Transwell cell invasion counting analysis device and analysis method

Abstract

The invention discloses a Transwell cell invasion counting analysis device and analysis method. The Transwell cell invasion counting analysis device comprises a pneumatic test fixture table body, a power interface and a data interface, the pneumatic test fixture table body sequentially comprises a pneumatic pressing test device, a pneumatic sliding table clamp, a pneumatic test fixture table upper cover plate and a pneumatic test fixture table bakelite box from top to bottom, and the frame structure is constructed by bakelite plates; a pneumatic pressing test device and a pneumatic sliding table clamp are mounted above the pneumatic test fixture table upper cover plate, and the pneumatic sliding table clamp is used for clamping a cell culture experiment base; the mechanical action of the double-shaft sliding table cylinder and the pressing test device is controlled through induction instead of manual pressing test, so that the labor intensity is reduced, and convenience is brought to operators.

Description

Technical Field

[0001] The invention relates to the technical field of cell migration and invasion detection, and in particular to a Transwell chamber invasion counting analysis device and an analysis method. Background Art

[0002] The Transwell assay is an experimental technique that can evaluate both cell migration and invasion abilities. It uses the commercialized Transwell chamber as a standard experimental device and simulates the behavior of cells crossing the basement membrane to study the migration and invasion behaviors of cells in a more physiological state. It is widely used in oncology, immunology, cell biology and other fields.

[0003] Although the Transwell chamber can be used for a variety of experiments, the Transwell experiment based on the traditional optical counting method requires complex manual operations such as fixation, staining, wiping, drying, and counting. This method is invasive and harmful to cells. It is an endpoint detection method and cannot achieve real-time and continuous monitoring of the same sample group. It also has a low degree of automation. Secondly, the cells lose their activity after the experiment and cannot be used for subsequent applications. In addition, biochemical reagents are expensive, and the Transwell chamber cannot completely remove the reagents after the experiment is completed. They often have to be discarded, resulting in relatively high costs for Transwell experiments based on traditional optical counting methods.

[0004] Electrochemical impedance spectroscopy is considered to be a non-invasive, non-destructive, real-time automated quantitative detection tool, and is widely used in fields such as cell counting. However, the Transwell chamber in the traditional well plate cannot collect electrical signals, which limits the application of electrochemical analysis.

[0005] Therefore, combining and improving the Transwell experimental technology to develop a non-invasive, non-destructive, real-time Transwell chamber invasion automated counting and analysis device is of great value in improving the experimental efficiency of the Transwell experiment, reducing costs, and enhancing the accuracy and reliability of data. Summary of the invention

[0006] To solve the above problems, the present invention provides a Transwell chamber invasion counting analysis device and analysis method. The device can simultaneously collect and dynamically analyze the non-labeled electrochemical impedance spectroscopy information of all cells cultured in the Transwell and cells after invasion by switching electrode pairs in the experiment, and calculate the number of cells by theoretical model deduction, thereby evaluating the invasion ability of cells and realizing non-destructive and automated detection of cells.

[0007] The technical solution of the present invention is as follows:

[0008] A chamber invasion counting analysis device based on Transwell technology includes a pneumatic test fixture table, a cell culture experimental base, a gold-plated probe electrode, a forked electrode, a power module, a main controller module, an impedance spectrum acquisition module and an electrode switching module.

[0009] The pneumatic test fixture table includes a bakelite bottom frame, on which a test bracket for fixing a downward pressure test device and a pneumatic slide fixture is installed. All electrical function modules are integrated in the bakelite bottom frame, including a power module, a main controller module, an impedance spectrum acquisition module and an electrode switching module.

[0010] Furthermore, the pneumatic test fixture is equipped with a pair of test button switches and an emergency stop button. During the test, the experimenter needs to press the test button switch with both hands at the same time to start the device and start the test process. If only a single button is operated, the device will not start. This design effectively prevents misoperation. In the event of equipment out of control or other emergency situations, the experimenter can also stop the device by pressing the emergency stop button, thereby enhancing the safety of the experiment.

