CN111500417B

CN111500417B - High-throughput cell sorting and enriching device and using method thereof

Info

Publication number: CN111500417B
Application number: CN202010239836.2A
Authority: CN
Inventors: 赵阳; 程洁; 黄成军
Original assignee: Institute of Microelectronics of CAS
Current assignee: Institute of Microelectronics of CAS
Priority date: 2020-03-30
Filing date: 2020-03-30
Publication date: 2023-01-03
Anticipated expiration: 2040-03-30
Also published as: CN111500417A

Abstract

Aiming at the technical defects of low screening flux, large damage, easy loss and pollution of recovered cells and the like of target cells in the prior cell research technology, the invention provides a high-flux cell sorting and enriching device and a using method thereof, wherein the device 1) integrates cell enrichment, recovery, capture and culture in a closed manner, thereby solving the problems of cell loss and pollution in the link; 2) In the cell separation module, the inclined cell filter membrane and the fluid channel are combined, and the flux of target cells is improved on the basis of keeping the cell activity; 3) In a single cell (/ cell mass) capturing and culturing observation module, the difficult problem of cell tracking and positioning detection is solved, and the proliferating cells are effectively cultured. The device is simple and convenient to operate, and provides equipment technical support for preparation and amplification of rare cell samples and accurate single cell research in biomedical and clinical diagnosis research.

Description

High-throughput cell sorting and enriching device and using method thereof

Technical Field

The invention belongs to the technical field of biology, and particularly provides a high-throughput cell sorting and enriching device and a using method thereof.

Background

Cells are the basic unit of a living body, tissues or body fluids of higher living bodies (such as human bodies) are formed by mixing various cells, and the separation and purification of target cells from mixed cell samples is an important step in the current cell research, and has important significance for biological research and clinical diagnosis and analysis (such as tumor liquid biopsy). The detection and accurate evaluation of the enriched tumor cells are clinically important, and can guide the stage and clinical management of cancer patients; the clinical diagnosis of tumor cells is currently mainly carried out by cytological methods, the sensitivity of which depends to a large extent on the quantity and quality of the liquid and on the experience of the cytopathologist, and invasive biopsy methods, some mesothelial cells reducing the cytological sensitivity by being similar to malignant cells, which are relatively painful for the patient. Cell filter membranes prepared based on porous membranes of different materials and processes are currently common cell screening methods. The method has been applied to the removal of large-particle impurities and the separation of large and small cells in cell suspension, and has a good effect on the enrichment of tumor cells, however, the existing porous membrane cell sieve with a horizontal plane structure has the defects of low flow rate and low flux in the use process, and the pores are easy to block in the filtering process, so that the application of the porous membrane cell sieve in the aspects of cell biological research and clinical diagnosis is limited; in addition, various cost factors such as the yield in practical application are also important obstacles for the popularization of the cell screening technology.

For the follow-up detection of the recovered cells, the traditional biological cell experiment is mostly finished in a pore plate, and then the cells are directly dripped on a glass slide for observation, so that the time and the labor are wasted, the reagent dosage is large, and the tracking observation of single cells and the uniform microenvironment are difficult to realize. Under the development of micro-nano technology, a micro-nano array structure prepared from silicon-based micro-pits, glass micro-pits or other materials provides a thought for developing a novel cell detection platform. The cell microarray can capture single cells or cell groups in specific array positions in a chemical or physical mode, so that a uniform microenvironment is conveniently applied and continuous observation is facilitated, and the technology has important significance in cell transfection, drug screening, cell culture and differentiation research.

Disclosure of Invention

In order to overcome the problems in the background art, the present invention provides a high throughput cell sorting and enriching apparatus, comprising: the cell filter membrane is fixed on the channel structure, a microfluidic channel is formed between the cell filter membrane and the channel structure, and a liquid inlet and a liquid outlet are formed at two ends of the microfluidic channel respectively.

Preferably, the support structure comprises a centrifuge tube, the centrifuge tube is provided with a PBS buffer solution, and the cell filter membrane and the channel structure are soaked in the PBS buffer solution.

