A cyclone array chip and cell screening device and method

Abstract

The application provides a cyclone array chip and a cell screening device and method, the cyclone array chip comprises four cyclone units, which are one three-pipe cyclone unit and three two-pipe cyclone units; the cell screening device comprises the cyclone array chip, a solution collector, an electron microscope, a syringe pump and a suction pump, the cyclone array chip is installed on the solution collector and is connected with the syringe pump and the suction pump, and the electron microscope is used for collecting cell image information; the cell screening method is that cells are output from the bottom microchannel of the three-pipe cyclone unit, the cells are captured under the action of low pressure of a cyclone stagnation point, and a target cell is identified by using an image analysis technology, the target cell is induced to the two-pipe cyclone unit through the bottom microchannel of the two-pipe cyclone unit and suction, so that cell screening is realized. The application realizes the integration of cell screening, can identify cells with different sizes, shapes and physiological phenotype characteristics, has no mechanical damage to the cells, and improves the success rate of experiments.

Description

Technical Field

[0001] This invention relates to the field of cell screening technology, and more specifically to a cell screening device and method based on a vortex array chip. Background Technology

[0002] In the field of biological detection, it is necessary to screen and collect target cells from mixed cell solutions containing various cell types to facilitate further observation or testing of the target cells. Microfluidic chips are widely used in cell screening due to their advantages such as reagent saving, ease of integration, low cost, and miniaturization, and show promising development prospects.

[0003] Currently, cell screening methods based on microfluidic chip technology are divided into passive screening and active screening. Passive screening utilizes the combined effects of microchannel structural design and fluid dynamics to achieve screening. Common channel microstructures include micropillar arrays, sheath flow aggregation microstructures, and microporous filter structures (Yang Yang, Zhuang Ziyun. Cell Screening Chips, Cell Screening Systems and Methods [P]: CN112226336A. 2021-01-15), which have advantages such as high throughput and easy control. However, they require redesign when target cell parameters change, and the fabrication of these microstructures is quite difficult. Active screening, on the other hand, applies an external force field to target cells in a mixed cell solution, thereby screening out the target cells. Common external force fields include dielectric electrophoresis field, light radiation field, magnetic field, and sound field (Cheng Jingmeng. Method and experimental study of laser screening of cells in microchannels [D]: Hebei University of Technology, 2017). Active screening method has the advantages of good controllability and high accuracy, but the effect of the applied external force field on cell activity has not yet been clarified, and additional external force field generating equipment is required, which is costly. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, the present invention provides a vortex array chip and a cell screening device and method, which can screen and collect target cells in a mixed cell solution containing multiple cells.

[0005] The present invention is achieved by at least one of the following technical solutions.

[0006] A swirl array chip has a cell screening area at its center, surrounded by a first cell collection area, a second cell collection area, and a third cell collection area. The chip has a three-tube swirl unit and three two-tube swirl units. The three-tube swirl unit is located in the cell screening area, and the three two-tube swirl units are located in the first cell collection area, the second cell collection area, and the third cell collection area, respectively.

[0007] Furthermore, the three-tube cyclone unit includes three microtube channels and a bottom microchannel. The three microtube channels are 120° apart, and the axes of the three microtube channels are parallel to the corresponding sides of the cell screening area. The endpoint of the inner wall of the outlet of each microtube channel coincides with the corresponding vertex of the cell screening area. The inlet of each microtube channel is located at the edge of the chip. The bottom microchannel is located at the center of the cell screening area and is perpendicular to the bottom surface of the chip.

[0008] Furthermore, the area enclosed by the line connecting the endpoints of the inner wall surfaces of the three microchannel outlets is a characteristic triangle, which is an equilateral triangle; the ratio of the side length of the characteristic triangle to the cell diameter is 2:1 to 20:1.

[0009] Furthermore, each two-tube cyclone unit includes two microchannels and a bottom microchannel; the outlets of the two microchannels are arranged opposite each other in parallel, and the outlet end faces of the two microchannels and the axis of the microchannel outlets form a characteristic square, with the midpoints of the outlet end faces of the two microchannels coinciding with the vertices on the corresponding diagonals of the characteristic square; the inlets of the two microchannels are both located on the outside of the chip; the bottom microchannel is located at the center of the characteristic square and is perpendicular to the bottom surface of the chip.

