maximum extent and improve the enrichment rate and the detection rate of malignant pleural effusion tumor cells is helpful for clearly diagnosing the pleural effusion diseases and avoiding further traumatic examination.

In recent years, with the development of micro/nano technology, the sensitivity of recovering specific cells from clinical fluids has been significantly improved. Known liquid biopsy techniques include microfluidics-based liquid biopsy techniques and filtration-based liquid biopsy techniques (e.g., microcolumns, microbeads, microwells). Filtration-based liquid biopsy techniques are considered promising approaches for high throughput cell enrichment. Many microporous filters have uniform pore size and pore-to-pore space and have achieved efficient cell enrichment. However, the porosity of these filters is still low, only filtration fluxes lower than 2mL/min can be achieved, and the enrichment process of large volumes of clinical fluid cannot be completed.

Disclosure of Invention

The present application provides a malignant pleural effusion tumor cell separation device and a separation method, which solve the above-mentioned problems.

According to one aspect of the invention, a malignant pleural effusion tumor cell separation device is provided, which comprises a holding tank, a vibrating filter screen, a drainage funnel and a filter cup, wherein the drainage funnel, the vibrating filter screen and the filter cup are all positioned in the holding tank.

Wherein, the vibration filter sieve includes lid shape filter house and the elastic support frame that is equipped with the oscillator, the centre of lid shape filter house is equipped with first millipore filtration, it includes cup and the second millipore filtration of lid on the cup to filter the cup, filter the cup setting and in the elastic support frame, make second millipore filtration position aim at first millipore filtration, the lower extreme of drainage funnel stretches into lid shape filter house and is located the top of first millipore filtration, its end opening aims at first millipore filtration, the first portion of filtering the cup is equipped with the vacuum aspiration ware that is used for drainage with higher speed.

Wherein the pore diameter of the micropores of the first microfiltration membrane is smaller than the diameter of the large cells to be removed, and the pore diameter of the micropores of the first microfiltration membrane is larger than the diameter of the thymus tumor cells and the diameter of the small cells to be removed. The micropore diameter of the second microporous filter membrane is smaller than the diameter micropore of the thymus tumor cell and larger than the diameter of the small cell to be removed, so that the pleural effusion containing the cell to be removed is prevented from waiting to remove the large cell and passing through the first microporous filter membrane when passing through the first microporous filter membrane, and meanwhile, the thymus tumor cell and the small cell to be removed pass through the first microporous filter membrane and are remained on the second microporous filter membrane.

Wherein, the first microporous filter membrane and the second microporous filter membrane are both made of Parylene C.

Wherein the distance between the micropores of the first microfiltration membrane and the distance between the micropores of the second microfiltration membrane are both smaller than the diameter of the non-capture target.

Wherein the shape of the micropores of the first microporous filter membrane and the second microporous filter membrane is regular hexagon or square.

Wherein, drainage funnel's upper portion outer wall and the inner wall butt that holds the jar form the level spacing to drainage funnel, and the last mouthful of lid shape filter house forms horizontal location with drainage funnel's lower extreme outer wall butt, and the bottom of elastic support frame and the inner wall bottom butt that holds the jar form the level spacing to elastic support frame.

Wherein, the elastic support frame includes base and four support columns that are equipped with the spring, and the bottom of every support column respectively is equipped with a micro oscillator, and the upper end of filter cup stretches into the end opening to the filter house, forms the spacing of horizontal direction.

Wherein the large cells to be removed include cellulose-like substances, and the small cells to be removed include exfoliated mesothelial cells, lymphocytes, neutrophils, and blood cells.

According to another aspect of the present invention, there is provided a separation method based on the malignant pleural effusion tumor cell separation device, comprising the following steps: step S1, opening the micro oscillator and the negative pressure aspirator; step S2, placing the container filled with the pleural effusion above the containing tank, and slowly dumping the container; and step S3, after the pleural effusion completely passes through the filtering device, closing the oscillator and the negative pressure aspirator, and taking out the lower layer of membrane to obtain tumor cells enriched on the membrane.