Preferably, the cell filtration membrane comprises a nucleopore membrane made of a polymeric material.

Preferably, the cell filtration membrane comprises a polycarbonate sieve membrane, a poly-p-phthalic acid sieve membrane, a silicon-based sieve membrane, a polydimethylsiloxane sieve membrane or a C-type poly-p-xylene filtration membrane.

Preferably, the microfluidic channel shape is at least one of: spiral shape, fishbone structure, array structure.

Preferably, the channel structure comprises a substrate and a cell detection capture array disposed on a surface of the channel structure.

Preferably, the cell detection capture array comprises a microwell array or a micropillar array.

Preferably, the depth of the micro well array or the height of the micro column array is 5 μm to 5000 μm.

Preferably, the cell filter membrane is sealed on the channel structure by cutting, folding and heat sealing, and is matched with the shape of the support structure.

The invention also provides a use method of the high-throughput cell sorting and enriching device, the high-throughput cell sorting and enriching device comprises the high-throughput cell sorting and enriching device, and the method comprises the following steps:

washing the cell filter membrane by PBS cell buffer solution through the channel structure to enable the cell filter membrane to be in an infiltrated state;

injecting a biopsy sample into the channel structure;

washing the cell filter membrane and the channel structure by using the PBS cell buffer solution, and recovering the cells enriched on the cell filter membrane;

horizontally placing the high-throughput cell sorting and enriching device, and removing the cell filter membrane and the support structure;

staining the enriched and recovered target cells, and then paving the target cells on the channel structure for detection;

and adding a cell culture solution to the channel structure to continue culture observation.

Advantageous effects

The invention designs a sandwich type cell sorting device based on a gravity field and an inclined cell filter membrane based on a micro-fluidic technology and a mechanical principle, provides a high-throughput cell sorting, enriching, detecting and culturing integrated addressable single cell (/ cell mass) culturing and continuous observing method, and has the following innovation benefit effects:

(1) Compared with a common plane filtering and sorting structure, the sorting device has the advantages that the same amount of samples have higher liquid level, so that a small amount of samples can realize cell sorting operation, the flux is improved, meanwhile, the cell filter membrane lower part is soaked in liquid, the liquid tension on the surface of the cell filter membrane can be increased, the pressure required by the liquid passing through the micron holes is reduced, the speed of the liquid passing through the filter membrane is accelerated, and the flux is assisted to be improved;

(2) Compared with the common microfluidic control device for realizing cell separation, the device has the advantages that the size exceeds micron level, the shearing force to cells is small, the activity rate of the separated cells is high, and the continuous culture and proliferation can be realized;

(3) The inclined filter membrane and the surface physical structure thereof enable the convection and convolution of the fluid to generate the tangential flow of the fluid on the screen surface, so that more small cells can pass through the surface of the filter membrane until being filtered out, and large cells are washed on the surface of the filter membrane to avoid staying, thereby effectively solving the problem that the cells/microparticles in the screen pores are blocked and agglomerated, and realizing the sorting and enrichment of cells with high purity;

(4) The gravity field is used as the driving force, an additional device is not needed for providing power, and meanwhile, the device is easy to process and simple and convenient to operate, the integration of multiple layers of filter membranes can be realized, and the complexity of the device and the experimental operation can be simplified;

(5) Because the cell filtering membrane used in the method is compatible with the commercial nucleopore membrane, and the device can be processed by adopting a common processing or 3D printing method, compared with the traditional microfluid control device, the preparation cost is reduced, and the method is beneficial to clinical application;

(6) The cell is detected after cell enrichment, multiple dyeing and complex operation are not needed, and a uniform microenvironment is conveniently applied and continuous observation is conveniently carried out through cells which are conveniently addressed and distributed and recovered by the cell pit array. Cells with vigorous metabolism are selected from the angle of cell metabolism to serve as alternative tumor cells, so that the enrichment of the tumor cells is completed, the operation is simple and quick, and the clinical quick detection is facilitated;

(7) Compared with the existing cell sorting method based on filtration and separation, most methods only have one-time filtration to sort out cells in two size ranges.