[0010] Two unloading grooves are provided around the two microchannels.

[0011] Furthermore, the ratio of the side length of the characteristic square to the cell diameter is 1:1 to 20:1.

[0012] Furthermore, the microchannel outlet cross-section is square, and the ratio of the square side length to the cell diameter is 1:2 to 10:1.

[0013] Furthermore, the bottom microchannel cross-section is circular, with the ratio of the circle diameter to the cell diameter being 5:4 to 5:1.

[0014] A cell screening device containing the aforementioned swirl array chip includes a swirl array chip, a solution collector, an electron microscope, a syringe pump, and a suction pump. The swirl array chip is mounted on the solution collector. The solution collector is connected to the suction pump via a silicone tubing. The microchannel inlet of the swirl array chip is connected to the syringe pump via a microfluidic connector and a silicone tubing. The bottom microchannel of the three-tube swirl unit of the swirl array chip is connected to the syringe pump via a microfluidic needle and a silicone tubing. The bottom microchannel of the two-tube swirl unit of the swirl array chip is connected to the suction pump via a microfluidic needle and a silicone tubing. The electron microscope is located above the swirl array chip and is used to collect cell image information.

[0015] Furthermore, the total flow rate of the syringe pump is equal to the total flow rate of the pump, so that the liquid level in the cell screening device remains constant; the flow rates of both the syringe pump and the pump are 1–300 mL / h.

[0016] The cell screening method of the aforementioned cell screening device comprises a cell screening zone enclosed by three swirling units and a cell collection zone enclosed by two swirling units. Before cell screening, the initial height of the liquid level in the solution collector is adjusted so that the liquid level is flush with the outer side of the upper wall of the swirling array microtube channel. The cell screening includes the following steps:

[0017] (1) Run the injection pump connected to the microchannel of the cell screening area. The solution is sprayed at the same speed through the three microchannels to form a vortex. The vortex stagnation point is located at the center of the cell screening area. The solution enters the vortex array chip through the microchannel, flows out of the vortex array chip through the unloading tank and is collected by the solution collector. The pump draws the solution from the solution collector.

[0018] (2) Cells are output from the microchannel at the bottom of the cell screening area. Under the action of low pressure at the stagnation point of the three tubes, the cells are captured. The size, shape and physiological phenotypic characteristics of the cells are analyzed by electron microscopy, and the target cell collection area to be moved is determined.

[0019] (3) Change the flow rate of the three microtube channels, adjust the position of the stagnation point of the three tubes, and shift the cells held by the stagnation point toward the target cell collection area;

[0020] (4) Run the injection pump connected to the microchannel of the target cell collection area. The solution is sprayed at the same speed in the two microchannels to form a swirling flow. Run the pump connected to the microchannel at the bottom of the target cell collection area to form a low pressure in the target cell collection area. The cells move quickly to the target cell collection area and are enriched under the action of the low pressure at the stagnation point of the two tubes, thereby achieving cell screening.

[0021] Compared with the prior art, the present invention has significant advantages:

[0022] (1) The traditional cell screening steps such as cell capture, cell screening, and cell enrichment are integrated into a microfluidic chip, realizing an integrated cell screening operation. The structure is simple, the manufacturing cost is low, and it can identify cells of different sizes, shapes and physiological phenotypes without causing any mechanical damage to the cells, thus improving the success rate of the experiment.

[0023] (2) By using image recognition technology, not only can cells be screened based on their size and shape, but cell defects can also be identified through the physiological phenotypic characteristics of cells, thus improving the range of cell screening.

