Microfluidic cell sorting device, tumor stem cell sorting method and anticancer drug screening method

Abstract

The present invention relates to a microfluidic cell sorting device, a method for sorting tumor stem cells and a method for screening anticancer drugs, wherein the microfluidic cell sorting device comprises a first sorting chip and a second sorting chip; the first sorting chip is provided with channels of different sizes for sorting cells, the first sorting chip is provided with a first inlet and a first outlet, the first inlet is used for injecting samples; the second sorting chip is provided with a second inlet and a second outlet, the second inlet is connected to the first outlet, so that the sample flowing out of the first outlet enters the second sorting chip for sorting, and the inner surface of the second sorting chip is covered with a matrix glue layer. The microfluidic cell sorting device of the present invention is sorted based on cell flexible deformation and cell adhesion, and can accurately and efficiently sort out a tumor stem cell group with high deformability, low adhesion and good cell activity, providing a guarantee for downstream experiments, and having important significance for the treatment and prognosis of cancer for the later drug efficacy verification.

Description

Technical Field

[0001] The present invention relates to the technical field of cell biology, and in particular to a microfluidic cell sorting device, a tumor stem cell sorting method and an anticancer drug screening method. Background Art

[0002] Cancer stem cells (CSCs) are considered to be the key factor in tumor resistance and recurrence. It is of great significance to develop effective methods for isolating and identifying CSCs. Existing methods for isolating CSCs can be divided into cell surface marker sorting and cell surface marker-independent sorting. Cell surface marker sorting is to separate CSCs with the help of known special cell surface markers, using immunomagnetic beads (Magnetic activated cell sorting, MACS) or fluorescent "labels" (Fluorescence-activated cell sorting, FACS) that can bind to surface markers. This method has high cell purity, but it affects the biological characteristics of the cells themselves and is not conducive to subsequent experiments. In addition, the cell surface marker sorting method is not only based on expensive equipment support and relatively complex operations, but also in many types of cancer, the specific surface markers of CSCs are still unclear, which limits the scope of application of the cell surface marker sorting method. The main methods that do not rely on cell surface marker sorting include side population analysis (SP), serum-free culture and drug resistance screening. Although they make full use of the characteristics of CSCs for separation, they have certain limitations due to their low throughput and low purity.

[0003] Microfluidic chips have the advantages of low cost, short analysis time, high sensitivity, and strong operability. Researchers have organically combined the biological characteristics of cells with fluid mechanics operations to develop a cell separation method based on microfluidic chips. However, general microfluidic chips have low accuracy and poor sorting efficiency when used for tumor stem cell sorting. Summary of the invention

[0004] Based on this, it is necessary to provide a microfluidic chip with high accuracy for sorting tumor stem cells.

[0005] A microfluidic cell sorting device comprises a first sorting chip and a second sorting chip; the first sorting chip is provided with channels of different sizes for sorting cells, the first sorting chip is provided with a first inlet and a first outlet, the first inlet is used for injecting samples; the second sorting chip is provided with a second inlet and a second outlet, the second inlet is connected to the first outlet, so that the sample flowing out of the first outlet enters the second sorting chip for sorting, the inner surface of the second sorting chip is covered with a matrix glue layer, and the second outlet is used for the sample after sorting from the second sorting chip to flow out.

[0006] In one embodiment, the matrix gel layer is formed by solidifying 1 mg / mL to 5 mg / mL of matrix gel.

[0007] In one embodiment, a capture column is provided in the second sorting chip, and a plurality of first protrusions are provided on the side wall of the capture column.

[0008] In one embodiment, a flow mixing structure is provided in the second sorting chip, the flow mixing structure extends from the second inlet to the second outlet, and a side wall of the flow mixing structure is provided with a plurality of second protrusions from the second inlet to the second outlet.

[0009] In one embodiment, there are multiple flow mixing structures, and there are multiple capture columns. The multiple flow mixing structures are arranged relative to each other at intervals, and the multiple capture columns are distributed between two adjacent flow mixing structures.

[0010] In one embodiment, a plurality of interception columns are provided in the first sorting chip, and every two or more interception columns are arranged in a row to form an interception unit, and the plurality of interception units are arranged in sequence and spaced apart from each other along the direction from the first inlet to the first outlet; the gap between two adjacent interception columns in each interception unit forms a channel for sorting cells, and the gap gradually decreases along the direction from the first inlet to the first outlet.

[0011] In one of the embodiments, the gap between two adjacent interception columns in the interception unit is 2 μm to 30 μm.

[0012] The present invention also provides a method for sorting tumor stem cells, comprising the following steps: performing a first sorting on the sample using a first sorting chip provided with channels of different sizes for sorting cells, and then performing a second sorting on the cells obtained by the first sorting using a second sorting chip having an inner surface covered with a matrix gel layer, to obtain the tumor stem cells.

[0013] In one embodiment, the flow rate in the first sorting chip is controlled to be 45 μL / min to 75 μL / min, and the flow rate in the second sorting chip is controlled to be 0.1 mL / h to 0.7 mL / h.

[0014] The present invention also provides a method for screening anticancer drugs, comprising the following steps:

[0015] The cancer cell samples treated with the drug to be tested are sorted using a first sorting chip and a second sorting chip in sequence, wherein the first sorting chip is provided with channels of different sizes for sorting cells, and is used to sort cells according to the softness and hardness of the cells, and the inner surface of the second sorting chip is covered with a matrix glue layer, and is used to sort cells according to the cell adhesion ability;

[0016] Count the number of cells obtained by sorting or the distribution of cells in the first sorting chip and the second sorting chip.

