Single cell distribution system and distribution method

Abstract

The invention relates to a single cell distribution system and a single cell distribution method, and relates to the technical field of cell culture, the distribution system comprises a cell sensor, the cell sensor comprises a conical tube, a microwell plate and electrodes, the microwell plate is connected with the end part of the conical tube, the number of the electrodes is two, and the two electrodes are respectively arranged inside and outside the conical tube; a flow control device that causes the cell sensor to suck in or discharge cells; the movement device is connected with the cell sensor and drives the cell sensor to reach the cell culture dish at a specified position; the control device comprises a touch screen and a circuit control system, and the circuit control system is connected with the electrodes, collects signals of the cell sensor and sends signals to the flow control device and the movement device, so that the flow control device and the movement device perform corresponding actions. According to the single cell distribution system, the accuracy and efficiency of cell distribution can be improved, and the influence on cell activity is reduced.

Description

Technical Field

[0001] The present application relates to the field of cell culture technology, and in particular to a single cell distribution system and distribution method. Background Art

[0002] Single cell analysis is the focus of current medical diagnosis. Cells are heterogeneous. Traditional cell analysis techniques ignore the uniqueness of single cells. Therefore, single cell analysis has important research significance. Single cell capture is the first key step in single cell analysis. Existing single cell distribution methods include: (1) limited dilution method, in which the cell group suspension is diluted with liquid to reduce its cell concentration, and then the diluted suspension is quantitatively distributed to each culture dish. The probability of a single cell appearing in each culture dish is low. This method has a random component and cannot guarantee that there is only one cell in each culture dish. The efficiency is low. (2) flow cytometer distribution method, which requires fluorescent labeling and laser irradiation to stimulate fluorescence, which has an adverse effect on cell activity. (3) image recognition method, Chinese patents CN118652743A and CN118325717A adopt the following method: first prepare a cell suspension, then the spray device sprays out tiny droplets, and the image recognition system determines whether the droplet contains a single cell. If it is a single cell, the droplet is sent to the culture dish. If it is a droplet of 0 cells or multiple cells, it is sent to the waste liquid pool. This method has a complex structure and requires a microscope to take pictures and an image recognition system. The differences in cell morphology and the angle of the cells in the droplet will affect the accuracy of the image recognition system. Summary of the invention

[0003] The purpose of the present application is to provide a single cell distribution system and distribution method to solve the inaccuracy of existing distribution methods and the impact on cell activity.

[0004] The single cell distribution system provided in this application adopts the following technical solution:

[0005] A single cell dispensing system, the dispensing system comprising:

[0006] A cell sensor for receiving cell signals, the cell sensor comprising a conical tube, a microporous plate and an electrode, the microporous plate being connected to the end of the conical tube, and two electrodes being arranged inside and outside the conical tube respectively;

[0007] A fluid control device, used to control the pressure of the fluid entering the cell sensor, so that the cell sensor can absorb cells or spit out cells;

[0008] a motion device, connected to the cell sensor, to drive the cell sensor to a cell culture dish at a designated position; and

[0009] The control device includes a touch screen and a circuit control system. The circuit control system is connected to the electrodes to collect signals from the cell sensors and send signals to the flow control device and the motion device to make the flow control device and the motion device perform corresponding actions.

[0010] As a preferred technical solution of the present application, the conical tube is configured to be an insulating material, the upper end is configured to be a wide-mouthed end and is equipped with an end cap, the microporous plate is configured to be at the narrow-mouthed end of the conical tube, the electrode includes an inner electrode and an outer electrode, the inner electrode is coaxially configured to be inside the conical tube, and the outer electrode is fixedly configured to be outside the conical tube.

[0011] As a preferred technical solution of the present application, the flow control device includes a micropump, a solenoid valve and a pipeline, the pipeline is connected to the end cover of the conical tube, the micropump and the solenoid valve are electrically connected to the control device, and the control device changes the pressure in the pipeline by controlling the micropump and the solenoid valve.