[0011] The pressure test device comprises a double-axis slide cylinder, a linear optical axis, a sliding bearing, and a pressure plate, on which a test probe and a spring pressure rod matching the test sample are arranged. During the test, the double-axis slide cylinder moves, driving the pressure plate to descend and ascend, so that the probe contacts the sample to complete the test.

[0012] Furthermore, the pressure plate is provided with slots matching the annular buckle at the top of the spring pressure rod at different positions and intervals, which can facilitate the experimenter to remove or install the spring pressure rod from the slot of the pressure plate to meet the clamping requirements of cell culture experimental bases of different types and sizes in actual experiments.

[0013] Preferably, the surface of the test probe electrode is gold-plated, which has excellent electrical properties and good conductivity, can reduce the contact impedance between the electrode and the sample to be tested, reduce measurement errors, and has good anti-interference performance and long-term stability.

[0014] The spring pressure rod comprises a structure of an annular buckle, a rubber gasket, a pressure sheet, a spring pressure rod housing, a pressure rod core, a spring, a limiting boss, a pressure rod body and a conical rubber pressure head.

[0015] The spring pressure rod housing is equipped with a pressure plate, a pressure rod core, a spring and a limiting boss, wherein the pressure plate is located between the annular buckle and the spring, one end of the pressure rod core moves in the housing and cooperates with the spring, and the limiting boss cooperates with one end of the pressure rod body to prevent the pressure rod body from separating from the housing. Rubber gaskets and conical rubber pressure heads are respectively installed at both ends of the pressure rod core to provide buffering to reduce the impact on the cell culture experimental base when the pressure plate is pressed down, thereby protecting the experimental base. In addition, the spring pressure rod is easy to install, and the position or model can be quickly changed through the annular buckle without disassembling the machine module.

[0016] The pneumatic slide fixture includes a double linear guide slide cylinder, a loading platform with groove positioning, and a loading clamp with a clamp handle. During the test, the double linear guide slide cylinder moves to drive the loading platform to move horizontally on the linear guide to complete the sample loading or sample removal.

[0017] Furthermore, the structure of the loading platform is similar to that of a microscope stage, and the cell culture medium is placed in a positioning groove of the loading platform. The loading clamp with a clamp handle is more convenient for the experimenter to operate with one hand than an ordinary loading clamp.

[0018] The cell culture experiment base includes a glass substrate, a PDMS (polydimethylsiloxane, hereinafter referred to as PDMS) base body and interdigital electrodes placed on the surface of the glass substrate. At least one Transwell plug-in hole structure is arranged in the base body, and its shape and size are designed according to the selected Transwell and are adapted to the outer contour and size of the selected Transwell. The Transwell chamber can be inserted into the mounting hole and fixed in the Transwell plug-in hole.

[0019] The interdigital electrodes are made by metal sputtering process, and thin metal layers such as gold and platinum with electrode shapes are deposited on the glass substrate by means of magnetic control, electric field and other technical means. The electrodes have excellent electrical properties, good conductivity, good anti-interference performance and long-term stability.

[0020] Optionally, the device can be provided with an Ethernet interface and a GPIB parallel interface to connect with various other instruments to form a small- to medium-scale automatic test system, or communicate with a host computer such as a computer to achieve fast data transmission. Furthermore, the present invention can also combine the connector with the use of a robotic arm to achieve automated replacement of the Transwell chamber.

[0021] The present invention discloses an innovative low-cost method for manufacturing a PDMS base body, based on any of the above-mentioned cell culture experimental bases, and further includes the following contents:

[0022] First, use CAD (computer-aided design, hereinafter referred to as CAD) software to construct the microchannel in the cell culture experimental base required for the experiment, and export the CAD model; load the CAD model into the 3D printing equipment for printing, and the printing material can be low-cost consumables such as PLA (polylactic acid, hereinafter referred to as PLA); use the printed CAD model as a mold, prepare the fixative and PDMS in a ratio of 1:10, pour them in the mold, and send them into a vacuum box for vacuum degassing; heat and cure the PDMS base; remove the cured PDMS base from the mold, and send it into a plasma cleaning machine together with the glass substrate for plasma treatment; take out the treated PDMS and glass, and close them to complete the bonding.