[0017] When the microfluidic cell sorting device of the present invention performs cell sorting, the cell suspension sample can be injected from the first inlet, and the channels of different sizes of the first sorting chip can be used to sort out the cells with high flexibility and easy deformation from the cell group passed in, and the cells that are not easy to deform are intercepted by the first sorting chip and remain in the chip. The cells passing through the first sorting chip reach the second inlet through the first outlet and enter the second sorting chip. The inner surface of the chamber of the second sorting chip is covered with a matrix glue layer, which can simulate cell-matrix adhesion and the natural cell microenvironment in the body, so that cells with high adhesion characteristics can be captured, and finally a stem cell group with high flexibility, easy deformation and low adhesion characteristics can be obtained from the second outlet. In the occurrence and metastasis of tumors, cells with high deformability and low adhesion tend to metastasize to distant places and show a higher degree of malignancy, and have tumor stem cell characteristics. The microfluidic cell sorting device of the present invention performs sorting based on flexible deformation and cell adhesion, and can accurately and efficiently sort out tumor stem cell groups that have high deformability, low adhesion and good cell activity, providing guarantees for downstream experiments (drug screening, discovery of new targets for tumor stem cells), and is of great significance for the subsequent efficacy verification of cancer treatment and prognosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 A schematic diagram of the structure of a microfluidic cell sorting device according to an embodiment of the present invention;

[0019] Figure 2 The proportion of collected cells (a) and the expression level of stem cell-related proteins (b) of the MS-Chip at different flow rates in Example 1;

[0020] Figure 3 This is a fluorescence microscopic image of the cells in MS-Chip in Example 1;

[0021] Figure 4 The proportion of collected cells (a) and the expression level of stem cell-related proteins (b) of the HCA-Chip at different flow rates in Example 1;

[0022] Figure 5 This is a fluorescence microscopic image of the cells in the HCA-Chip in Example 1;

[0023] Figure 6 The number of cells perfused in Example 1 is 1×10 4 The number of cells collected after passing through MS-Chip, HCA-Chip and MS-HCA-Chip respectively;

[0024] Figure 7 The results of the CFSE / PI cell viability experiment in Example 2;

[0025] Figure 8 The results of the trypan blue staining experiment in Example 2, (a) is a micrograph, and (b) is the statistical data of cell viability;

[0026] Fig. 9 The results of the CCK-8 experiment in Example 2;

[0027] Fig.10 The results of the Transwell in vitro migration experiment in Example 3, (a) is a micrograph, and (b) is the statistical data of cell migration;

[0028] Fig.11 The results of the wound healing experiment in Example 3;

[0029] Fig.12 The results of Western Blot in Example 3;

[0030] Fig.13 are micrographs of cells before and after sorting in the logarithmic growth phase in Example 3;

[0031] Fig.14 The results of the plate cloning experiment in Example 4, (a) is a plate photo, (b) is the statistical data of the number of clones;

[0032] Fig.15 The results of the cell counting experiment in Example 4;

[0033] Fig.16 The results of the spherical balloon formation experiment in Example 4, (a) is a microscopic photo, and (b) is the statistical data of the number of spherical balloons;

[0034] Fig.17 is the result of flow cytometry in Example 4;

[0035] Fig.18 The results of Western Blot in Example 4;

[0036] Fig.19 The results of the cell resistance experiment in Example 4;

[0037] Fig. 20 The results of the nude mouse tumor formation experiment in Example 4;

[0038] Fig.21 The results of the CCK-8 experiment in screening the active ingredients of Licorice in Example 5;

[0039] Fig. 22 The results of the CCK-8 experiment for detecting the IC50 of isoliquiritigenin in Example 5;

[0040] Fig.23 The results of the plate cloning experiment in Example 5, (a) is a plate photo, (b) is the statistical data of the number of clones;

[0041] Fig.24 The results of Western Blot in Example 5;

[0042] Fig.25 The results of the spherical balloon formation experiment in Example 5, (a) is a microscopic photo, and (b) is the statistical data of the number of spherical balloons;

[0043] Fig.26 The results of the Transwell in vitro migration experiment in Example 5, (a) is a micrograph, and (b) is the statistical data of cell migration;

[0044] Fig. 27 The results of cell cycle detection in Example 5;

[0045] Fig.28 The results of the in vivo experiment in Example 5, (a) is a tumor photo, (b) is the tumor weight data, (c) is the mouse body weight change data, and (d) is the change data of the stemness protein level in the tumor. DETAILED DESCRIPTION

[0046] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below.

[0047] In the description of the present invention, it should be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", "clockwise", "counterclockwise", "axial", "radial", "circumferential" and the like indicate positions or positional relationships based on the positions or positional relationships shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first" and "second" are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of "multiple" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined. In the present invention, unless otherwise clearly defined and limited, the terms "installed", "connected", "connected", "fixed" and the like should be understood in a broad sense, for example, it can be fixed connection, detachable connection, or integrated; it can be mechanical connection or electrical connection; it can be directly connected or indirectly connected through an intermediate medium, it can be the internal connection of two elements or the interaction relationship between two elements, unless otherwise clearly defined. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances. In the present invention, unless otherwise clearly defined and limited, the first feature "on" or "below" the second feature can be that the first and second features are in direct contact, or the first and second features are in indirect contact through an intermediate medium. Moreover, the first feature "above", "above" and "above" the second feature can mean that the first feature is directly above or obliquely above the second feature, or simply means that the first feature is higher in level than the second feature. The first feature "below", "below" and "below" the second feature can mean that the first feature is directly below or obliquely below the second feature, or simply means that the first feature is lower in level than the second feature. It should be noted that when an element is referred to as "fixed on" or "set on" another element, it can be directly on the other element or there can be an element in the middle. When an element is considered to be "connected" to another element, it may be directly connected to another element or there may be a central element at the same time. The terms "vertical", "horizontal", "upper", "lower", "left", "right" and similar expressions used herein are for illustrative purposes only and are not intended to be the only implementation method.

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0049] like Figure 1 As shown, a microfluidic cell sorting device 100 according to an embodiment of the present invention includes a first sorting chip 10 and a second sorting chip 20 .

[0050] The first sorting chip 10 is provided with channels of different sizes for sorting cells, and is used to sort cells in the sample according to the softness and hardness of the cells. Cells that are not easily deformed (harder) will be intercepted by the first sorting chip 10. The first sorting chip 10 is provided with a first inlet 11 and a first outlet 12, and the first inlet 11 is used for injecting samples. The second sorting chip 20 is provided with a second inlet 21 and a second outlet 22, and the second inlet 21 is connected to the first outlet 12 so that the sample flowing out of the first outlet 12 enters the second sorting chip 20 for sorting. The inner surface of the second sorting chip 20 is covered with a matrix glue layer, and the second outlet 22 is used for the sample to flow out after sorting from the second sorting chip 20.