Compared with the prior art, the method has the following advantages:

the separation device for malignant pleural effusion tumor cells provided by the invention is based on a double-layer microporous filter membrane, and is used for enriching and recovering tumor cells in pleural effusion, and the size difference and the deformability difference between the tumor cells and other cells in body fluid are mainly relied on, so that the tumor cells are selectively separated and enriched aiming at different targets.

The separation device for applying malignant pleural effusion tumor cells provided by the invention has the advantages of quickly and efficiently separating and enriching tumor cells from pleural effusion, can provide high-purity and high-concentration samples for downstream detection and identification of pleural tumor cells, greatly improves the time and accuracy of pathogen detection, and can complete cell separation of the samples within 3 minutes.

The separation device and the separation method for malignant pleural effusion tumor cells provided by the invention can efficiently remove larger-size cells (such as cellulose-like substances) and smaller-size cells (mainly exfoliated mesothelial cells, lymphocytes, neutrophils and blood cells) in body fluid, thereby efficiently separating malignant tumor cells in pleural effusion, and the capture sensitivity is as high as 80% (the capture sensitivity of the traditional cell centrifugation method is only about 45%).

The separation device applying malignant pleural effusion tumor cells, provided by the invention, has the advantages that the microporous filter membrane has good flexibility and high compatibility with various downstream detections. Large field of view scanning imaging of captured cells on the filter membrane can be achieved, and interpretation/prescreening of tumor cells is performed through AI-based machine learning and identification.

Drawings

FIG. 1 is a schematic structural diagram of a malignant pleural effusion tumor cell separation device in accordance with the present invention;

fig. 2 is a comparison graph of the detection results of the separation device and separation method of malignant pleural effusion tumor cells and the cell centrifugation method of the present invention on bronchoalveolar lavage fluid (BALF) shed tumor cells, wherein fig. 2a is a comparison graph of the detection rates of tumor cells of the above two methods, fig. 2b is a comparison graph of the detection sensitivity of tumor cells of the above two methods, and fig. 2c is a comparison graph of the detection rates of the separation device and separation method of malignant pleural effusion tumor cells of the present invention on lavage fluids with different viscosities;

FIG. 3 is an electron microscope image of the separation result of the malignant pleural effusion tumor cell separation device in the invention;

FIG. 4 is a graph showing the results of the filtration throughput of the malignant pleural effusion tumor cell separation device of the present invention;

FIG. 5 is a scan image of the separation result of the malignant pleural effusion tumor cell separation device in the invention.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present application more comprehensible, the present application is described in further detail with reference to the accompanying drawings and the detailed description.

As shown in figure 1, the malignant pleural effusion tumor cell separation device comprises a holding tank 1, a vibration filter sieve 2, a drainage funnel 3 and a filter cup 4, wherein the vibration filter sieve 2, the drainage funnel 3 and the filter cup (4) are all positioned in the holding tank 1.

Wherein, the vibration filter sieve 2 includes lid shape filter house and the elastic support frame that is equipped with the oscillator, and the centre of lid shape filter house is equipped with first microfiltration membrane 210, and filter cup 4 includes cup 410 and the second microfiltration membrane 420 of lid on cup 410, and filter cup 4 sets up in the elastic support frame for second microfiltration membrane 420 is located first microfiltration membrane 210's below, and the position aims at first microfiltration membrane 210, and the lower extreme of drainage funnel 3 stretches into lid shape filter house and is located the top of first microfiltration membrane 210, and its end opening aims at first microfiltration membrane 210, and the first half of filter cup 4 is equipped with the vacuum aspiration ware 430 that is used for drainage with higher speed to be used for drainage with higher speed.

The upper portion outer wall of drainage funnel 3 and the inner wall butt that holds jar 1 form the level spacing to drainage funnel 3, make drainage funnel 3 fixed in the horizontal direction position. The upper port of the cover-shaped filtering part is abutted against the outer wall of the lower end of the drainage funnel 3 to horizontally limit the cover-shaped filtering part and limit the movable distance of the cover-shaped filtering part in the horizontal direction. The bottom of the elastic support frame is abutted against the bottom of the inner wall of the containing tank 1 to horizontally position the elastic support frame, so that the elastic support frame is fixed in the horizontal direction.