[0024] (3) Using fluid pressure and viscosity to screen cells without causing any mechanical damage to the cells, thus improving the success rate of the experiment;

[0025] (4) This swirl array has a simple structure and can be prepared by photopolymerization 3D printing technology, resulting in low manufacturing cost. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the swirl array chip structure;

[0027] Figure 2 This is a cross-sectional view of the swirl array chip on a horizontal plane;

[0028] Figure 3 Schematic diagram of a cell screening device

[0029] The image is labeled as follows:

[0030] 1-Swirl array chip;

[0031] 11-First cell collection area;

[0032] 110 - The first cell collection area is characterized by a square shape;

[0033] 111 - First cell collection area, first injection microtube channel;

[0034] 112 - First cell collection area, second injection microtube channel;

[0035] 113 - First cell collection area, first unloading tank;

[0036] 114 - First cell collection area, second unloading tank;

[0037] 115 - Microchannels on the bottom surface of the first cell collection area;

[0038] 12-Second cell collection area;

[0039] 120 - The second cell collection area is characterized by a square shape;

[0040] 121 - Second cell collection area, first injection microtube channel;

[0041] 122 - Second cell collection area, second injection microtube channel;

[0042] 123 - Second cell collection area, first unloading groove;

[0043] 124 - Second cell collection area, second unloading tank;

[0044] 125 - Microchannels on the bottom surface of the second cell collection area;

[0045] 13-Third cell collection area;

[0046] 130 - The third cell collection area is characterized by a square shape;

[0047] 131 - Third cell collection area, first injection microtube channel;

[0048] 132 - Third cell collection area, second injection microtube channel;

[0049] 133 - Third cell collection area, first unloading tank;

[0050] 134 - Third cell collection area, second unloading tank;

[0051] 135 - Microchannels on the bottom surface of the third cell collection area;

[0052] 14-Cell selection area;

[0053] 140 - Characteristic triangle of cell selection region;

[0054] 141 - First injection microtube channel in the cell selection area;

[0055] 142 - Second injection microtube channel in cell selection area;

[0056] 143 - Third injection microtube channel in cell selection area;

[0057] 144 - Microchannels on the bottom surface of the cell selection zone;

[0058] 2-Solution collector;

[0059] 3-Electron microscope;

[0060] 4-Injection pump;

[0061] 5-Liquid pump. Detailed Implementation

[0062] The specific implementation of the present invention will be further described below with reference to the accompanying drawings, but the implementation and protection scope of the present invention are not limited thereto.

[0063] like Figure 1 As shown, a swirl array chip structure is presented. The center of the swirl array chip 1 is a cell screening area 14, and the cell screening area 14 is surrounded by a first cell collection area 11, a second cell collection area 12, and a third cell collection area 13. The chip 1 is provided with a three-tube swirl unit and three two-tube swirl units. The three-tube swirl unit is located in the cell screening area 14, and the three two-tube swirl units are located in the first cell collection area 11, the second cell collection area 12, and the third cell collection area 13, respectively.

[0064] The area enclosed by the lines connecting the endpoints of the inner walls of the three microchannel outlets in the cell screening zone 14 is the characteristic triangle 140 of the cell screening zone, which is an equilateral triangle. The first cell collection zone 11, the second cell collection zone 12, and the third cell collection zone 13 are distributed around the characteristic triangle 140 of the cell screening zone.

[0065] like Figure 2As shown, in the first cell collection area 11, the second cell collection area 12, and the third cell collection area 13, the regions enclosed by the corresponding two microchannel outlet end faces and the two axes perpendicular to the corresponding two microchannel outlets are respectively the first cell collection area characteristic square 110, the second cell collection area characteristic square 120, and the third cell collection area characteristic square 130.

[0066] like Figure 2 As shown, the cell selection zone 14 includes a first injection microchannel 141, a second injection microchannel 142, a third injection microchannel 143, and a bottom microchannel 144. The first, second, and third injection microchannels 141, 142, and 143 are at 120° angles to each other, and their axes are parallel to the corresponding sides of the characteristic triangle 140 of the cell selection zone. The endpoints of the inner walls of the outlets of the three microchannels (141, 142, 143) coincide with the vertices of the characteristic triangle 140. The outlet cross-sections of the three microchannels (141, 142, 143) are square, with the ratio of the square's side length to the cell diameter ranging from 1:2 to 10:1. The ratio of the side length of the characteristic triangle 140 to the cell diameter ranges from 2:1 to 20:1. The inlets of the three microchannels (141, 142, 143) are located at the edge of chip 1; the cross-sections of the inlets of the three microchannels (141, 142, 143) are circular, and the diameter of the circle is the assembly size of the microfluidic connector. The microchannel 144 on the bottom surface of the cell screening area is located at the center of the characteristic triangle 140 of the cell screening area, perpendicular to the bottom surface, and its cross-section is circular, with the ratio of the circle diameter to the cell diameter being 5:4 to 5:1.