[0051] When the microfluidic cell sorting device 100 (MS-HCA-Chip, mechanical separation-high-throughput cell adhesion chip) of the present invention performs cell sorting, the cell suspension sample can be injected from the first inlet 11, and the first sorting chip 10 can be used to sort out the cells with high flexibility and easy deformation from the cell group passed through the channels of different sizes, and the hard cells that are not easy to deform are intercepted by the first sorting chip 10 and remain in the chip. The cells passing through the first sorting chip 10 reach the second inlet 21 through the first outlet 12 and enter the second sorting chip 20. The inner surface of the chamber of the second sorting chip 20 is covered with a matrix glue layer, which can simulate cell-matrix adhesion and the natural cell microenvironment in the body, so that cells with high adhesion characteristics can be captured, and finally a stem cell group with high flexibility, easy deformation and low adhesion characteristics can be obtained from the second outlet 22. In the occurrence and metastasis of tumors, cells with high deformability and low adhesion tend to metastasize to distant places and show a higher degree of malignancy, and have tumor stem cell characteristics. The microfluidic cell sorting device 100 of the present invention performs sorting based on cell flexible deformation and cell adhesion, and can accurately and efficiently sort out tumor stem cell groups that have high deformability, low adhesion and good cell activity, providing guarantees for downstream experiments (drug screening, discovery of new targets for tumor stem cells), and is of great significance for the subsequent drug efficacy verification for cancer treatment and prognosis.

[0052] Understandably, Figure 1 It is only a schematic diagram, and the size, proportion and quantity of each structural feature have been adjusted and simplified for the convenience of explanation, which should not be understood as a limitation to the present invention.

[0053] In a specific example, the above-mentioned matrix gel layer is formed by solidifying 1 mg / mL to 5 mg / mL of matrix gel, preferably 3 mg / mL of matrix gel, which has better biological activity. The thawed matrix gel can be injected into the pre-cooled chip through a hose, and then solidified at room temperature for 30 minutes to form a matrix gel layer.

[0054] In a specific example, a capture column 23 is provided in the second sorting chip 20, and a plurality of first protrusions are provided on the sidewall of the capture column 23. The capture column 23 can cause a certain disturbance to the fluid, more realistically simulating the in vivo environment, thereby improving the accuracy of separation and improving the capture performance of highly adherent cells. Preferably, the cross-section of the capture column 23 is in the shape of a triangular propeller, but is not limited thereto.

[0055] In a specific example, a mixing flow structure 24 is provided in the second sorting chip 20, and the mixing flow structure 24 extends from the second inlet 21 to the second outlet 22, and a plurality of second protrusions are provided on the sidewall of the mixing flow structure 24 from the second inlet 21 to the second outlet 22. In this way, the mixing flow is increased by the mixing flow structure 24, so that the contact probability between the cell and the capture column 23 can be increased, and the capture performance of the highly adherent cells can be further improved. Preferably, the cross section of the mixing flow structure 24 is fishbone-shaped, and the length of the mixing flow structure 24 extending is 0.8 mm to 1.2 mm, and the width is 30 μm to 50 μm.

[0056] In a specific example, there are multiple flow mixing structures 24 and multiple capture columns 23 . The multiple flow mixing structures 24 are arranged relative to each other at intervals, and the multiple capture columns 23 are distributed between two adjacent flow mixing structures 24 .

[0057] In a specific example, a plurality of interception columns 13 are provided in the first sorting chip 10, and every two or more interception columns 13 are arranged in a row to form an interception unit, and the plurality of interception units are arranged in sequence along the direction from the first inlet 11 to the first outlet 12; the gap between two adjacent interception columns 13 in each interception unit forms a channel for sorting cells, and the gap gradually decreases along the direction from the first inlet 11 to the first outlet 12. In this way, when the cell suspension flows through the first sorting chip 10, cells that are not easily deformed are gradually blocked by the interception columns 13 according to the hardness of the cells, and relatively soft and easily deformed cells reach the first outlet 12, and cells stuck in the chip can also be recoiled to achieve the reuse of the chip. Optionally, the cross-section of the interception column 13 is circular, but not limited to this, for example, it can also be rectangular, elliptical, etc. It can be understood that the size of the channel for sorting cells in each interception unit can be different, and there can also be a situation where the size of the channel for sorting cells in multiple interception units is the same, as long as a trend of gradually decreasing channels is formed along the direction from the first inlet 11 to the first outlet 12.

[0058] Optionally, the interval between two mixing flow structures 24 is 200 μm to 300 μm, such as 240 μm. Optionally, the interval between two adjacent interception columns 13 in the interception unit is 2 μm to 30 μm, such as 5 μm, 7 μm, 10 μm, 12 μm, 15 μm, 20 μm, 25 μm, etc.

[0059] In a specific example, the first sorting chip 10 includes a plurality of sorting chambers, and the plurality of sorting chambers are respectively connected to the first inlet 11, and each sorting chamber is provided with the above-mentioned interception column 13. In a specific example, the second sorting chip 20 includes a plurality of sorting chambers, and the plurality of sorting chambers are respectively connected to the second inlet 21, and each sorting chamber is provided with the above-mentioned capture column 23 and mixed flow structure 24. In this way, sorting is performed simultaneously by multiple sorting chambers, and the flux can be increased while preventing blockage. It can be understood that the above-mentioned interception column 13, capture column 23, mixed flow structure 24, matrix glue layer, etc. are all arranged on the inner surface of the chip sorting chamber.

[0060] In a specific example, the sorting chamber of the first sorting chip 10 has a length of about 4 cm to 6 cm, a width of about 1.6 mm to 2.0 mm, and a height of 15 μm to 25 μm. An array formed by interception columns 13 is distributed throughout each sorting chamber, and the interval between two adjacent interception columns 13 in each column is 2 μm to 30 μm, and gradually decreases.

[0061] In a specific example, the sorting chamber of the second sorting chip 20 has a length of about 4.5 cm to 5.5 cm, a width of about 1 mm to 2 mm, and a height of 15 μm to 25 μm, and each sorting chamber is filled with mixed flow structures 24 and capture columns 23 located between the mixed flow structures 24. It can be understood that when used for sorting different tumor cells, the structural dimensions can be adjusted as needed to improve the sorting accuracy.

[0062] Optionally, the first sorting chip 10 and the second sorting chip 20 in the above-mentioned microfluidic cell sorting device 100 are prepared by the following method: spin-coat a negative photoresist such as SU-8 3025 on a clean silicon wafer, and after it solidifies, expose a chrome plate mask printed with a designed pattern on a photolithography machine, clean the silicon wafer to remove excess photoresist, and after it solidifies, pour PDMS (Polydimethylsiloxane), peel off the pattern after high-temperature curing and punch holes to inject liquid, clean the surface and bond it to a glass slide, and evacuate it before use.