Drawings

FIG. 1 is a schematic structural diagram of a high throughput cell sorting and enriching apparatus provided by the present invention;

FIG. 2 is a schematic view of an equivalent model of flow resistance of a channel structure and a cell filtration membrane in the high throughput cell sorting and enriching device provided by the present invention;

FIG. 3 is a schematic view of a streamline simulation of a fluid in a channel structure according to the present invention;

FIG. 4 is a schematic view of an embodiment 1 of the high throughput cell sorting and enriching apparatus provided in the present invention;

FIG. 5 is a flow chart of the operation of a high throughput cell sorting and enriching apparatus provided by the present invention;

FIG. 6 is a schematic diagram of a microfluidic channel in the high throughput cell sorting and enriching apparatus provided in the present invention;

FIG. 7 is a schematic view of an embodiment 2 of the high throughput cell sorting and enriching apparatus provided in the present invention;

FIG. 8 is a schematic view of an embodiment 3 of the high throughput cell sorting and enriching apparatus provided by the present invention;

FIG. 9 is a schematic view of an embodiment 4 of the high throughput cell sorting and enriching apparatus provided by the present invention;

FIG. 10 is a schematic view of an embodiment 5 of the high throughput cell sorting and enriching apparatus provided by the present invention;

FIG. 11 is a flow chart of a cell enrichment and subsequent observation and detection method of the present invention;

FIG. 12 is a diagram showing the effect of the distribution of malignant pleural effusion cells before and after enrichment in clinical trials;

FIG. 13 is a comparison of 2-NBDG staining fluorescence before and after the enrichment of malignant pleural effusion cells in the clinical examination of the invention;

FIG. 14 is a photograph of malignant pleural effusion cells enriched and cultured for 7 days in accordance with clinical verification of the present invention;

in the figure: 1. the device comprises a liquid inlet, a liquid outlet, a large cell, a cell filter membrane, a support structure, a channel structure, a micropore array, a microfluid channel, a substrate, a micro-column array, a micro-cell, a centrifuge tube and a PBS buffer solution, wherein the large cell is arranged in the large cell, the cell filter membrane is arranged in the cell filter membrane, the support structure is arranged in the cell filter membrane, the channel structure is arranged in the channel structure, the micro-pore array is arranged in the micro-column array, the microfluid channel is arranged in the micro-column array, the micro-column array is arranged in the micro-channel, the substrate is arranged in the micro-column array, the micro-cell is arranged in the micro-tube, and the PBS buffer solution is arranged in the centrifuge tube and the PBS buffer solution.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, preferred embodiments of the present invention will be described in detail below to facilitate understanding of the skilled person.

Example 1:

referring to fig. 4 and 5, for the high-throughput rare cell enrichment device based on the inclined filter membrane, besides providing channels for supporting the cell filter membrane 4, the channel structure 6 can also cause turbulence to the fluid, so that the cells are in a washing state without adhering to the surface of the cell filter membrane 4, as shown in fig. 6, and the microfluidic channels 8 with different shapes can achieve the effect.

The operation method of the integrated single cell (/ cell mass) culture observation module high-throughput rare cell enrichment device comprises the following steps:

(1) The method comprises the following steps of (1) preparing an experiment, firstly preprocessing a sample obtained by liquid biopsy, such as blood dilution, primary filtration of pleural effusion and the like, selecting a certain amount of biopsy cell sample according to actual requirements, taking an amount of 1-30ml, after packaging and disinfection of the device are completed, firstly injecting a small amount of PBS buffer solution 13 from a liquid inlet 1 through a pipette gun, and rinsing a channel structure 6 and a cell filter membrane 4, so as to reduce the surface tension of a subsequent sample and more easily realize filtration;

(2) Cell enrichment, after the rinse solution flows out of the device, the biopsy cell solution is slowly injected through a pipette gun, so that the overflow of the cell solution is avoided, and the cell solution needs to be slowly injected for multiple times until the sample completely enters the channel structure 6 and enrichment is realized; in order to improve the enrichment efficiency and avoid that some small cells 11 do not flow out of the upper channel structure 6 under high flux, a small amount of PBS buffer solution 13 is continuously injected from the liquid inlet 1 through the pipette gun to wash the channel structure 6 until the washing liquid flows out of the liquid outlet 2, at the moment, the enrichment operation of the cells is finished, and the enriched large cells 3 are positioned in the channel structure 6;