[0012] As a preferred technical solution of the present application, the motion device is configured as a three-axis motion platform, including an X-axis motion mechanism, a Y-axis motion mechanism and a Z-axis motion mechanism, the cell sensor is connected to the Z-axis motion mechanism, and the pipeline is connected to the Z-axis motion mechanism.

[0013] As a preferred technical solution of the present application, a working platform is also included, and the working platform includes:

[0014] Place countertops for mounting fluidics, motion devices, and controls;

[0015] A culture dish placement tray, placed on the placement table, for placing cell culture dishes; and

[0016] The culture dish conveying mechanism is arranged on the placing table and is used for driving the movement of the culture dish placing plate on the placing table.

[0017] As a preferred technical solution of the present application, the culture dish conveying mechanism comprises:

[0018] A driving roller is used to support and transport the culture dish placement plate, wherein a plurality of driving rollers are arranged in parallel and connected to the transport placement table;

[0019] A driving assembly connected to the driving roller and used to drive the driving roller to rotate; and

[0020] The contact switch is arranged on one side of the moving direction of the culture dish placement plate and is used for contacting the culture dish placement plate and closing the driving component.

[0021] The single cell distribution method provided in this application adopts the following technical solution:

[0022] A single cell distribution method comprises the following steps:

[0023] First, prepare the cell suspension, put the cell culture dish and culture dish tray in place, and put the cell culture solution in the culture dish;

[0024] The control device controls the micro pump and the electromagnetic valve to form a negative pressure in the conical tube of the cell sensor to suck in the cell suspension;

[0025] The cell sensor is moved into the cell culture dish through the motion device, and then the solenoid valve is controlled to form a positive pressure in the conical tube to spit out the cells;

[0026] The motion device drives the cell sensor to move to the next cell culture dish to continue spitting out cells until all designated cell culture dishes are assigned cells.

[0027] As a preferred technical solution of the present application, during the process of the cell sensor absorbing the cell suspension, the micropores on the microporous plate will be blocked. At this time, the resistance between the two electrodes will increase. After the circuit control system senses it, it controls the solenoid valve to switch the cell sensor to positive pressure to remove the blockage. After the resistance returns to normal, it switches to negative pressure to continue to absorb the cell suspension.

[0028] As a preferred technical solution of the present application, during the process of aspirating the cell suspension, due to misoperation, the cell suspension is not placed, resulting in the cell sensor aspirating air. Since air is not conductive, the electrode cannot detect the electrical signal, and the system will report an error to remind the user.

[0029] As a preferred technical solution of the present application, during the cell distribution process, impurities block the micropores of the microplate, and the circuit control system detects the resistance change and controls the solenoid valve to generate negative pressure recoil to remove the blockage.

[0030] In summary, the present application includes at least one of the following beneficial technical effects:

[0031] 1. The cell sensor of the present application has a simple structure, can improve the efficiency of single cell sorting, uses micropores to distribute cells, improves the accuracy of cell distribution, and combines with a fluid control device and a circuit control system to achieve flexible distribution of single cells or a fixed number of cells, reducing the impact on cell activity.

[0032] 2. Since cells are poor conductors of electricity, when cells pass through the micropores of the cell sensor, the resistance between the cell suspension in the conical tube of the cell sensor and the culture medium in the culture dish increases, and the resistance change reaches the circuit control system through the electrodes of the cell sensor. After the control device collects this signal, it controls the solenoid valve to change the pressure in the conical tube to zero, preventing the next cell from being spit out, and ensuring that this culture dish contains one cell or a fixed number of cells.