[0063] A method for sorting tumor stem cells according to an embodiment of the present invention comprises the following steps: performing a first sorting on a cell sample using a first sorting chip having channels of different sizes for sorting cells, and then performing a second sorting on the cells obtained by the first sorting using a second sorting chip having an inner surface covered with a matrix gel layer, to obtain tumor stem cells.

[0064] In a specific example, the flow rate of the sample in the first sorting chip is 45 μL / min to 75 μL / min, and the flow rate of the sample in the second sorting chip is 0.1 mL / h to 0.7 mL / h. Preferably, when the sample is a lung cancer cell line such as A549, the flow rate of the sample in the first sorting chip is 58 μL / min to 65 μL / min, and most preferably 60 μL / min, and the flow rate of the sample in the second sorting chip is 0.2 mL / h to 0.4 mL / h, and most preferably 0.3 mL / h. The sorting effect is significantly improved, indicating that the flow rate has a certain effect on cell sorting. Within a certain range, cells can pass through the chip through their own characteristics, and when the flow rate breaks through a certain limit, the fluid affects the cells. It can be understood that the flow rate can be adjusted according to the characteristics of different cells, so that the cells are separated by their own characteristics rather than the external flow rate, and it is appropriate to select the maximum flow rate that does not affect the separation accuracy.

[0065] For example, before use, vacuum and sterilize under ultraviolet light, and wet the channel with PBS containing 0.1% bovine serum albumin (BSA) to prevent nonspecific binding. Then, pre-cooled 3 mg / mL matrix glue is passed into the second sorting chip through a polyethylene hose, and after being placed at room temperature for 30 minutes, it is gently rinsed with serum-free medium. Use a syringe to draw the cell suspension back into the hose, and then fix it to the injection pump. Adjust the injection pump to push the cell suspension into the first sorting chip at a rate of 60 μL / min, and soft and deformable cells pass through the chip while cells with poor deformability are stuck in the chip. After that, the cells separated in the first step are enriched and passed into the second sorting chip coated with matrix glue at a flow rate of 0.3 mL / h, and cells with weak adhesion pass through the chip. After two sortings, the cells are cells with tumor stem cell phenotypes.

[0066] A method for screening anticancer drugs according to an embodiment of the present invention comprises the following steps:

[0067] The cancer cell samples treated with the drug to be tested are sequentially sorted using a first sorting chip and a second sorting chip, wherein the first sorting chip is provided with channels of different sizes for sorting cells, and is used to sort cells according to the softness and hardness of the cells, and the inner surface of the second sorting chip is covered with a matrix glue layer, and is used to sort cells according to the cell adhesion ability;

[0068] The number of cells obtained by sorting or the distribution of cells in the first sorting chip and the second sorting chip are counted.

[0069] In this way, the anti-cancer activity of the drug to be tested can be judged according to the number of cells. For example, the fewer the number of cells, the stronger the anti-cancer ability of the drug to be tested. Or in the first sorting chip, the fewer the number of cells distributed downstream and the fewer the cells accumulated at the outlet, the stronger the effect of the drug to be tested on the mechanical properties of the cells and the stronger the inhibitory effect on tumor stem cells; in the second sorting chip, the proportion of cells adhering to the chip is proportional to the inhibitory effect of the drug.

[0070] Optionally, the screening method may further include the following steps: using the first sorting chip and the second sorting chip to sort the cancer cell samples that have not been treated with the drug to be tested in sequence. In this way, the cancer cell samples that have not been treated with the drug to be tested are used as a control group, which can better reflect the anti-cancer ability of the drug to be tested.

[0071] For example, the cells sorted by MS-HCA-Chip can be exposed to the IC50 (half maximal inhibitory concentration) of each candidate drug for the same time, and the cells are collected and passed into the chip in the same number. Evaluation: 1) The number of cells collected after passing through the MS-HCA-Chip. The collected cells are cells with a tumor stem cell phenotype. Compared with the control group (PBS treatment), the lower the collection ratio, the stronger the inhibitory effect of the drug on the cells. 2) The distribution of cells in the chip system. In the first sorting chip, the fewer the number of downstream cells and the fewer cells accumulated at the outlet, the stronger the effect of the drug on the mechanical properties of the cells and the stronger the inhibitory effect on tumor stem cells. In the second sorting chip, the proportion of cells adhering to the chip is proportional to the inhibitory effect of the drug. Combining the two chips, the biophysical properties of the cells are comprehensively evaluated to evaluate the collection ratio and distribution of cells after the drug action, reflecting the inhibitory effect of the components on the cells, screening drugs for targeting tumor stem cells, and finally screening the active ingredient of licorice, isoliquiritigenin (ISL), for subsequent experiments.

[0072] The present invention utilizes multidisciplinary cross-disciplinary knowledge to construct a microfluidic cell sorting device that fully considers cell heterogeneity for the sorting of tumor stem cells, and uses the sorted cells to achieve high-throughput targeted tumor stem cell drug screening. Due to its high sensitivity, it is particularly suitable for the efficacy evaluation of trace and difficult-to-extract components, combining cell sorting and drug screening to achieve integration, convenience and efficiency.

[0073] The present invention will be further described in detail below mainly in conjunction with specific embodiments and drawings.

[0074] Example 1: Exploring the ratio of separation flow rate to enriched cells

[0075] 1. Separation flow rate of the first mechanical separation chip (MS-Chip)

[0076] (1) Seal the MS-Chip with liquid before use.

[0077] (2) Human lung cancer A549 cells were counted after normal digestion and the cell number was adjusted to 1×10 4 / mL, take 1mL of cells into a 1.5mL centrifuge tube, pipette to make a single-cell suspension, and draw the cell suspension into the hose connected to the syringe.

[0078] (3) The syringe connected to the hose is fixed to the syringe pump. The other end of the hose is connected to the inlet of the MS-Chip with a metal head for introducing liquid into the chip. The outlet of the chip is used to collect cells sorted by the MS-Chip.

[0079] (4) Adjust the syringe pump to the "infusion" mode, adjust the flow rate to 45 μL / min, and start perfusing the cell suspension.