[0067] like Figure 2 As shown, the first cell collection area 11 includes a first injection microchannel 111, a second injection microchannel 112, a first unloading groove 113, a second unloading groove 114, and a bottom microchannel 115. The outlets of the first injection microchannel 111 and the second injection microchannel 112 are arranged parallel to each other, with their axes parallel to the corresponding sides of the characteristic square 110 of the first cell collection area. The midpoints of the outlet end faces of the two microchannels (111, 112) coincide with the vertices of the corresponding diagonals of the characteristic square 110 of the first cell collection area.

[0068] In a preferred embodiment, the outlet cross-section of the microchannels (111, 112) is square, with the ratio of the square's side length to the cell diameter being 1:2 to 10:1. The ratio of the side length to the cell diameter of the first cell collection area's characteristic square 110 is 1:1 to 20:1. The inlet of the microchannels (111, 112) is located at the edge of chip 1, and the inlet cross-section of the microchannels (111, 112) is circular, with the diameter of the circle being the assembly size of the microfluidic connector. The bottom microchannel 115 of the first cell collection area is located at the center of the first cell collection area's characteristic square 110, perpendicular to the bottom surface, and its cross-section is circular, with the ratio of the circle's diameter to the cell diameter being 5:4 to 5:1.

[0069] like Figure 2 As shown, the second cell collection area 12 includes a first injection microchannel 121, a second injection microchannel 122, a first unloading groove 123, a second unloading groove 124, and a bottom microchannel 125. The outlets of the first injection microchannel 121 and the second injection microchannel 122 in the second cell collection area are arranged parallel to each other, with their axes parallel to the corresponding sides of the characteristic square 120 of the second cell collection area. The midpoints of the outlet end faces of the two microchannels (121, 122) coincide with the vertices of the corresponding diagonals of the characteristic square 120 of the second cell collection area. The outlet cross-section of the microchannels (121, 122) is square, with a side length to cell diameter ratio of 1:2 to 10:1. The side length to cell diameter ratio of the characteristic square 120 of the second cell collection area is 1:1 to 20:1. The inlets of the microchannels (121, 122) are located at the edge of chip 1, and the inlet cross-section of the microchannels is circular, with the diameter of the circle being the assembly dimension of the microfluidic connector. The bottom microchannel 125 of the second cell collection area is located at the center of the characteristic square 120 of the second cell collection area, perpendicular to the bottom surface, and its cross-section is circular, with a diameter to cell diameter ratio of 5:4 to 5:1.

[0070] like Figure 2As shown, the third cell collection area 13 includes a first injection microchannel 131, a second injection microchannel 132, a first unloading groove 133, a second unloading groove 134, and a bottom microchannel 135. The outlets of the first injection microchannel 131 and the second injection microchannel 132 in the third cell collection area are arranged parallel to each other, with their axes parallel to the corresponding sides of the characteristic square 130 of the third cell collection area. The midpoints of the outlet end faces of the two microchannels (131, 132) coincide with the vertices of the corresponding diagonals of the characteristic square 130 of the third cell collection area. The outlet cross-section of the microchannels (131, 132) is square, with the ratio of the square's side length to the cell diameter being 1:2 to 10:1. The ratio of the side length to the cell diameter of the characteristic square 130 of the third cell collection area is 1:1 to 20:1. The inlet of the microchannel is located at the edge of chip 1, and the inlet cross-section of the microchannels (131, 132) is circular, with the diameter of the circle being the assembly dimension of the microfluidic connector. The bottom microchannel 135 of the third cell collection area is located at the center of the characteristic square 130 of the third cell collection area, perpendicular to the bottom surface, and its cross-section is circular, with the ratio of the circle's diameter to the cell diameter being 5:4 to 5:1.