(3) Capturing or recovering cells, wherein the support structure 5 is positioned above the cell capture or recovery device by the turnover device, the channel structure 6 is positioned below the cell capture or recovery device, the microcolumn array 10 in the channel structure 6 captures the large cells 3 under a gravity field, and the PBS buffer solution 13 in the channel between the support structure 5 and the cell filter membrane 4 flows into the channel structure 6 to wash the small cells 11 which are not enriched; horizontally placing the overturning device, removing the supporting structure 5 after the large cells 3 are settled, exposing the cell filter membrane 4, flushing the membrane surface by using a liquid transfer gun, and settling the cells adhered to the lower surface of the cell filter membrane 4 into the channel structure 6, so that the subsequent cell detection and culture can be carried out; if the micro-column array 10 is not arranged in the channel structure 6, the PBS buffer solution 13 in the channel between the supporting structure 5 and the cell filter membrane 4 flows into the channel structure 6, and the liquid in the channel structure 6 is recovered, so that the target cell can be recovered;

(4) And (3) cell culture and observation, namely, flatly paving the sorted and enriched cells in the micro-column array 10, dripping cell culture solution, growing the cells in a proper cell culture environment, and tracking and observing the cells in the array through a microscope.

Example 2:

in this embodiment, referring to fig. 7, the device also includes a three-layer sandwich type enrichment device composed of a cell filtration membrane 4, a channel structure 6 above the membrane and a support structure 5 below the membrane, similar to embodiment 1, except that the cell filtration membrane 4 can realize multi-layer fixation; fig. 7 shows that two layers of cell filtration membranes 4 are fixed, the size of the upper layer filtration membrane micropore array is large, large cells 4 can be retained above the upper layer filtration membrane, medium and small cells 11 flow into a channel above the second layer filtration membrane, the medium cells are enriched again, finally the small cells 11 flow out through the second layer filtration membrane, the channel formed by the support structure 5 below the membrane and the second layer cell filtration membrane 4 realizes washing and cell recovery through the PBS buffer solution 13, and the principle is consistent with that of a single-layer enrichment device.

The operation method of the high throughput cell sorting and enriching device of the embodiment comprises the following steps:

(2) Cell enrichment, after the rinse solution flows out of the device, the biopsy cell solution is slowly injected through a pipette gun to avoid overflow of the cell solution, the cell solution needs to be slowly injected for multiple times until the sample completely enters the channel structure 6 and enrichment is realized, then in order to improve the enrichment efficiency, a few small cells 11 under high flux are prevented from not flowing out of the upper channel structure 6, a small amount of PBS buffer solution 13 is continuously injected from a liquid inlet 1 through the pipette gun to wash the channel structure 6 until washing liquid flows out of a liquid outlet 2, the enrichment operation of the cells is completed at this moment, and the enriched large cells 3 are positioned in the channel structure 6;

(3) The cell is captured or recovered, the support structure 5 is positioned above the turnover device, the channel structure 6 is positioned below the turnover device, the PBS buffer solution 13 in the channel of the support structure 5 flows into the channel structure 6, the enriched cell is washed until the channel of the channel structure 6 and the two layers of cell filter membranes 4 is filled with liquid, and the liquid in the channel of the channel structure 6 and the two layers of cell filter membranes 4 is directly sucked out by a pipette respectively, so that the recovery is realized; if cell capture is needed, the overturned structure is required to be horizontally placed, after the cells are settled, the supporting structure 5 is removed, the cell filter membrane 4 is exposed, the surface of the membrane is washed by a pipette, the cells adhered to the lower surface of the cell filter membrane 4 are settled in the channel structure 6, and subsequent cell examination and culture can be carried out after settlement.