[0080] (5) After all the cell suspension has passed through the MS-Chip, collect the cells at the collection port and count them.

[0081] (6) The number of cells collected at the collection port was compared with the number of cells injected to obtain the ratio of collected cells when the flow rate was 45 μL / min in the MS-Chip.

[0082] (7) The above experiment was repeated with the flow rate adjusted to 50, 55, 60, 65, 70, and 75 μL / min in sequence. The results were repeated three times at each flow rate.

[0083] (8) Arrange the results and make a graph, such as Figure 2 The figure shows the relationship between the proportion of collected cells and the flow rate in the MS-Chip. It can be seen that the proportion of collected cells gradually increases when the separation flow rate in the MS-Chip increases; when the separation flow rate is 60 μL / min, the stemness of the tumor cells sorted is the strongest.

[0084] A549 single cell suspension labeled with GFP green fluorescence was selected, and the cells were perfused from the inlet to the MS-Chip at a flow rate of 60 μL / min. The image of the cells in the chip was taken with a fluorescence microscope. Figure 3 shown.

[0085] 2. Separation flow rate of the second sorting chip (high-throughput cell adhesion chip, HCA-Chip)

[0086] (1) Before use, the HCA-Chip was sealed with liquid and matrix gel was perfused into the chip to simulate the in vivo microenvironment.

[0087] (2) Human lung cancer A549 cells were counted after normal digestion and the cell number was adjusted to 1×10 4 / mL, take 1mL of cells into a 1.5mL centrifuge tube, and pipette to make a single cell suspension. Pipette the cell suspension into the hose connected to the syringe.

[0088] (3) The syringe connected to the hose is fixed to the syringe pump. The other end of the hose is connected to the inlet of the HCA-Chip with a metal head for introducing liquid into the chip. The outlet of the chip is used to collect cells sorted by the HCA-Chip.

[0089] (4) Adjust the syringe pump to the "infusion" mode, adjust the flow rate to 0.1 mL / h, and start perfusing the cell suspension.

[0090] (5) After all the cell suspension has passed through the HCA-Chip, collect the cells at the collection port and count them.

[0091] (6) Compare the number of cells collected at the collection port with the number of cells injected to obtain the ratio of collected cells when the flow rate is 0.1 mL / h in the HCA-Chip.

[0092] (7) The above experiment was repeated with the flow rate adjusted to 0.2, 0.3, 0.4, 0.5, 0.6, and 0.7 mL / h in sequence. The results were repeated three times at each flow rate.

[0093] (8) Arrange the results and make a graph, such as Figure 4 The figure shows the relationship between the proportion of collected cells and the flow rate in the HCA-Chip. It can be seen that the proportion of collected cells gradually increases when the separation flow rate in the HCA-Chip increases; when the separation flow rate is 0.3 mL / h, the stemness of the tumor cells sorted is the strongest.

[0094] A GFP green fluorescent labeled A549 single cell suspension was selected and the cells were perfused from the inlet to the HCA-Chip at a flow rate of 0.3 mL / h. The images of the cells in the chip were taken using a fluorescence microscope. Figure 5 As shown, the green fluorescence in the figure represents cells adhered to the chip.

[0095] When the number of perfused cells is 1×10 4 The number of cells collected after MS-Chip, HCA-Chip and MS-HCA-Chip was repeated three times. Figure 6 shown.

[0096] The above experiments show that MS-HCA-Chip can be used to separate cells, and the separation flow rate in MS-Chip is preferably 60 μL / min, and the separation flow rate in HCA-Chip is preferably 0.3 mL / h.

[0097] Example 2 Effect of MS-HCA-Chip on Cell Viability

[0098] Through the CFSE / PI cell viability experiment (live cells are green fluorescence, dead cells are red fluorescence), trypan blue staining experiment, and CCK8 cell activity experiment, it was proved that the chip sorting had basically no effect on cell viability and the cells could be used for subsequent experiments.

[0099] 1.CFSE / PI cell viability assay

[0100] (1) A549 cells were resuspended into a single cell suspension and then passed into the MS-Chip using a hose.

[0101] (2) The cells were passed through the chip at a flow rate of 60 μL / min and collected at the collection port.

[0102] (3) The cells collected from the collection port and the cells injected into the chip were stained with the live cell stain CFSE (calcein green Invitrogen C34852) and the dead cell stain PI (propidium iodide, Sigma).

[0103] (4) The stained cells were photographed under a fluorescence microscope. Green fluorescence indicates live cells, and red fluorescence indicates dead cells, reflecting the effect of the chip on cell viability after passing through the MS-Chip.

[0104] (5) Similarly, the cells were passed through the HCA-Chip and MS-HCA-Chip at the optimal flow rate, and the collected cells and injected cells (sort in) were stained. The experiment was repeated three times, and the results are shown in Figure 7 shown.

[0105] 2. Trypan blue staining experiment

[0106] (1) The collected cells and the cells injected into the chip were stained with 0.4% trypan blue stain (Solabol C0040).

[0107] (2) Use a 10 μL pipette to inject the stained cells into a blood cell counting chamber and then take photos under a microscope.

[0108] (3) According to the principle of trypan blue stain, dead cells are stained blue while live cells are not stained.

[0109] (4) The ratio of the number of unstained cells to the number of cells injected into the blood cell counting chamber was counted as the cell viability after passing through the MS-Chip, HCA-Chip, and MS-HCA-Chip, respectively. The experiment was repeated three times, and the results were as follows: Figure 8 shown.

[0110] 3.CCK-8 Experiment

[0111] (1) Resuspend the cells before sorting and after sorting using MS-Chip, HCA-Chip, and MS-HCA-Chip to 4×10 4 / ml single cell suspension was seeded in a 96-well plate at a density of 100 μL / well.

[0112] (2) After 48 hours of culture, CCK8 cell activity was measured (10 μL of CCK8 was added to the wells and incubated in the dark for 40 min, and the absorbance was measured at 450 nm on a microplate reader). The experiment was repeated three times, and the results were as follows: Fig. 9 shown.

[0113] Example 3 Detection of migration characteristics of cells after sorting

[0114] Through Transwell experiments, scratch experiments and Western blot detection of migration-related proteins (E-cadherin, N-cadherin, Vimentin) and other experiments, the migration characteristics of cells sorted by MS-HCA-Chip and cells sorted only by MS-Chip or HCA-Chip were compared, indicating that the cell migration ability was enhanced after combined chip sorting, indicating the feasibility of chip sorting and the effectiveness of combined use.