[0071] like Figure 3 As shown, a cell screening device includes a cyclone array chip, a solution collector 2, an electron microscope 3, a syringe pump 4, and a suction pump 5. The cyclone array chip is mounted on the solution collector 2. The solution collector 2 is connected to the suction pump 5 via a silicone tubing. The microchannel inlet of the cyclone array chip is connected to the syringe pump 4 via a microfluidic connector and a silicone tubing. The bottom microchannel of the three-tube cyclone unit is connected to the syringe pump 4 via a microfluidic needle and a silicone tubing. The bottom microchannel of the two-tube cyclone unit is connected to the suction pump 5 via a microfluidic needle and a silicone tubing. The electron microscope 3 is located above the cyclone array chip and is used to collect cell image information. The total flow rate of the syringe pump 4 is equal to the total flow rate of the suction pump 5, keeping the liquid level in the cell screening device constant.

[0072] In a preferred embodiment, the flow rates of the syringe pump 4 and the pump 5 are 1 to 300 mL / h.

[0073] Based on the swirl array chip and cell screening equipment provided by this invention, a cell screening method is also provided. To make the advantages and effects of this method more apparent and understandable, the following embodiments are provided in detail:

[0074] In this embodiment, target cells are screened into the first cell collection area 11. Before cell screening, the initial height of the liquid level in the solution collector 2 is adjusted so that the liquid level is flush with the outer side of the upper wall of the microchannel of the vortex array chip. Cell screening includes the following steps: (1) The corresponding injection pump 4 in the cell screening area 14 is run, and the solution is sprayed at the same speed in the first injection microchannel 141, the second injection microchannel 142, and the third injection microchannel 143 of the cell screening area to form a vortex. The vortex stagnation point is located at the center of the cell screening area 14. The solution enters the vortex array chip through the microchannel, flows out of the vortex array chip through the unloading tank and is collected by the solution collector 2. The pump 5 draws the solution from the solution collector 2; (2) Cells are output from the microchannel 144 at the bottom of the cell screening area 14. Under the action of the low pressure of the three-tube vortex stagnation point, the cells are captured. The size, shape and physiological phenotypic characteristics of the cells are analyzed by the electron microscope 3, and the cells to be moved to the first cell collection area 11 are determined; (3) The injection flow rates of the first injection microchannel 141 and the second injection microchannel 142 in the cell screening area remain unchanged, while the injection flow rate of the third injection microchannel 143 in the cell screening area increases. The vortex stagnation point clamps the cells and shifts them toward the first cell collection area 11; (4) The corresponding injection pumps 4 of the first injection microchannel 111 and the second injection microchannel 112 in the first cell collection area are run. The two microchannels (111, 112) spray the solution at the same speed to form a vortex. The pump 5, which is connected to the microchannel 115 at the bottom of the first cell collection area, is operated to create a low pressure in the first cell collection area 11. The cells move quickly to the first cell collection area 11 and are enriched under the action of the low pressure at the stagnation point of the two tubes, thereby achieving cell screening.

[0075] The present invention provides a swirl array chip, cell screening device and method, which can achieve stable cell screening. The swirl array chip can be prepared by photopolymerization 3D printing technology, thus achieving simplicity, convenience and low manufacturing cost, making it of great commercial application value.

[0076] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A swirl array chip, characterized in that, The system includes a substrate, with a cell screening zone at its center. Surrounding the cell screening zone are a first cell collection zone, a second cell collection zone, and a third cell collection zone. The substrate is equipped with a three-tube vortex unit and three two-tube vortex units. The three-tube vortex unit is located within the cell screening zone, and the three two-tube vortex units are located within the first, second, and third cell collection zones, respectively. Each three-tube vortex unit includes three microchannels and a bottom microchannel. The three microchannels are at 120° intervals, and their axes are parallel to the corresponding edges of the cell screening zone. The endpoint of the inner wall of the outlet of each microchannel coincides with the corresponding vertex of the cell screening zone. The inlet of each microchannel is located at the edge of the substrate. The bottom microchannel is located in the center of the cell selection area, perpendicular to the bottom surface of the substrate; Each two-tube cyclone unit includes two microtube channels and a bottom microchannel; the outlets of the two microtube channels are arranged opposite each other in parallel, and the outlet end faces of the two microtube channels and the axis of the microtube channel outlets form a characteristic square, with the midpoints of the outlet end faces of the two microtube channels coinciding with the vertices on the diagonals of the characteristic square; the inlets of the two microtube channels are both located on the outside of the substrate; the bottom microchannel is located at the center of the characteristic square and is perpendicular to the bottom surface of the substrate. Two unloading grooves are provided around the two microchannels.