[0023] The present invention also discloses an analysis method of a Transwell chamber invasion counting analysis device, based on any of the above-mentioned Transwell chamber invasion counting analysis devices with electrode pair switching function, further comprising the following contents:

[0024] In a Transwell chamber cell invasion experiment, the Transwell chamber was placed vertically in a cell culture base, and the cells were migrating and invading from top to bottom. The observer could use the polycarbonate semipermeable membrane at the bottom of the Transwell chamber as a boundary to distinguish between cells that had invaded and those that had not. The interdigitated electrodes were placed directly below the Transwell chamber to detect the impedance response of the cells below the polycarbonate semipermeable membrane during the attachment process on the electrodes. The external probe electrode was fixed vertically above the Transwell chamber, and the conductive needle at the end of the probe electrode was immersed in the tissue fluid to form a circuit with any of the interdigitated electrodes at the bottom to detect the impedance response of the entire tissue fluid to be tested. During the experiment, the device can select different electrodes to connect to the test circuit through electrical switching. The above test electrodes only differ in appearance characteristics, and there is no difference in electrical function.

[0025] For the convenience of describing the principle, the total number of cells in the tissue fluid sample to be tested in the experimental device is recorded as N total , above the polycarbonate semipermeable membrane, the number of cells that did not invade was recorded as N up , below the polycarbonate semipermeable membrane, the number of cells that have invaded is recorded as N down , the results of electrochemical impedance spectroscopy are recorded as Z 1 and Z 2 . Then the number of cells in the experimental device can be expressed as formula (1).

[0026] N total =N up +N down (1)

[0027] At this time, the tissue fluid sample to be tested is directly tested through a pair of interdigital electrodes at the bottom. During the test, the device applies weak AC excitation to the sample through the interdigital electrodes. Since the electric field is concentrated on the electrode surface, the current circulation path is: one side interdigital electrode-tissue fluid near the bottom interdigital electrode-the other side interdigital electrode. When the area of ​​the interdigital electrode is appropriate, the electrochemical impedance test result can be derived from the theoretical model to approximately reflect the number of cells in the area below the polycarbonate semipermeable membrane, that is, the number of cells that have invaded. The function f(N) represents the specific algebraic formula of the impedance model, where N refers to the number of cells. The result of the electrochemical impedance test can be simply expressed in the form of formula (2).

[0028] Z 1 =f(N down ) (2)

[0029] The electrode pair is electrically switched so that the external test probe electrode and any one of the two interdigitated electrodes are electrically connected to the test circuit. At this time, the two electrodes are far apart, and the current circulation path is: any interdigitated electrode-part of the tissue fluid below the polycarbonate semipermeable membrane-polycarbonate membrane-part of the tissue fluid above the polycarbonate semipermeable membrane-probe electrode. Therefore, the electrochemical impedance test result at this time can be derived from the theoretical model to approximately reflect the sum of the number of cells in the area below the polycarbonate semipermeable membrane and the number of cells in the area above the polycarbonate semipermeable membrane, that is, the number of cells that have invaded. The function g(N) is used to represent the specific algebraic formula of the impedance model, where N refers to the number of cells. The result of the electrochemical impedance test at this time can be simply expressed in the form of formula (3).

[0030] Z 2 =g(N total ) (3)

[0031] In summary, through this theoretical analysis method of Transwell chamber cell counting analysis, experimenters can simultaneously test the number of cells that have undergone invasive behavior and the total number of cells in one test, and deduce the invasive ability of the cells.

[0032] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0033] Compared with the existing magnetic beads and fluorescent dyes that mark cells through cell surface proteins (biochemical means), which will cause cell death, the present invention uses electrochemical means, that is, accurate identification is achieved through electrical impedance. Different types of cells cause different electric field disturbances in the detection area, and there is no need for biochemical reagent labeling treatment, so their counting and identification can be achieved non-destructively.

[0034] The present invention integrates the Transwell chamber, sample processing system, electrode switching system, and electrical impedance detection system into a fixture test bench, greatly improving the integration of the entire system and avoiding the expensive and time-consuming defects of tumor cell identification based on biochemical methods, making tumor cell enrichment and detection easier to achieve, and ordinary personnel can operate it after simple training.