[0115] 1. Transwell in vitro migration assay

[0116] (1) The cells before and after serum removal were sorted at 2×10 4 The number of cells per well was inoculated on the upper layer of the Transwell chamber, and the culture medium containing 10% serum was added to the lower layer.

[0117] (2) After 24 hours, use a cotton swab to gently wipe off the cells that have not passed through the upper chamber, place them in 4% paraformaldehyde for fixation for 15 minutes, and then stain with crystal violet.

[0118] (3) Take photos of the cells that have passed through under a microscope and count them. The experiment was repeated three times. The results are as follows: Fig.10 As shown, the migratory properties of the sorted cells were increased.

[0119] 2. Wound healing assay

[0120] (1) Inoculate the cells before and after sorting in a six-well plate, and then scratch the plate when the cells have grown to 60% of the well.

[0121] (2) Use a 200 μL pipette tip to streak the wells of a six-well plate, then wash the cell debris with PBS, take pictures under a microscope, and replace the wells with serum-free medium to continue culturing for 24 hours.

[0122] (3) Take a photo at the same location as the photo taken at 0h and record the status 24 hours later.

[0123] (4) Data processing and comparison of scratch widths. When the initial width is the same, the stronger the cell migration, the narrower the scratch width after 24 hours. The results are as follows: Fig.11 As shown, it was demonstrated that the migration properties of cells increased after sorting.

[0124] 3. Western Blot

[0125] (1) Collect cells before and after sorting and perform protein extraction and quantification.

[0126] (2) Experimental operation. Electrophoresis: After protein loading, perform electrophoresis at 120V. Transfer: Transfer the protein to PVDF membrane and transfer at 300mA for 60min. Blocking: Block with skimmed milk powder to remove non-specific binding. Primary antibody: Add antibodies representing cell migration characteristics such as E-cadherin, N-cadherin, Vimentin, and internal reference antibodies. Incubate at 4°C overnight. Secondary antibody: After washing the PVDF membrane with TBST, incubate with secondary antibody for 2 hours. Washing and exposure: Wash the unbound secondary antibody with TBST, and expose the bands with ECL luminescent solution. The results are as follows. Fig.12 As shown, it was demonstrated that the migration properties of cells increased after sorting.

[0127] 4. Cell morphology detection after sorting

[0128] Cells before sorting in the logarithmic growth phase and cells after sorting by MS-HCA-Chip were photographed under a microscope. Fig.13 As shown, it can be seen that the cells after sorting are more in the shape of long strips. After sorting with MS-HCA-Chip, the migration characteristics of cells are increased compared with those before sorting, and compared with sorting with a single chip, the migration characteristics of cells after combined chip sorting are increased.

[0129] Example 4 Detection of high stemness of cells after sorting

[0130] The CSC characteristics of the sorted cells were identified by experiments such as clone formation, in vitro spheroid formation, and chemotherapy drug resistance. Flow cytometry and Western blot were used to detect the expression changes of stem cell-related proteins (CD133, CD44, SOX2). Cell counting and other experiments were used to compare the changes in cell proliferation levels before and after sorting. In the in vivo tumor formation experiment of nude mice, cells before and after the sorting platform with different gradients were subcutaneously inoculated after one week of feeding, and the formation and volume changes of tumors under limiting dilution were observed. When 10 4 When the cells were sorted, the cells could still form tumors after sorting, while the cells before sorting could not, which showed that the stemness of the cells was enhanced after sorting.

[0131] 1. Plate cloning experiment

[0132] (1) The cells before and after sorting were seeded in a six-well plate at a density of 200 cells / well and cultured for 10 days.

[0133] (2) After washing three times with PBS, the cells were fixed with 4% paraformaldehyde for 15 minutes and then stained with crystal violet for 15 minutes.

[0134] (3) Take photos under a microscope and count the number of clones formed to assess the ability of cells to survive independently. Fig.14 As shown, the number of cell clones formed after sorting increased and their stemness was enhanced.

[0135] 2. Cell counting experiment

[0136] (1) The cells before and after sorting were divided into the same number (1×10 5 The cells were inoculated at a density of 1 / well in six-well plates for culture.

[0137] (2) The cells before and after sorting were digested and counted at the same time on the second, third, and fourth days.

[0138] (3) Count the number of cells for four consecutive days and analyze the results. Fig.15 As shown, the number of cells after sorting increased compared with that before sorting at the same time point, indicating faster proliferation.

[0139] 3. Balloon Formation Assay

[0140] (1) The cells before and after sorting were divided into 3×10 4 The cells were seeded in a low-adhesion six-well plate at a number of 100 cells / well and cultured in DMEM / F12K supplemented with cell growth factors, with the medium changed every three days.

[0141] (2) On the tenth day, take photos and count the number of mammospheres larger than 50 μm. In a serum-free and low-adhesion culture environment, tumor stem cells can form mammospheres. The size and number of mammospheres reflect the strength of cell stemness. Fig.16 As shown, after sorting, the number of cells forming spheroids increased and their stemness was enhanced.

[0142] 4. Flow Cytometry

[0143] The cells before and after sorting were resuspended in pre-cooled PBS at a concentration of 1×10 6 After adding 3 μL of CD133 antibody to each cell, the cells were incubated in the dark for 30 minutes. The changes in cell stemness before and after sorting were detected using the tumor stem cell surface marker CD133. Fig.17 As shown, it indicates that the stemness of cells is enhanced after sorting.

[0144] 5.Western Blot

[0145] (1) Collect cells before and after sorting and perform protein extraction and quantification.

[0146] (2) Experimental operation. Electrophoresis: After protein loading, perform electrophoresis at 120V. Transfer: Transfer the protein to PVDF membrane and transfer at 300mA for 60min. Blocking: Block with skim milk powder to remove non-specific binding. Primary antibody: Add antibodies indicating cell stemness such as CD133, CD44, SOX2, and internal reference antibodies. Incubate at 4°C overnight. Secondary antibody: After washing the PVDF membrane with TBST, incubate with secondary antibody for 2 hours. Washing and exposure: Wash the unbound secondary antibody with TBST, and expose the bands with ECL luminescent solution. The results are as follows. Fig.18 As shown, it shows that the stemness indicators such as cell stemness markers CD133 / CD44 / SOX2 are enhanced after sorting.