Example 3:

in this embodiment, referring to fig. 8, similarly to embodiment 1, the three-layer sandwich type enrichment device including the cell filter membrane 4, the channel structure 6 and the support structure 5 below the membrane is also included, and the difference is that the cell filter membrane 4 is bent in the curved flow channel, and the non-planar flow channel formed by the cell filter membrane 4 itself causes the disturbance of the fluid in the channel above the membrane, so as to accelerate the process of cell enrichment.

The operation method of the high-throughput cell sorting and enriching device comprises the following steps:

(2) Cell enrichment, namely slowly injecting biopsy cell sap through a pipette gun after the cell sap flows out of the device to avoid overflow of the cell sap, and slowly injecting the cell sap for multiple times until a sample completely enters the channel structure 6 to realize enrichment, and then continuously injecting a small amount of PBS (phosphate buffered saline) buffer solution 13 from the liquid inlet 1 through the pipette gun to wash the channel structure 6 until washing liquid flows out of the liquid outlet 2 in order to improve the enrichment efficiency and prevent small cells 11 under high flux from not flowing out of the upper channel structure 6, wherein the enriched large cells 3 are positioned in the channel structure 6 after the enrichment operation of the cells is finished;

(3) Capturing or recovering cells, wherein the supporting structure 5 is positioned above and the channel structure 6 is positioned below by the overturning device, the PBS buffer solution 13 in the channel of the supporting structure 5 flows into the channel structure 6 to wash the enriched cells until the space between the channel structure 6 and the cell filter membrane 4 is filled with liquid, and the liquid between the channel structure 6 and the cell filter membrane 4 is respectively sucked out by a pipette, so that the recovery is realized; if cell capture is needed, the overturned structure is required to be horizontally placed, the supporting structure 5 is removed after the cells settle into the channel structure 6, the cell filter membrane 4 is exposed, the cells adhered to the lower surface of the cell filter membrane 4 are washed by a pipette at the moment and are settled into the channel structure 6, and subsequent cell examination and culture can be carried out after settlement.

Example 4:

in this embodiment, please refer to fig. 9, which has the same principle as that of embodiment 1, and the structure of the three-layer sandwich type enrichment device includes the cell filtration membrane 4, the channel structure 6 and the support structure 5 below the membrane, except that the fixation of the upper and lower two-layer structures of the filtration membrane is not required by a clamp or other fixation methods in the three-dimensional device, and the cell filtration membrane 4 needs to be cut, folded and heat sealed to form a shape and a size that are matched with the support structure 5 below.

(1) The method comprises the following steps of (1) preparing an experiment, namely, pretreating a sample obtained by liquid biopsy, such as blood dilution, primary filtering of pleural effusion and the like, selecting a certain amount of biopsy cell sample according to actual requirements, taking an amount of 1-30ml, after packaging and disinfection of the device are completed, injecting a small amount of PBS buffer solution 13 from a liquid inlet 1 through a pipette gun, rinsing a channel structure 6 and a cell filter membrane 4, and then flowing out from a liquid outlet 2, so that the surface tension of a subsequent sample is reduced, and the filtering is easier to realize;

(3) Cell recovery, cell recovery need remove access structure 6, makes cell filtration membrane 4 expose, injects PBS buffer solution 13 through the pipette many times and washes the membrane surface until the flush fluid flows out, injects PBS buffer solution 13 at last and washes, needs the pipette to collect the flush fluid fast, places in recovery tube or cultivation, and the large cell 3 of enrichment is collected, carries out subsequent cell inspection and cultivation.

(4) Cell culture and observation, wherein the recovered cell sap is collected in a test tube, and the cells recovered after enrichment are tiled after specific dyeing steps and detected in a micro-column array 10; or dripping cell culture solution, growing the cells in a proper cell culture environment, and tracking and observing the cells in the array through a microscope.