[0035] The impedance measurement part uses the four-wire Kelvin test method for precise impedance measurement. Combined with the design of switchable electrode interface, the system can adapt to different measurement environments and different interdigital electrodes, ensuring the accuracy, stability and repeatability of the measurement.

[0036] Compared with the operation steps that rely on manual intervention, such as the scratch test and microscopic counting, the present invention reduces the need for manual operation, reduces the subjective factors that may cause deviations in the experimental data, and significantly enhances the repeatability of the experiment, except for the necessary key control.

[0037] Compared with traditional manual fixture test instruments, the present invention adopts pneumatic method to realize the automated testing process. Through the above-mentioned pneumatic test fixture table, the operator can replace the manual pressing test by sensing and controlling the mechanical action of the dual-axis slide cylinder and the downward pressure test device after placing the cell culture experimental base, thereby reducing labor intensity and facilitating the operator.

[0038] In addition, the cell culture experimental base of the present invention is manufactured based on an innovative 3D printing mold and PDMS casting process. Furthermore, the material of the interdigitated electrode substrate can be replaced with PET (polyethylene terephthalate, hereinafter referred to as PET), and the low-cost electrode membrane products produced in batches can be directly bonded to the glass substrate using a simple process to further reduce costs.

[0039] The cell culture experiment base adopts a structure that can directly plug and replace the Transwell invasion chamber, which improves the reuse rate of the Transwell chamber. The consumables of each component are low in cost, and the processing technology is simple and easy to mass produce. At the same time, it realizes a disposable and disposable design, which makes up for the expensive shortcomings of existing integrated microfluidic chips. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 It is a schematic diagram of the structure of the pneumatic test fixture of the present invention.

[0041] Figure 2 This is a schematic structural diagram of the pneumatic downward pressure testing device of the present invention.

[0042] Figure 3 This is a schematic diagram of the spring pressure rod structure of the present invention.

[0043] Figure 4This is a schematic diagram of the structure of the pneumatic slide fixture of the present invention.

[0044] Figure 5 This is a schematic diagram of the structure of the cell culture experimental base of the present invention.

[0045] Figure 6 It is a schematic diagram of the test state of the pneumatic test fixture of the present invention.

[0046] Figure 7 This is a schematic diagram of the pneumatic test fixture of the present invention when replacing a sample.

[0047] Figure 8 This is a functional block diagram of the electrode pair switching principle of the present invention.

[0048] Main parts marking:

[0049] 1-Pneumatic test fixture body, 2-Pneumatic down-pressure test device, 3-Pneumatic slide fixture, 4-Cell culture experiment base, 5-Pneumatic test fixture cover, 6-Emergency stop button, 7-Folding hand, 8-Pneumatic test fixture bakelite box, 9-Test button switch, 10-Rubber foot pad, 21-Pneumatic test bench test bracket, 22-Dual-axis slide cylinder, 23-Linear optical axis, 24-Sliding bearing, 25-Pressing plate, 26-Test probe, 27-Spring pressure rod, 261-Ring buckle, 262-Rubber gasket, 263-Pressure Sheet, 264-spring pressure rod housing, 265-pressure rod core, 266-spring, 267-limiting boss, 268-pressure rod body, 269-conical rubber pressure head, 31-carrier clamp with clamp handle, 32-carrier platform with groove positioning, 33-linear guide slide cylinder, 41-PDMS base body, 42-Transwell invasion chamber body, 421-neck, 422-cup body, 423-polycarbonate semipermeable membrane with micropores, 44-glass substrate, 45-interdigitated electrode, 451-pad, 452-interdigitated. DETAILED DESCRIPTION

[0050] The technical solution of the present invention will be described clearly and completely below in conjunction with specific embodiments and drawings. In the description of the present invention, if there are terms indicating orientation or positional relationship such as "upper", "lower", "inner", "outer", etc., they are based on the orientation or positional relationship 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 "include" and "have" and any variations thereof are intended to cover non-exclusive inclusions, but may include other units that are not clearly listed or inherent to these products or devices.