[0147] 6. Cellular drug resistance experiment

[0148] (1) Resuspend the cells before and after MS-HCA-Chip sorting at 4×10 4 / ml single cell suspension. Inoculate in 96-well plates at a density of 100 μL / well.

[0149] (2) After the cells adhered, doxorubicin (DOX) was added to give final concentrations of 0 μM, 20 μM, 40 μM, and 60 μM, respectively.

[0150] (3) After 48 hours of culture, CCK8 cell activity was measured (10 μL of CCK8 was added to the wells and incubated in the dark for 40 min, and the absorbance was measured at 450 nm on a microplate reader). The experiment was repeated three times. Fig.19 As shown, the results showed that compared with the cells before sorting, the sorted cells showed higher drug resistance at the same concentration of doxorubicin, and the increase in drug resistance is one of the characteristics of enhanced stemness of tumor stem cells.

[0151] 7. Nude mouse tumor formation experiment

[0152] (1) The cells before and after sorting were divided into 1×10 6 , 1×10 5 , 1×10 4 The density was subcutaneously inoculated into 4-week-old nude mice.

[0153] (2) Four weeks later, the mice were killed and the tumor tissues were collected.

[0154] The results are as follows Fig. 20 As shown in the figure, the tumor volume formed by the cells after sorting is larger than that formed by the cells before sorting. 4 In this case, the cells before sorting could no longer form tumors, while the cells after sorting could still form tumors, indicating that the cells after sorting had stronger tumor-forming ability.

[0155] Example 5 Drug screening of liquorice active ingredients

[0156] CCK8 cell viability experiment was used to screen drugs targeting tumor cells and tumor stem cell signaling pathways, and components that had inhibitory effects on cells sorted by MS-HCA-Chip were selected as candidate drugs. The sorted cells were treated with candidate drugs, and the effects on tumor cell stemness before and after enrichment and after drug action were compared through classic biological experiments such as plate cloning experiment, balloon formation experiment, and Western Blot detection of stemness-related proteins. The tumor volume and weight of the drug ISL treatment group and the PBS control group were compared when the same cells were inoculated by in vitro nude mouse tumor formation experiment. Flow cytometry verified that the drug had an inhibitory effect by regulating the cell cycle. The successful enrichment of tumor stem cell-like phenotype cells and the targeting of the candidate drug ISL to tumor stem cells were verified.

[0157] 1.CCK-8 Experiment

[0158] (1) Fourteen active ingredients of licorice with tumor inhibitory effects (glycyrrhizic acid, glycyrrhizin, isoliquiritigenin, isoliquiritigenin, glabridin, hydroxychalcone, glycyrrhizic acid, glycyrrhizin, glycyrrhizinone, glycyrrhizin, glabridin, glycyrrhizin A, licorice chalcone, and licorice glycoside) were selected for drug screening.

[0159] (2) Resuspend the cells sorted by MS-HCA-Chip to 4×10 4 / ml single cell suspension was seeded in a 96-well plate at a density of 100 μL / well.

[0160] (3) After the cells adhered to the wall, the active ingredients of licorice were added to the concentrations equal to the IC50 of the drugs, and a control group was set up.

[0161] (4) After 48 hours of culture, CCK8 cell activity was measured (10 μL of CCK8 was added to the wells and incubated in the dark for 40 min, and the absorbance was measured at 450 nm on a microplate reader). The experiment was repeated three times.

[0162] like Fig.21 As shown, Control is the control group and Ingredient is the experimental group. The results show that isoliquiritigenin (ISL) has a good tumor stem cell inhibitory effect, the drug has a low effective concentration and good repeatability.

[0163] (5) Resuspend the cells sorted by MS-HCA-Chip to 4×10 4 / ml single cell suspension. Inoculate in 96-well plates at a density of 100 μL / well.

[0164] (6) After the cells adhered to the wall, isoliquiritigenin (ISL) was added to the solution at concentrations of 0 μM, 15 μM, 20 μM, and 25 μM, respectively.

[0165] (7) After 48 hours of culture, CCK8 cell activity was measured (10 μL of CCK8 was added to the wells and incubated in the dark for 40 min, and the absorbance was measured on a microplate reader at 450 nm). The experiment was repeated three times.

[0166] like Fig. 22 As shown, the results showed that isoliquiritigenin had a certain inhibitory effect on the cells with a tumor stem cell-like phenotype after sorting, and its IC50 was 20 μM.

[0167] 2. Plate cloning experiment

[0168] (1) The cells before and after sorting were seeded in a six-well plate at a density of 200 cells / well and cultured for 10 days. The sorted cells were seeded in the same number and ISL was added to the plate after they adhered to the wall to a final concentration of 20 μM.

[0169] (2) After washing three times with PBS, the cells were fixed with 4% paraformaldehyde for 15 minutes and then stained with crystal violet for 15 minutes.

[0170] (3) Take photos under a microscope and count the number of formed colonies to assess the ability of cells to survive independently.

[0171] like Fig.23 As shown in the figure, the number of cells that form clones increases after sorting, and their stemness is enhanced. However, the ability of cells to form clones weakens after drug treatment, indicating that ISL has a certain inhibitory effect on tumor stem cells.

[0172] 3. Western Blot

[0173] (1) Collect cells before and after sorting and after ISL drug treatment and perform protein extraction and quantification.

[0174] (2) Experimental operation. Electrophoresis: After the protein is loaded, electrophoresis is performed at 120V. Transfer: Transfer the protein to the PVDF membrane and transfer the membrane at 300mA for 60min. Blocking: Block with skim milk powder to remove non-specific binding. Primary antibody: Add antibodies indicating cell stemness such as CD133, CD44, SOX2 and internal reference antibodies respectively, and shake at 4°C overnight. Secondary antibody: After washing the PVDF membrane with TBST, incubate with secondary antibody for 2 hours. Washing and exposure: Wash the unbound secondary antibody with TBST, and expose the bands with ECL luminescent solution.

[0175] like Fig.24 As shown, the results showed that the stemness indicators such as cell stemness markers CD133 / CD44 / SOX2 were enhanced after sorting, and the stemness of the sorted cells was weakened after ISL drug treatment.