[0033] 3. In the present application, the cell culture dishes can be placed on a culture dish placement tray for unified placement, and then uniformly transported by a driving roller. After the cells are distributed, they are uniformly transported away from the work surface, which can greatly improve the efficiency of cell distribution and culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is a schematic diagram of the principle structure of an embodiment of the present application;

[0035] Figure 2 is a schematic diagram of the structure of the cell sensor in the embodiment of the present application;

[0036] Figure 3 It is a schematic diagram of the structure of the system device of the embodiment of the present application;

[0037] Figure 4 It is a schematic diagram of the top view of the structure on the working table in the embodiment of the present application;

[0038] Figure 5 is a schematic diagram of the side view structure on the working table in the embodiment of the present application;

[0039] In the figure, 1. cell sensor; 11. conical tube; 12. microplate; 13. inner electrode; 14. outer electrode; 2. fluid control device; 21. micro pump; 22. solenoid valve; 23. pipeline; 3. motion device; 31. X-axis motion mechanism; 32. Y-axis motion mechanism; 33. Z-axis motion mechanism; 4. control device; 41. touch screen; 42. circuit control system; 5. work platform; 51. placement table; 52. culture dish placement tray; 53. drive roller; 54. drive assembly; 55. contact switch. DETAILED DESCRIPTION

[0040] The following is combined with Figure 1 -Attached Figure 5 , further details of this application are given.

[0041] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and should not be construed as limiting the present invention.

[0042] Example: This application proposes a single cell distribution system, referring to Figure 1-5The system includes a cell sensor 1, a fluid control device 2, a motion device 3, a control device 4 and a working platform 5. The cell sensor 1 is used to receive cell signals and distribute cells to the cell culture dish according to the required number; the fluid control device 2 is used to transport fluid into the cell sensor 1 and control the fluid pressure entering the cell sensor 1, so that the cell sensor 1 inhales or spits out cells; the motion device 3 drives the cell sensor 1 to move by displacement, and transports the cell sensor 1 to different cell culture dishes; the control device 4 is used to control the cell sensor 1, the fluid control device 2, the motion device 3 to work according to the set program; the cell sensor 1, the fluid control device 2, the motion device 3 and the control device 4 are all installed on the working platform 5, and multiple cell culture dishes are placed on the working platform 5, so that the cell sensor 1 can distribute cells conveniently.

[0043] The cell sensor 1 includes a conical tube 11, a microporous plate 12 and electrodes. The upper opening of the conical tube 11 is larger than the lower opening, and an end cap is installed on the upper end for connecting with the fluid control device 2. The microporous plate 12 is fixedly arranged at the lower end of the conical tube 11. The conical tube 11 is set to a non-conductive material, including but not limited to plastic, glass, and rubber. In order to facilitate observation of the internal situation of the conical tube 11, in this embodiment, the conical tube 11 adopts a transparent PP tube, and the end with a smaller diameter is ultrasonically welded with a PP film as the microporous plate 12. A micropore is opened at the center of the microporous plate 12 for the entry and exit of cells.

[0044] Two electrodes are provided, which are respectively arranged inside and outside the conical tube 11. The electrodes include an inner electrode 13 and an outer electrode 14. The inner electrode 13 is arranged inside the conical tube 11, and the outer electrode 14 is arranged outside the conical tube 11. In this embodiment, the inner electrode 13 is fixed on the inner wall of the conical tube 11, and the distance between the lower end of the inner electrode 13 and the microporous plate 12 is 2 mm. The outer electrode 14 is fixed on the outer wall of the conical tube 11, and the lower end of the outer electrode 14 is flush with the lower surface of the microporous plate 12.

[0045] The fluid control device 2 includes a micro pump 21, a solenoid valve 22 and a pipeline 23. The micro pump 21 and the solenoid valve 22 are both installed on the pipeline 23. The end of the pipeline 23 is connected to the end cover of the tapered tube 11 and communicated with the tapered tube 11. The micro pump 21 and the solenoid valve 22 are both electrically connected to the control device 4, so that the control device 4 changes the pressure in the pipeline 23 by controlling the micro pump 21 and the solenoid valve 22.