Example 5:

in this embodiment, referring to fig. 10, a two-dimensional planar inclined filter membrane structure is optimized to a three-dimensional conical structure, which has the same principle as that of the embodiment 1, and the structure also includes a cell filter membrane 4 and a channel structure 6, but a support structure 5 below the membrane is replaced by a PBS buffer solution 13 standing in a centrifuge tube 12, that is, the cell filter membrane 4 and the channel structure 6 are directly immersed in the PBS buffer solution 13, separation is achieved by diffusion of cells in liquid, small cells can enter an external solution through a micropore array, and large cells 3 cannot diffuse out, so that separation is achieved; PBS buffer solution 13 in the centrifuge tube 12 can be updated regularly to ensure that the concentration of the extracellular minicells is far lower than that of the minicells 11 in the channel structure 6, so that the diffusion rate is ensured, and the enrichment efficiency is improved.

(1) The method comprises the following steps of (1) preparing an experiment, namely, firstly preprocessing a sample obtained by liquid biopsy, such as blood dilution, primary filtering of pleural effusion and the like, selecting a certain amount of biopsy cell samples according to actual requirements, wherein the amount of the biopsy cell samples can be 1-30ml, and after packaging and disinfection of the device are completed, injecting a PBS (phosphate buffered saline) buffer solution 13 into a centrifuge tube to soak a channel structure 4 and a cell filter membrane 4, so that the surface tension of a subsequent sample is reduced, and the filtering is easier to realize;

(2) Cell enrichment, namely after the rinse solution flows out of the device, slowly injecting biopsy cell fluid through a pipette, opening a waste liquid outlet to avoid overflowing of the cell fluid, and simultaneously opening a buffer solution inlet until all samples enter a channel to realize enrichment; in order to improve the enrichment efficiency, the device filtration can be carried out on a low-speed centrifuge;

(3) Cell recovery, wherein the channel structure 6 needs to be removed to expose the cell filter membrane 4, cells attached to the upper surface of the cell filter membrane 4 are washed by utilizing the osmotic pressure of a PBS buffer solution 13 below the cell filter membrane 4, and meanwhile, a pipette is needed to quickly collect cell sap on the membrane and place the cell sap in a recovery tube or a pore plate;

(4) Cell culture and observation, wherein the recovered cell sap is collected in a test tube, and the cells recovered after enrichment are subjected to a specific dyeing step and then are tiled and detected in a micro-column array 10; or dripping cell culture solution, growing the cells in a proper cell culture environment, and tracking and observing the cells in the array through a microscope.

Clinical validation example:

the method of the invention realizes the enrichment and detection of tumor cells in malignant pleural effusion of clinical lung cancer patients. The experimental materials and equipment are as follows.

60mL of pleural fluid samples are taken from a patient, 30mL of pleural fluid cell fluid is obtained by firstly pretreating the pleural fluid for centrifugal resuspension, then enrichment and recovery of cells are carried out, and a 10-micron filter membrane is selected as the cell filter membrane 4. After the device is packaged and sterilized, the channel structure 6 is firstly rinsed by 2mL of PBS buffer solution 13 from the liquid inlet, the breast water cell fluid is slowly injected after the rinsing fluid flows out of the device until the sample completely enters the channel structure 6 to realize enrichment, the 2mL of PBS buffer solution 13 is continuously injected to rinse the channel structure 6 until the rinsing fluid flows out of the liquid outlet 2, and the enrichment operation of cells is completed. The channel structure 6 was removed, the membrane surface was washed with 2mL PBS buffer 13, and the filter wash was rapidly collected and placed in a recovery tube.

The original cell sap and the recovered cell droplets were applied to a slide glass, and the cell size distribution was observed. The cell size distribution before and after enrichment and recovery is shown in fig. 12, and it can be seen that only a very small number of large cells can be seen in the original sample, while the small cells 11 in the recovered sample are obviously reduced, most of the large cells are 3, and the cell clusters are enriched, so that the enrichment effect is good;