[0176] 4. Balloon Formation Assay

[0177] (1) The cells before and after sorting and the cells after drug addition were respectively divided into 3×10 4 The number of cells per well was inoculated into a low-adhesion six-well plate and cultured in DMEM / F12K and an environment supplemented with cell growth factors. The sorted cells were inoculated in the same number and ISL was added to a final concentration of 20 μM.

[0178] (2) On the tenth day, take photos and count the number of mammospheres larger than 50 μm. In a serum-free and low-adhesion culture environment, tumor stem cells can form mammospheres, and the size and number of mammospheres reflect the strength of cell stemness.

[0179] like Fig.25 As shown, the number of cells that form mammospheres increases and their stemness is enhanced after sorting, while the ability of cells to form mammospheres decreases after drug treatment.

[0180] 5. Transwell in vitro migration assay

[0181] (1) The cells before and after the serum removal were sorted at 2×10 4 The number of cells per well was inoculated on the upper layer of the Transwell chamber, and a medium containing 10% serum was added to the lower layer. The sorted cells were inoculated in the same number, and ISL was added to a final concentration of 20 μM.

[0182] (2) After 24 hours, use a cotton swab to gently wipe off the cells that have not passed through the upper chamber, place them in 4% paraformaldehyde for fixation for 15 minutes, and then stain with crystal violet.

[0183] (3) Take photos of the cells that have passed through under a microscope and count them. Repeat the experiment three times. Fig.26 As shown, the migration properties of cells increased after sorting, while the migration properties of cells decreased after drug treatment.

[0184] 6. Cell cycle detection

[0185] (1) The sorted cells and the cells treated with ISL drugs were collected and washed with pre-cooled PBS, and the supernatant was removed after centrifugation.

[0186] (2) Resuspend the cells to make a single-cell suspension, add pre-cooled anhydrous ethanol, and fix at -20°C.

[0187] (3) Wash with the same volume of pre-cooled PBS, centrifuge and remove the supernatant, digest with RNase in a water bath, and then stain with PI in the dark for 30 minutes.

[0188] (4) Detect red fluorescence at 488 nm on a flow cytometer and analyze the DNA content.

[0189] like Fig. 27As shown, the results showed that the G1 phase of cells treated with drugs was enhanced and ISL had an inhibitory effect on stem cells.

[0190] 7. In vivo experiments

[0191] (1) The sorted cells were divided into 1×10 5 / 100μL was subcutaneously inoculated into nude mice.

[0192] (2) Ten days later, the mice were randomly divided into two groups and intraperitoneally administered with 200 μL / mouse PBS or ISL (25 mg / kg) at intervals, and the body weights of the mice were measured.

[0193] (3) After 28 days, the mice were killed and the tumors were weighed. The tumor tissues were ground and subjected to cell protein quantification and Western Blot experiments to detect changes in the levels of stemness proteins in the tumors.

[0194] (4) Draw a line graph of the weight of mice over the days and count the results. Fig.28 As shown, (a) is a tumor photo, (b) is the tumor weight data, (c) is the mouse weight change data, and (d) is the change data of the level of stem protein in the tumor. It can be seen that ISL has almost no effect on the weight of mice and has a certain inhibitory effect on tumors.

[0195] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0196] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for isolating tumor stem cells, characterized in that: The sorting method comprises the following steps: using a first sorting chip in a microfluidic cell sorting device to perform a first sorting on a sample containing tumor stem cells, and then using a second sorting chip in the microfluidic cell sorting device to perform a second sorting on the cells obtained by the first sorting to obtain the tumor stem cells; The sample is a lung cancer cell line A549, the flow rate in the first sorting chip is controlled to be 45 μL / min to 75 μL / min, and the flow rate in the second sorting chip is controlled to be 0.2 mL / h to 0.4 mL / h; The microfluidic cell sorting device comprises a first sorting chip and a second sorting chip; the first sorting chip is provided with channels of different sizes for sorting cells, the first sorting chip is provided with a first inlet and a first outlet, the first inlet is used for injecting samples, and the channel gradually decreases in the direction from the first inlet to the first outlet; the second sorting chip is provided with a second inlet and a second outlet, the second inlet is connected to the first outlet, so that the sample flowing out of the first outlet enters the second sorting chip for sorting, the inner surface of the second sorting chip is covered with a matrix glue layer, the matrix glue layer is used to sort cells according to cell adhesion ability, and the second outlet is used for the sample sorted from the second sorting chip to flow out; The matrigel layer is formed by solidifying 3 mg / mL matrigel; The second sorting chip is provided with a capture column, and the side wall of the capture column is provided with a plurality of first protrusions; the cross section of the capture column is in the shape of a triangular propeller; A mixed flow structure is provided in the second sorting chip, the mixed flow structure extends from the second inlet to the second outlet, and a plurality of second protrusions are provided on the sidewall of the mixed flow structure from the second inlet to the second outlet; the cross section of the mixed flow structure is fishbone-shaped, the length of the mixed flow structure extending is 0.8 mm to 1.2 mm, and the width is 30 μm to 50 μm; There are multiple flow mixing structures, there are multiple capture columns, the multiple flow mixing structures are arranged relatively at intervals, and the multiple capture columns are distributed between two adjacent flow mixing structures; the interval between two flow mixing structures is 200 μm to 300 μm; The first sorting chip is provided with a plurality of interception columns, and every two or more interception columns are arranged in a row to form an interception unit, and the plurality of interception units are arranged in sequence and at intervals from the first inlet to the first outlet; The gap between two adjacent interception columns in each of the interception units forms a channel for sorting cells; The gap between two adjacent interception columns in the interception unit is 2 μm to 15 μm, and the gap gradually decreases in the direction from the first inlet to the first outlet; when the cell suspension flows through the first sorting chip, cells that are not easily deformed are gradually blocked by the interception columns according to the hardness of the cells, and relatively soft and easily deformed cells reach the first outlet; The sorting chamber in the first sorting chip has a length of 4cm to 6cm, a width of 1.6mm to 2.0mm, and a height of 15μm to 25μm; the sorting chamber in the second sorting chip has a length of 4.5cm to 5.5cm, a width of 1mm to 2mm, and a height of 15μm to 25μm, and each sorting chamber is filled with the mixed flow structure and the capture column located between the mixed flow structures.

2. The sorting method according to claim 1, characterized in that: The flow rate in the first sorting chip is controlled to be 58 μL / min to 65 μL / min.

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