[0051] Combination Figures 1 to 5 As shown, the embodiments disclose a Transwell chamber invasion counting analysis device (hereinafter referred to as "the invasion analysis device"). Figure 1 As shown, the invasion analysis device is mainly composed of a pneumatic test fixture table body (1), a pneumatic downward pressure test device (2), a pneumatic slide fixture (3), a cell culture experiment base (4), a pneumatic test fixture table cover (5), an emergency stop button (6), a foldable handheld device (7), a pneumatic test fixture table bakelite box (8), a test key switch (9), a rubber foot pad (10), an internal circuit module, a power interface and a data interface.

[0052] The pneumatic test fixture table body (1) comprises a pneumatic downward pressure test device (2), a pneumatic slide fixture (3), a pneumatic test fixture table upper cover (5), and a pneumatic test fixture table bakelite box (8); the frame structure is constructed of bakelite boards and has good insulation properties.

[0053] The front face of the pneumatic test fixture bakelite box (8) is equipped with an emergency stop button (6) and a pair of test key switches (9). The emergency stop button (6) is used to shut down the device in an emergency when the device is out of control or in other emergency situations, and the test key switches (9) are used to start the device to start the test process.

[0054] The two side surfaces of the pneumatic test fixture bakelite box (8) are symmetrically equipped with folding handles (7), which are convenient for experimenters to move the device and can be kept in a folded state when there is no need to move it, thereby reducing the occupied space.

[0055] The bottom of the pneumatic test fixture bakelite box (8) is provided with four rubber foot pads (10) for reducing vibration.

[0056] The pneumatic test fixture bakelite box (8) integrates all electrical function modules, including a power supply module, a main controller module, an impedance spectrum acquisition module and an electrode switching module.

[0057] The rear surface of the pneumatic test fixture bakelite box (8) is provided with a power interface and a data interface for external power supply and external communication of the device.

[0058] A pneumatic downward pressure test device (2) and a pneumatic slide table clamp (3) are installed above the cover plate (5) on the pneumatic test fixture table, and the pneumatic slide table clamp clamps the cell culture experiment base (4).

[0059] Combination Figure 2 As shown, the pneumatic downward pressure test device (2) mainly includes a pneumatic test bench test bracket (21), a dual-axis slide cylinder (22), a linear optical axis (23), a sliding bearing (24), a pressure plate (25), a test probe (26), and a spring pressure rod (27).

[0060] A double-axis slide cylinder (22) and a pair of linear optical axes (23) are installed on the crossbeam of the pneumatic test bench test bracket (21). Through the coordinated control of an external air source and an air valve switch, the pneumatic push rod in the double-axis slide cylinder (22) realizes telescopic movement, thereby driving the pressing plate (25) to move in a vertical direction. The pressing plate (25) is inlaid with a sliding bearing (24), and the linear optical axis (23) serves to guide the sliding bearing (24) to ensure that the pressing plate (25) realizes vertical linear movement along the guidance of the linear optical axis (23).

[0061] A test probe (26) and a spring pressure rod (27) are mounted on the pressure plate, wherein the test probe (26) is used to form a second test current loop outside the interdigital electrode pair to detect the overall electrochemical impedance of the sample, and the spring pressure rod (27) is used to press the cell culture experimental base (4) to minimize the vibration of the device during the test process, which may cause the cell culture experimental base (4) to deviate, cause the electrode to shift, and cause measurement errors.

[0062] Combination Figure 3 As shown, the spring pressure rod (27) comprises an annular buckle (261), a rubber gasket (262), a pressure sheet (263), a spring pressure rod housing (264), a pressure rod core (265), a spring (266), a limiting boss (267), a pressure rod body (268), and a conical rubber pressure head (269).