2-NBDG can be used as a glucose transport probe to quickly detect suspected tumor cells with high metabolism, referring to the flow in FIG. 11, a cell detection platform is utilized to carry out starvation treatment on recovered cells for 10min on samples before and after enrichment, 2-NBDG solution is dripped for dyeing, the treated cells are coated above a micro-column array 10 until the micro-column array 10 is completely filled, a detection module is placed under a microscope for observation, fluorescent cells are counted, suspected tumor cells are picked out for subsequent single cell sequencing and other operations, and the cells are further analyzed; as shown in fig. 13, it can be seen from the comparison of the numbers of the fluorescent cells that the number of the fluorescent cells is significantly increased by the enrichment operation, i.e., the suspected tumor cells in the original sample are separated by the enrichment operation, and other interfering cells such as white blood cells, red blood cells, etc. are effectively removed; in addition, the partially enriched pleural effusion sample is placed in a cell well plate for culture, as shown in fig. 14, the pleural effusion cells cultured for 7 days after enrichment have good survival state and can grow and proliferate adherently, and the influence of the method on the cell activity is verified to be low. Therefore, the method is easy to operate and is suitable for separating and enriching cells in the pleural fluid sample.

The above embodiments can be combined to fully demonstrate the using approach and the beneficial effect of the present invention, and in addition, it should be understood that the above is only the preferred embodiment of the present invention, and although the present specification is described by the embodiments, not every embodiment includes only one independent technical solution, and this description of the specification is only for the sake of clarity, and those skilled in the art should make the specification as a whole, and the technical solutions in the embodiments can also be combined appropriately to form other embodiments that can be understood by those skilled in the art.

Claims

1. A high throughput cell sorting enrichment apparatus, comprising: the cell filtration membrane structure comprises a cell filtration membrane (4), a support structure (5) and a channel structure (6), wherein the support structure (5) is matched with the channel structure (6) in shape and jointly embeds the cell filtration membrane (4) between the two, the cell filtration membrane (4) is a two-dimensional plane inclined structure or a three-dimensional cone structure and is a nuclear pore membrane made of a polymer material, the polymer material comprises a polycarbonate material sieve membrane, a poly (terephthalic acid) material sieve membrane, a silicon-based sieve membrane, a polydimethylsiloxane material sieve membrane or a C-type poly (p-xylylene) filtration membrane, the cell filtration membrane (4) is fixed on the channel structure (6), a microfluidic channel (8) is formed between the cell filtration membrane (4) and the channel structure (6), two ends of the microfluidic channel (8) are respectively a liquid inlet (1) and a liquid outlet (2), and the shape of the microfluidic channel (8) is at least one of the following shapes: spiral shape, fishbone structure, array structure.

2. The high throughput cell sorting enrichment device according to claim 1, wherein the support structure (5) comprises a centrifuge tube (12), the centrifuge tube (12) is provided with a PBS buffer (13), and the cell filter membrane (4) and the channel structure (6) are soaked in the PBS buffer (13).

3. The high throughput cell sorting enrichment device of claim 1 or 2, wherein the channel structure (6) comprises a substrate (9) and a cell detection capture array disposed on the surface of the channel structure (6).

4. The high throughput cell sorting enrichment device of claim 3, wherein the cell detection capture array comprises a microwell array (7) or a micropillar array (10).

5. The high throughput cell sorting enrichment device of claim 4, wherein the depth of the micro well array (7) or the height of the micro column array (10) is 5 μm to 5000 μm.

6. The high-throughput cell sorting and enriching device according to claim 1 or 2, wherein the cell filtration membrane (4) is sealed on the channel structure (6) by cutting, folding and heat sealing, and is matched with the shape of the support structure (5).

7. A method for using a high-throughput cell sorting and enriching device, wherein the high-throughput cell sorting and enriching device comprises the high-throughput cell sorting and enriching device as claimed in any one of claims 1 to 6, and the method comprises the following steps:

rinsing the cell filter (4) with a PBS cell buffer (13) through the channel structure (6) to leave the cell filter (4) in an infiltrated state;

-injecting a biopsy sample into the channel structure (6);

washing the cell filter membrane (4) and the channel structure (6) with the PBS cell buffer (13) to recover cells enriched on the cell filter membrane (4);

horizontally placing the high-throughput cell sorting and enriching device, and removing the cell filter membrane (4) and the support structure (5);

staining the enriched and recovered target cells, and then paving the target cells on the channel structure (6) for detection;

adding cell culture fluid to the channel structure (6) to continue culture observation.