[0046] The cell sensor 1 is mounted on the moving device 3 , and the moving device 3 drives the cell sensor 1 to the cell culture dish at the designated position and distributes cells into the cell culture dish. In this embodiment, the motion device 3 is configured as a three-axis motion platform, including an X-axis motion mechanism 31, a Y-axis motion mechanism 32 and a Z-axis motion mechanism 33; the X-axis motion mechanism 31 is configured as two parallel guide rails, the two parallel guide rails are installed on both sides above the working platform 5 through pillars, and four pillars are fixed at the top angle positions of the upper surface of the working platform 5; the Y-axis motion mechanism 32 includes a linear guide rail, a traveling vehicle connected to both ends of the linear guide rail, and a driving motor, the traveling vehicle is installed on the two parallel guide rails of the X-axis motion mechanism 31, and is driven by the driving motor, the driving motor is connected to the control device 4 and is controlled by the control device 4; the Z-axis motion mechanism 33 includes a vertical guide rail, a traveling vehicle, a driving motor and a lifting seat, the traveling vehicle is connected to the upper end of the vertical guide rail, the traveling vehicle translates on the linear guide rail of the Y-axis motion mechanism 32 and is driven by the driving motor, the lifting seat is set on the vertical guide rail, the cell sensor 1 is connected to the lifting seat and moves with the lifting seat.

[0047] The control device 4 includes a touch screen 41 and a circuit control system 42. The touch screen 41 is installed on one side of the work platform 5 to facilitate the operator to control. The circuit control system 42 is connected to the electrodes of the cell sensor 1 to collect the signals sensed by the cell sensor 1. The circuit control system 42 is electrically connected to the micropump 21 and the solenoid valve 22 to control the start and stop of the micropump 21 and the solenoid valve 22. The circuit control system 42 is electrically connected to the motion device 3 to control the translation of the Y-axis motion mechanism 32, the translation of the Z-axis motion mechanism 33 and the lifting and lowering of the cell sensor 1.

[0048] The working platform 5 includes a placement table 51 , a culture dish placement tray 52 and a culture dish conveying mechanism, and the culture dish conveying mechanism includes a driving roller 53 , a driving assembly 54 and a contact switch 55 .

[0049] The placement table 51 is used to install the fluid control device 2, the motion device 3 and the control device 4. A groove is provided on the top of the placement table 51 for installing the culture dish conveying mechanism and placing the culture dish placement tray 52. ​​The placement table 51 is rectangular, and the groove provided on its upper surface is also rectangular. One side of the groove is open and penetrates to one side of the placement table 51, which is used for the entry and exit of the culture dish placement tray 52. ​​In this embodiment, the culture dish placement tray 52 is configured as a rectangular tray, and a plurality of placement stations for cell culture dishes are evenly distributed on the upper surface, which can place the cell culture dishes to slide.

[0050] The culture dish conveying mechanism is disposed on the placement table 51 and is used to drive the culture dish placement plate 52 to move on the placement table 51 . In this embodiment, a plurality of driving rollers 53 are arranged in parallel, and the two ends of the driving rollers 53 are rotatably connected to the two opposite side walls of the groove, and the axial direction of the driving rollers 53 is parallel to the opening side of the groove. The culture dish placement plate 52 is placed on the driving rollers 53 and is driven to move by the driving rollers 53. In this embodiment, the driving assembly 54 adopts a combination of a servo motor and a synchronous pulley, and the ends of all the driving rollers 53 are equipped with synchronous pulleys, which are connected by synchronous belts. The servo motor is connected to a driving roller 53 at the end to drive the driving roller 53 to rotate. The contact switch 55 is arranged on a side wall of the groove where the driving roller 53 is installed and away from the opening of the groove. When the culture dish placement plate 52 moves into the groove, it will contact the contact switch, and the contact switch 55 will close the driving assembly 54. After the cell sensor 1 distributes the cells, the circuit control system 42 controls the servo motor to reverse, and the driving roller 53 pushes the culture dish placement plate 52 out of the groove in the opposite direction. After the cell culture dishes are rearranged, the culture dish placement plate 52 is sent back into the groove.