[0063] The spring pressure rod housing (264) is provided with a pressure plate (263), a pressure rod core (265), a spring (266), and a limiting boss (267); the pressure plate (263) is arranged between the annular buckle (261) and the spring (266); one end of the pressure rod core (265) is movably fitted in the spring pressure rod housing (264) and cooperates with the spring (266); the spring pressure rod housing (264) is provided with a limiting boss (267) which cooperates with one end of the pressure rod body (268); the setting of the limiting boss (267) limits the pressure rod body (268) to prevent the pressure rod body (268) from being separated from the spring pressure rod housing (264). The spring pressure rod housing (264) is also provided with a rubber gasket (262) and a conical rubber pressure head (269) on the outside to provide a buffering effect, thereby reducing the impact caused by the spring pressure rod (27) contacting the cell culture experiment base (4) when the pressure plate (25) is pressed down during the test process, thereby better protecting the cell culture experiment base (4).

[0064] In addition, the spring pressure rod (27) is easy to install, and the position or model of the spring pressure rod (27) can be changed by disengaging and fastening the annular buckle (261) on the spring pressure rod (27) without disassembling the machine module.

[0065] Combination Figure 4 As shown, the pneumatic slide fixture (3) comprises a pair of carrier clamps (31) with clamping handles, a carrier platform (32) with groove positioning, and a pair of linear guide slide cylinders (33).

[0066] The loading platform (32) with groove positioning is installed on the slide of a pair of linear guide slide cylinders (33). Through the coordinated control of an external air source and an air valve switch, the pneumatic slider in the linear guide slide cylinder (33) realizes linear motion, thereby driving the loading platform (32) with groove positioning to move horizontally on the linear guide. At the same time, a pair of loading clamps (31) with clamp handles are installed on the groove edge of the loading platform (32) with groove positioning, which are used to clamp and tighten the cell culture experiment base (4) after placing the cell culture experiment base (4) in the groove of the loading platform (32), so as to minimize the vibration of the device caused by mechanical action during the test process, so as to cause the cell culture experiment base (4) to deviate, so that the electrode is displaced, and the measurement error occurs.

[0067] Combination Figure 5 As shown, the cell culture experimental base (4) in the embodiment includes a PDMS base body (41), a Transwell invasion chamber body (42), a Transwell plug-in hole (43), a glass substrate (44), and interdigital electrodes (45).

[0068] The main structure of the cell culture experimental base (4) is composed of a glass substrate (44) bonded to a PDMS base body (41). A Transwell plug-in hole (43) structure is cast in the PDMS base body (41). Its shape and size are designed according to the selected Transwell invasion chamber body (42) model, and are compatible with the outer contour and size of the selected Transwell invasion chamber body (42). The Transwell invasion chamber body (42) can be inserted into the Transwell plug-in hole (43) and fixed therein. The Transwell invasion chamber body (42) can be directly purchased as a Transwell standard part according to demand, or it can be customized according to demand. The Transwell invasion chamber body (42) usually has a polycarbonate semipermeable membrane (423) with micropores, a cup body (422) and a neck (421) connected in sequence from bottom to top. Among them, the cup body (422) is mainly used for culturing cells; the cup body (422) is usually a cylindrical structure, the side wall of which is made of polypropylene, and the bottom is a polycarbonate semipermeable membrane (423) with micropores, and the pore size of the semipermeable membrane is about 0.4μm. The polycarbonate semipermeable membrane (423) with micropores realizes the material exchange between the cup body (422) and the cell culture fluid perfusion channel below the semipermeable membrane, as well as the invasion and migration behavior of the cells in the cup.

[0069] The bottom of the PDMS base body (41) is a forked electrode (45), including a pad (451) and a forked finger (452), wherein the pad (451) is used for connecting the circuit of the test loop, and the forked finger (452) is used for sensing the cell impedance. The forked electrode (45) is a low-cost electrode film finished product produced in batches by depositing a metal thin layer of gold, platinum or the like in an electrode shape on a glass substrate through a sputtering process, or using PET material as a substrate, and is directly bonded to the glass substrate (44) by a simple process when in use.

[0070] Reference Figure 8 As shown, the Transwell chamber invasion counting analysis device of the present invention is composed of the following electrical functional modules: a power supply module, a main controller module, an impedance spectrum acquisition module and an electrode switching module.