In a specific embodiment, the elastic support frame comprises a base and four support columns provided with springs, the bottom of each support column is provided with a micro oscillator 2310, and the upper end of the filter cup 4 extends into the lower opening of the cover-shaped filter part to form a limit in the horizontal direction and limit the movable distance of the filter cup 4 in the horizontal direction.

In another specific embodiment, the elastic support frame comprises a micro-vibrator, a base and four support columns provided with springs, the micro-vibrator is arranged on the outer edge of the modified filtering part, and the four support columns are respectively connected with the cover-shaped filtering part and the base from four directions and support the cover-shaped filtering part.

Wherein the pore diameter of the first microporous filter membrane 210 is smaller than the diameter of the large cell to be removed, and the pore diameter of the first microporous filter membrane 210 is larger than the diameter of the thymus tumor cell and the diameter of the small cell to be removed; the pore diameter of the second microfiltration membrane 420 is smaller than the diameter of the thymic tumor cells, so that the pleural effusion containing the cells to be removed avoids the large cells to be removed from passing through the first microfiltration membrane 210 when passing through the first microfiltration membrane 210, and simultaneously, the thymic tumor cells and the small cells to be removed pass through the first microfiltration membrane 210, and then the thymic tumor cells are remained on the second microfiltration membrane 420.

Both the first microfiltration membrane 210 and the second microfiltration membrane 420 are made of Parylene C. The first microporous filter membrane 210 and the second microporous filter membrane 420 are made of Parylene C based on a micro electro mechanical system (Parylene MEMS) process, and have flexibility and high mechanical strength. The Parylene C has the characteristics of high temperature resistance, good chemical stability and direct compatibility with a subsequent Polymerase Chain Reaction (PCR) detection technical system.

In the present invention, the pore diameters of the micropores of the two layers of the first microfiltration membrane 210 and the second microfiltration membrane 420 are different, and the pore diameter of the micropores of the first microfiltration membrane 210 is larger than that of the micropores of the second microfiltration membrane 420. The first microporous filter membrane 210 is a micro-oscillator, so that tumor cells and cells with smaller sizes to be removed (mainly exfoliated mesothelial cells, lymphocytes, neutrophils and blood cells) rapidly pass through the first microporous filter membrane 210; the pore size of the second microporous filter 420 is smaller, and the second microporous filter is matched with a negative pressure device to capture and recover target tumor cells in the body fluid, so that the target tumor cells are retained on the second microporous filter 420.

In a specific example 1, the first microfiltration membrane 210 is a square filter membrane with a side length of 17 mm; the second microfiltration membrane 420 is a square filter membrane with a side length of 10 mm; the working area of the body fluid passing through the first microporous filter membrane 210 is 28.3mm²(ii) a The working area of the blood passing through the second microporous filter membrane 420 is 28.3mm². The first microfiltration membrane 210 and the second microfiltration membrane 420 provided in this embodiment have a large working area.

The first microfiltration membrane 210 and the second microfiltration membrane 420 can precisely control the micropore size and the micropore-micropore spacing, and the micropore size of the first microfiltration membrane 210 is smaller than the diameter of larger-sized cells (such as cellulose-like substances) to be removed; the pore size of the micropores of the second microfiltration membrane 420 is smaller than the diameter of the target tumor cells and larger than the diameter of the small cells to be removed (e.g., exfoliated mesothelial cells, lymphocytes, neutrophils, and blood cells). Meanwhile, the first microfiltration membrane 210 and the second microfiltration membrane 420 can precisely control the topology of micropores. The first microfiltration membrane 210 and the second microfiltration membrane 420 have a membrane thickness comparable to the characteristic pore size.