[0051] In some embodiments of the present application, the control device includes but is not limited to a touch screen 41, a non-touch screen, an external computer, etc. The number of cells allocated to the culture dish can be one, or two, three, etc.

[0052] It should be noted that when cells pass through, the basic principle of signal generation is the Coulter principle. The cell culture fluid is conductive, and cells are poor conductors of electricity. When the cell sensor 1 enters the culture dish for distribution, the inner and outer electrodes of the conical tube 11 conduct electricity through the cell culture fluid; when the cells pass through the micropores, the conductive channel is temporarily blocked, so the resistance of the two electrodes suddenly changes, and the control system can sense this signal.

[0053] The present application also proposes a single cell distribution method, comprising the following steps:

[0054] First, prepare the cell suspension, transport the cell culture dish and the culture dish placement plate 52 to the position by using the servo motor and the driving roller 53, and put the cell culture liquid into the cell culture dish;

[0055] The control device 4 controls the micro pump 21 and the electromagnetic valve 22 to form a negative pressure in the conical tube 11 of the cell sensor 1 to suck in the cell suspension;

[0056] The cell sensor 1 is moved into the cell culture dish by the motion device 3, and then the solenoid valve 22 is controlled to operate so that a positive pressure is formed in the conical tube 11 to spit out the cells;

[0057] The motion device 3 drives the cell sensor 1 to move to the next cell culture dish to continue spitting out cells until all designated cell culture dishes are allocated with cells.

[0058] In some embodiments, during the process of the cell sensor 1 absorbing the cell suspension, the micropores on the microporous plate 12 will be blocked, and the resistance between the two electrodes will increase. After the circuit control system 42 senses this, it controls the solenoid valve 22 to operate, so that the cell sensor 1 is converted into positive pressure to remove the blockage. After the resistance returns to normal, it is converted into negative pressure to continue to absorb the cell suspension.

[0059] In some embodiments, during the process of aspirating the cell suspension, if the cell suspension is not placed due to misoperation, resulting in the cell sensor 1 aspirating air, the electrode cannot detect the electrical signal because air is not conductive, and the system will report an error to remind the user.

[0060] In some embodiments, due to the high cell concentration, multiple cells may be spit out simultaneously, and the control device 4 may detect multiple resistance change values ​​and report an error on the touch screen 41 .

[0061] In some embodiments, after the control device 4 collects the signal of the cell passing, the pressure in the conical tube 11 becomes a negative value, preventing the cell suspension in the conical tube 11 from flowing out due to gravity.

[0062] In some embodiments, during the cell distribution process, impurities block the micropores of the micropore plate 12, and the circuit control system 42 detects the resistance change and controls the solenoid valve 22 to generate negative pressure recoil to clear the blockage.

[0063] The embodiments of this specific implementation are all preferred embodiments of the present application, and are not intended to limit the protection scope of the present application. The same components are represented by the same figure marks. Therefore, any equivalent changes made based on the structure, shape, and principle of the present application should be included in the protection scope of the present application.

Claims

1. A single cell distribution system, characterized in that: The distribution system includes: A cell sensor (1) for receiving cell signals, the cell sensor (1) comprising a conical tube (11), a microporous plate (12) and an electrode, the microporous plate (12) being connected to the end of the conical tube (11), and two electrodes being arranged inside and outside the conical tube (11) respectively; A fluid control device (2) for controlling the pressure of the fluid entering the cell sensor (1) so that the cell sensor (1) can absorb cells or expel cells; A moving device (3) connected to the cell sensor (1) to drive the cell sensor (1) to a cell culture dish at a designated position; and The control device (4) comprises a touch screen (41) and a circuit control system (42). The circuit control system (42) is connected to the electrode, collects the signal of the cell sensor (1), and sends a signal to the flow control device (2) and the motion device (3), so that the flow control device (2) and the motion device (3) perform corresponding actions.