[0071] Among them, the main function of the power module is power conversion, which is responsible for providing power to all modules; the main controller module is responsible for communicating with the computer host, controlling the logic switching of the electrode switching module and the acquisition of the impedance spectrum acquisition module; the principles of the electrode switching module and the impedance acquisition module mainly involve the following components: switching circuit, analog front-end circuit and corresponding A / D and D / A conversion circuit. Among them, the switching circuit is responsible for switching the electrical connection of the interdigital electrode or the probe electrode into the test loop; the analog front-end circuit is at the signal input end, and is composed of an I / V transimpedance amplifier circuit, a post-stage amplifier circuit and a differential amplifier circuit, which is responsible for the circuit module of analog signal conditioning; the A / D and D / A conversion circuit is responsible for realizing the conversion between analog signals and digital signals, among which the A / D conversion circuit converts the analog signal into a digital signal for subsequent digital signal processing; the D / A conversion circuit converts the digital signal used for control into an analog signal to apply excitation to the test sample.

[0072] Combination Figure 6 , Figure 7 , Figure 8 As shown, in the experiment, the operation process of the Transwell chamber invasion counting analysis device of the present invention is as follows:

[0073] The specific steps include:

[0074] First, the device is connected to a power source and started up, the circuit system of the device starts initialization, and then the mechanical structure is driven to enter a reset state.

[0075] like Figure 7 As shown, in the reset state, no compressed air enters the dual-axis slide cylinder (22), the pneumatic push rod shrinks to the initial state, the pressure plate (25) is at the top, and at the same time, no compressed air enters the linear guide slide cylinder (33), the cylinder piston is in the initial position, and the slider is fixed at the starting point of the slide rail, that is, the side close to the edge of the cover plate (5) on the pneumatic test fixture table.

[0076] When starting the experiment, the experimenter prepares the cell culture experimental base (4), inserts the Transwell invasion chamber body (42) into the Transwell plug hole (43), adds the upper culture fluid into the Transwell invasion chamber body (42), and adds the lower culture fluid into the Transwell plug hole (43). The cells to be studied are planted in the Transwell invasion chamber body (42), and the cells are cultured for a period of time.

[0077] Next, the experimenter gently places the cell culture experiment base (4) in the positioning groove of the carrier platform (32) with a groove positioning, and rotates a pair of carrier clamps (31) with clamp handles counterclockwise so that the clamp arms of the carrier clamps (31) are pressed against the glass substrate (44) of the cell culture experiment base (4), thereby clamping and fastening the cell culture experiment base (4).

[0078] After being ready, the experimenter presses a pair of test button switches (9) located on the front surface of the pneumatic test fixture bakelite box (8) with both hands at the same time to start the device and start the test process.

[0079] The valve of the linear guide slide cylinder (33) is opened, compressed air enters, and the cylinder piston moves to drive the pneumatic slider in the linear guide slide cylinder (33) to achieve linear motion, thereby pushing the loading platform (32) with groove positioning to move horizontally on the linear guide until it moves to the end position of the slide rail and is fixed. Figure 1 As shown, at this time, the cell culture experimental base (4) is located below the pressure plate (25), and the Transwell invasion chamber body (42) and the test probe (26) are coaxial.

[0080] Next, the valve of the dual-axis slide cylinder (22) is opened to allow compressed air to flow in. This allows the pneumatic push rod to extend and drive the pressure plate (25) to move in the vertical direction. The pressure plate (25) is equipped with a sliding bearing (24), and the function of the linear optical axis (23) is to provide a guide for the sliding bearing (24) to ensure that the pressure plate (25) can move linearly downward along the guide direction of the linear optical axis (23). This process will continue until the pneumatic push rod of the dual-axis slide cylinder (22) is in place and the pressure plate (25) smoothly reaches its set position.

[0081] During the process of the pressing plate (25) moving downward in a straight line, the conical rubber pressing head (269) of the spring pressing rod (27) first contacts the glass substrate (44) on the cell culture experimental base (4). As the pressing plate (25) continues to move downward, the spring (266) in the spring pressing rod housing (264) begins to be compressed, and the elastic force generated thereby acts on the conical rubber pressing head (269) to provide additional downward pressure thereon. This design helps to more firmly fix the cell culture experimental base (4) and minimize vibrations caused by mechanical movement, which may cause the cell culture experimental base (4) to deviate, thereby causing changes in electrode position and measurement errors.