The micropores of the first microfiltration membrane 210 are regular hexagons, the length of the diagonal line of the regular hexagons is 30-80 μm, and the thickness of the first microfiltration membrane 4 is about 5-12 μm. The micropores of the second microfiltration membrane 420 are square, the side length of the square is about 5-12 μm, the thickness of the second microfiltration membrane 8 is about 2-3 μm, the pore spacing of the first microfiltration membrane 210 is 6-8 μm, and the pore spacing of the second microfiltration membrane 420 is 1-3 μm.

In example 1, the micropores of the first microfiltration membrane 210 are regular hexagons, the diagonal length of each regular hexagon is 40 μm, and the thickness of the first microfiltration membrane 4 is about 7 μm. The micropores of the second microfiltration membrane 420 are square, the side length of the square is about 5 μm, the thickness of the second microfiltration membrane 8 is 3 μm, the pore spacing of the first microfiltration membrane 210 is 6 μm, and the pore spacing of the second microfiltration membrane 420 is 1 μm.

The spacing between the micropores of the first microfiltration membrane 210 and the spacing between the micropores of the second microfiltration membrane 420 are both smaller than the diameter of the non-captured target (i.e., the spacing between the micropores is smaller than the diameter of the cells to be removed), so that the non-captured target is prevented from falling on the gap between the micropores as much as possible to form "non-specific adhesion". Non-specific adhesion means: if the gap between the micropores is too large (mainly referring to the first microfiltration membrane 210), the smaller-sized cells to be removed (for example, exfoliated mesothelial cells, lymphocytes, neutrophils, and blood cells) and tumor cells smaller than the micropores of the first microfiltration membrane 210 are more likely to fall on the gap and thus cannot pass through the first microfiltration membrane 210, i.e., "non-specific adhesion" is formed (since the first microfiltration membrane 210 should specifically remove the larger-sized cells to be removed (for example, cellulose-like substances) and pass the smaller-sized cells to be removed (for example, exfoliated mesothelial cells, lymphocytes, neutrophils, and blood cells), tumor cells).

Wherein the non-capture targets refer to, relative to the first microporous filter membrane 210: targets other than "larger size cells to be removed"; non-capture targets relative to the second microfiltration membrane 420 refer to: targets other than "tumor cells".

Wherein, the process of the first layer of filtration is as follows:

placing the body fluid sample into a drainage funnel; under the drive of gravity and negative pressure, the effusion sample permeates downwards; because the pore diameter of the first microfiltration membrane 210 is smaller than the diameter of the larger cells to be removed, and the pore diameter of the first microfiltration membrane 210 is larger than the diameters of the tumor cells and the smaller cells to be removed, the arrangement of the oscillation device prevents the micropores from being blocked by the fiber-like substances or cell clusters, so the cellulose-like substances to be removed can be "intercepted" on the first microfiltration membrane 210, and the tumor cells, the smaller cells to be removed and the rest of the effusion can pass through the first microfiltration membrane 210;

the second filtration process was as follows:

the liquid sample filtered through the first microfiltration membrane 210 falls onto the surface of the second microfiltration membrane 420. The residual body fluid belongs to fluid and can directly pass through the micropores of the second microfiltration membrane 420, and the small cells to be removed can also fall into the cup body of the filter cup through the micropores of the second microfiltration membrane 420 due to the existence of negative pressure and the filter membrane gaps. The tumor cells will remain on the second microfiltration membrane 420, and the tumor cells enriched in the second microfiltration membrane 11 will be taken down for morphological, immunoomics, genomics, functionality and other studies. After the sample filtration is finished, the upper and lower double-layer microporous filter membranes are replaced, and the enrichment of the next pleural effusion sample can be carried out.

The separation method based on the malignant pleural effusion tumor cell separation device specifically comprises the following steps:

and step S1, opening the micro oscillator and the negative pressure suction device.

And step S2, placing the container filled with the pleural effusion above the containing tank, and slowly pouring.

And step S3, after the pleural effusion completely passes through the filtering device, closing the oscillator and the negative pressure aspirator, and taking out the lower layer of membrane to obtain tumor cells enriched on the membrane.