2. The swirl array chip according to claim 1, characterized in that, The area enclosed by the lines connecting the endpoints of the inner wall surfaces of the three microchannel outlets is a characteristic triangle, which is an equilateral triangle; the ratio of the side length of the characteristic triangle to the cell diameter is 5:1 to 20:

1.

3. The swirl array chip according to claim 2, characterized in that, The ratio of the side length of the characteristic square to the cell diameter is 5:1 to 20:

1.

4. The swirl array chip according to any one of claims 1 to 3, characterized in that, The microchannel outlet cross-section is square, and the ratio of the square side length to the cell diameter is 1:2 to 10:

1.

5. The swirl array chip according to claim 4, characterized in that, The bottom microchannel cross-section is circular, and the ratio of the circle diameter to the cell diameter is 5:4 to 5:

1.

6. A cell screening device comprising the swirl array chip of claim 5, characterized in that, The system includes a cyclone array chip, a solution collector, an electron microscope, a syringe pump, and a suction pump. The cyclone array chip is mounted on the solution collector. The solution collector is connected to the suction pump via a silicone tubing. The microchannel inlet of the cyclone array chip is connected to the syringe pump via a microfluidic connector and a silicone tubing. The bottom microchannel of the three-tube cyclone unit of the cyclone array chip is connected to the syringe pump via a microfluidic needle and a silicone tubing. The bottom microchannel of the two-tube cyclone unit of the cyclone array chip is connected to the suction pump via a microfluidic needle and a silicone tubing. The electron microscope is located above the cyclone array chip and is used to collect cell image information.

7. The cell screening device according to claim 6, characterized in that, The total flow rate of the syringe pumps is equal to the total flow rate of the pumps, ensuring that the liquid level in the cell screening device remains constant; the flow rates of both the syringe pumps and the pumps are 1~300mL / h.

8. A cell screening method for implementing the cell screening device of claim 6, characterized in that, The flow area enclosed by the three-tube cyclone unit is the cell screening zone, and the flow area enclosed by the two-tube cyclone unit is the cell collection zone. Before cell screening, the initial height of the liquid level in the solution collector is adjusted so that the liquid level is flush with the outer side of the upper wall of the cyclone array microtube channel. Cell screening includes the following steps: (1) Run the injection pump connected to the microchannel of the cell screening area. The solution is sprayed at the same speed through the three microchannels to form a vortex. The vortex stagnation point is located at the center of the cell screening area. The solution enters the vortex array chip through the microchannel, flows out of the vortex array chip through the unloading tank and is collected by the solution collector. The pump draws the solution from the solution collector. (2) Cells are output from the microchannel at the bottom of the cell screening area. Under the action of low pressure at the stagnation point of the three tubes, the cells are captured. The size, shape and physiological phenotypic characteristics of the cells are analyzed by electron microscopy, and the target cell collection area to be moved is determined. (3) Change the flow rate of the three microtube channels, adjust the position of the stagnation point of the three tubes, and shift the cells held by the stagnation point toward the target cell collection area; (4) Run the injection pump connected to the microchannel of the target cell collection area. The solution is sprayed at the same speed in the two microchannels to form a swirling flow. Run the pump connected to the microchannel at the bottom of the target cell collection area to form a low pressure in the target cell collection area. The cells move quickly to the target cell collection area and are enriched under the action of the low pressure at the stagnation point of the two tubes, thereby achieving cell screening.

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