2. A single cell distribution system according to claim 1, characterized in that: The conical tube (11) is made of insulating material, the upper end is a wide-mouthed end and is provided with an end cap, the microporous plate (12) is arranged at the narrow-mouthed end of the conical tube (11), the electrodes include an inner electrode (13) and an outer electrode (14), the inner electrode (13) is coaxially arranged in the conical tube (11), and the outer electrode (14) is fixedly arranged outside the conical tube (11).

3. A single cell distribution system according to claim 2, characterized in that: The fluid control device (2) comprises a micro pump (21), a solenoid valve (22) and a pipeline (23); the pipeline (23) is connected to the end cover of the tapered tube (11); the micro pump (21) and the solenoid valve (22) are electrically connected to the control device (4); and the control device (4) changes the pressure in the pipeline (23) by controlling the micro pump (21) and the solenoid valve (22).

4. A single cell distribution system according to claim 3, characterized in that: The motion device (3) is configured as a three-axis motion platform, comprising an X-axis motion mechanism (31), a Y-axis motion mechanism (32) and a Z-axis motion mechanism (33); the cell sensor (1) is connected to the Z-axis motion mechanism (33); and the pipeline (23) is connected to the Z-axis motion mechanism (33).

5. A single cell distribution system according to any one of claims 1 to 4, characterized in that: It also includes a working platform (5), wherein the working platform (5) includes: A table (51) is placed for installing the fluid control device (2), the motion device (3) and the control device (4); A culture dish placement tray (52), placed on the placement table (51), for placing a cell culture dish; and The culture dish conveying mechanism is arranged on the placement table (51) and is used to drive the culture dish placement plate (52) to move on the placement table (51).

6. A single cell distribution system according to claim 5, characterized in that: The culture dish conveying mechanism comprises: A driving roller (53) for supporting and conveying the culture dish placement plate (52), wherein a plurality of driving rollers (53) are arranged in parallel and connected to the conveying placement table (51); A driving assembly (54), connected to the driving roller (53), and used for driving the driving roller (53) to rotate; and The contact switch (55) is arranged on one side of the moving direction of the culture dish placement plate (52) and is used for contacting the culture dish placement plate (52) and closing the driving component.

7. A single cell distribution method, characterized in that: The following steps are involved: First, prepare a cell suspension, place a cell culture dish and a culture dish placement plate (52) in place, and put a cell culture solution into the culture dish; The control device (4) controls the micro pump (21) and the electromagnetic valve (22) to form a negative pressure in the conical tube (11) of the cell sensor (1) to suck in the cell suspension; The cell sensor (1) is moved into the cell culture dish by means of a motion device (3), and then the solenoid valve (22) is controlled to operate so that a positive pressure is formed in the conical tube (11) to eject the cells outwards; The motion device (3) drives the cell sensor (1) to move to the next cell culture dish to continue spitting out cells until all designated cell culture dishes are allocated cells.

8. A single cell distribution method according to claim 7, characterized in that: During the process of the cell sensor (1) absorbing the cell suspension, the micropores on the microporous plate (11) will be blocked. At this time, the resistance between the two electrodes will increase. After the circuit control system (42) senses it, it controls the solenoid valve (22) to operate, so that the cell sensor (1) is converted to positive pressure to remove the blockage. After the resistance returns to normal, it is converted to negative pressure to continue to absorb the cell suspension.

9. A single cell distribution method according to claim 7, characterized in that: During the process of aspirating the cell suspension, due to misoperation, the cell suspension is not placed, resulting in the cell sensor (1) aspirating air. Since air is not conductive, the electrode cannot detect the electrical signal, and the system will report an error to remind the user.

10. A single cell distribution method according to claim 7, characterized in that: During the cell distribution process, impurities block the micropores of the micropore plate (12), and the circuit control system (42) detects the resistance change and controls the solenoid valve (22) to operate to form a negative pressure recoil to clear the blockage.

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