Test example 1

The test method comprises the following steps:

1. a total of 39 pleural effusion specimens were selected.

Grouping standard: tumor incorporation-patients with massive pleural effusion; the test of accumulated liquid indicates the effusion.

2. The collected pleural effusion samples were divided equally into 2 equal portions.

(1) The first specimen was first centrifuged (3000r, 5min), the supernatant was discarded after centrifugation, the sediment was resuspended to a total volume of 1ml, and then ultra-thin cell smear and hematoxylin-eosin (HE) staining were performed.

(2) The second specimen was used to isolate cells using the double-layer filter apparatus of example 1 of the present application, 1, total amount and filtration time of each sample was recorded, cells collected by the filter were air-dried and fixed with 95% ethanol for 10min, and then HE staining was performed.

(3) And (4) observing and comparing the cells on the filter and the slide by a pathology expert with abundant experience, and determining a capture result.

3. Statistical analysis

The filtration apparatus of example 1 was compared with the centrifugal smear method for positive detection rate of tumor cells.

4. Test results

1. Research on 39 pleural effusion specimens shows that the detection rate of tumor cells exfoliated from bronchoalveolar lavage fluid (BALF) is increased to 80% by using the filter membrane separation device and the separation method of example 1, compared with that the detection rate of tumor cells exfoliated from bronchoalveolar lavage fluid (BALF) obtained by using the conventional cell centrifugation method is only 45%, the specific data is shown in fig. 2(a), and the separation result picture is shown in fig. 3.

2. The filter membrane separation device and the separation method of example 1 are greatly improved in detection sensitivity compared to the conventional cell centrifugation method, and specific data are shown in fig. 2 (b).

3. In the filter membrane separation device and the separation method of the embodiment 1, there is no difference in positive detection rate of BALF samples with different degrees of viscosity, and the universality of the liquid biopsy method based on the filter membrane separation device of the invention on practical clinical BALF samples is fully proved.

4. The separation device and method of example 1 achieved cell separation of 54.6% BALF sample within 3 minutes, and cell separation was achieved in 10 minutes for all the remaining samples (specific filtration flux, including average filtration flux and real-time filtration flux, results are shown in fig. 4), and the whole process from sample taking to cell separation, cell detection, and slide diagnosis could be achieved within 30 minutes.

5. The device has extremely high compatibility with various downstream detections. Large field-of-view scanning imaging of the captured cells on the second filter can be achieved (as shown in fig. 5).

Test example 2

To verify the cell recovery rate of the filter separation device of example 1, 10000A 549 cells in alveolar lavage fluid were tested. The test steps are as follows: a549 cells were purified by CellTracker Green (5. mu.M, C2925, Invitrogen)^TMThermoFisher, USA) and Hoechst33342 (5. mu.g/mL, H1399, Invitrogen)^TMThermo fisher, USA) to facilitate observation and counting of recovered cells under a fluorescent microscope. The A549 cells were separated by the membrane separation apparatus and the cell centrifugation method of example 1. After filtration and centrifugation, the separated cells on the filter were placed between a slide and a cover slip, observed and counted under a fluorescence microscope to obtain cell recovery, and the experiment was repeated three times.

The test results show that the filter membrane device of the example 1 achieves a high cell recovery rate of 89.8 +/-5.2%. The recovery rate of the cell centrifugation method is low and is only 13.6 +/-7.8%. The results show that the recovery rate of the A549 cells by the filter membrane device and the method is obviously higher than that of the traditional cell centrifugation method.

In conclusion, compared with the traditional cell centrifugation method, the malignant pleural effusion tumor cell separation device and the separation method have the advantages that the detection rate and the detection sensitivity of tumor cells are greatly improved, and the filtration flux is high, so that the device and the method have important significance for diagnosing the malignant tumor cells in clinical practice.

The present application provides a device, a system and a method for separating fungal spores based on a microporous filter membrane, which are described in detail above, and the principles and embodiments of the present application are explained herein by using specific examples, and the description of the above examples is only used to help understand the method and the core concept of the present application; meanwhile, for a person skilled in the art, according to the idea of the present application, there may be variations in the specific embodiments and the application scope, and in summary, the content of the present specification should not be construed as a limitation to the present application.

Claims (8)

1. The malignant pleural effusion tumor cell separation device is characterized by comprising a containing tank (1), a vibration filter sieve (2), a drainage funnel (3) and a filter cup (4), wherein the vibration filter sieve (2), the drainage funnel (3) and the filter cup (4) are all positioned in the containing tank (1);

the vibrating filter screen (2) comprises a cover-shaped filter part and an elastic support frame provided with an oscillator, a first microporous filter membrane (210) is arranged in the middle of the cover-shaped filter part, the filter cup (4) comprises a cup body (410) and a second microporous filter membrane (420) covered on the cup body (410), the filter cup (4) is arranged in the elastic support frame, so that the second microporous filter membrane (420) is aligned to the first microporous filter membrane (210), the lower end of the drainage funnel (3) extends into the cover-shaped filter part and is positioned above the first microporous filter membrane (210), the lower opening of the drainage funnel is aligned to the first microporous filter membrane (210), and the upper half part of the filter cup (4) is provided with a negative pressure aspirator (430) for accelerating drainage;

wherein the pore size of the first microporous filter membrane (210) is smaller than the diameter of the large cells to be removed, and the pore size of the first microporous filter membrane (210) is larger than the diameter of the thymus tumor cells and the diameter of the small cells to be removed; the pore diameter of the micropores of the second microfiltration membrane (420) is smaller than the diameter of the thymic tumor cells and larger than the diameter of the small cells to be removed, so that when pleural effusion containing the cells to be removed passes through the first microfiltration membrane (210), large cells to be removed are prevented from passing through the first microfiltration membrane (210), meanwhile, the thymic tumor cells and the small cells to be removed pass through the first microfiltration membrane (210), and then the thymic tumor cells are remained on the second microfiltration membrane (420).

2. The malignant pleural effusion tumor cell separation device of claim 1, wherein the first microfiltration membrane (210) and the second microfiltration membrane (420) are both made of Parylene C.

3. The malignant pleural effusion tumor cell separation device of claim 1, wherein the spacing between the pores of the first microfiltration membrane (210) and the spacing between the pores of the second microfiltration membrane (420) are both smaller than the diameter of a non-capture target.

4. The malignant pleural effusion tumor cell separation device of claim 1, wherein the first microfiltration membrane (210) and the second microfiltration membrane (420) have a shape of a regular hexagon or a square of micropores.

5. The device for separating malignant pleural effusion tumor cells of claim 1,

the upper portion outer wall of drainage funnel (3) with the inner wall butt that holds jar (1), it is right drainage funnel (3) form the level spacing, the last mouthful of lid shape filter house with the lower extreme outer wall butt of drainage funnel (3), it is right lid shape filter house forms the level spacing, the bottom of elasticity support frame with the inner wall bottom butt that holds jar (1), it is right the elasticity support frame forms horizontal location.

6. The device for separating malignant pleural effusion tumor cells of claim 1,

the elastic support frame comprises a base and four supporting columns provided with springs, a micro oscillator (2310) is arranged at the bottom of each supporting column, and the upper end of the filter cup (4) extends into the lower opening of the cover-shaped filter part to form horizontal limiting.

7. The device for separating malignant pleural effusion tumor cells of claim 1,

the large cells to be removed comprise cellulose-like substances, and the small cells to be removed comprise exfoliated mesothelial cells, lymphocytes, neutrophils and blood cells.

8. The method for separating malignant pleural effusion tumor cells according to any of claims 1-7, comprising the following steps:

step S1, opening the micro oscillator and the negative pressure aspirator;

step S2, placing the container filled with the pleural effusion above the containing tank, and slowly dumping the container;

and step S3, after the pleural effusion completely passes through the filtering device, closing the oscillator and the negative pressure aspirator, and taking out the lower layer of membrane to obtain tumor cells enriched on the membrane.

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