[0082] After the pneumatic push rod is fully extended and drives the pressing plate (25) to move downward to the set position, the valve of the double-axis slide cylinder (22) is closed, the movement is stopped and fixed. At this time, the top of the test probe (26) is inserted into the cup body (422), immersed in the cell culture fluid, close to the polycarbonate semipermeable membrane (423) with micropores but not in direct contact with it.

[0083] The final device test status is as follows Figure 6 shown.

[0084] Connect the computer to the device via a USB or other communication interface, start the host computer software, establish a communication link with the main controller module of the device, issue operating instructions to control the device to complete collection and other operations, and transmit the data collected by the device back to the computer.

[0085] The functional block diagram of the electrode pair switching principle of the present invention is as follows: Figure 8 As shown, after the host computer sends a test instruction to the main controller module, the main controller controls the electrode switching module to switch the circuit, and electrically connects the required test sample with the test circuit in the impedance spectrum acquisition module to form a test loop. The main controller controls the D / A conversion circuit in the impedance spectrum acquisition module to perform frequency sweep excitation, and the signal is drawn from interface 4 to the electrode switching port 2 and applied to the sample. After the weak excitation current flows through the sample, it returns from the electrode switching interface to the electrode switching interface 1, and is transmitted to the I / V transimpedance amplifier circuit in the impedance spectrum acquisition module through interface 1; and interface 2 and interface 3 respectively introduce the differential voltage between the electrode switching port 1 and the electrode switching interface 2, that is, the AC potential difference signal at both ends of the sample into the differential amplifier circuit in the impedance spectrum acquisition module.

[0086] The analog front-end circuit of the impedance spectrum acquisition module uses a differential voltage amplifier circuit and an IV transimpedance amplifier circuit to perform low-noise amplification on the AC potential difference signal generated at both ends of the sample after excitation and the weak AC current signal passing through the sample, and converts the response signal into a suitable voltage range for sampling by the subsequent A / D conversion circuit, thereby improving the signal-to-noise ratio.

[0087] Finally, the main controller processes the current and voltage sequence data sampled by the impedance spectrum acquisition module, and calculates and analyzes the electrochemical impedance spectrum information of the sample.

[0088] When the device is in the initial state, the switching circuit in the electrode switching module connects the interdigital electrode 1 and the interdigital electrode 2 to the main test circuit to form a current path 1, which is: interdigital electrode 1-tissue fluid near the bottom interdigital electrode-interdigital electrode 2. The electrochemical impedance test results can be deduced based on the theoretical model to approximately reflect the number of cells in the area below the polycarbonate semipermeable membrane, that is, the number of cells that have invaded.

[0089] After the test of the interdigital electrode pair is completed, the host computer sends a switching instruction to the main controller module to control the switching circuit action in the electrode switching module, and switches the electrode switching interface from the interdigital electrode 1 to the probe electrode. At this time, the interdigital electrode 2 and the probe electrode form a current path 2, and the specific path is: interdigital electrode 2-part of the tissue fluid below the polycarbonate semipermeable membrane-polycarbonate membrane-part of the tissue fluid above the polycarbonate semipermeable membrane-probe electrode. At this time, the electrochemical impedance test results can be derived from the theoretical model to approximately reflect the sum of the number of cells in the area below the polycarbonate semipermeable membrane and the number of cells in the area above the polycarbonate semipermeable membrane, that is, the number of cells that have invaded.

[0090] After all the test processes are completed, the experimenter once again presses a pair of test button switches (9) located on the front surface of the pneumatic test fixture bakelite box (8) with both hands to start the device reset operation and end the test process.

[0091] At this time, the main controller module transmits all the data results of measurement and analysis to the host computer. After the transmission is completed, the main controller module controls all electrical function modules to enter sleep mode.

[0092] The exhaust valve of the double-axis slide cylinder (22) is opened, compressed air flows out, and the pneumatic push rod can be retracted to drive the pressure plate (25) to move to the starting point reset state in the vertical direction and fix it. Then the exhaust valve of the linear guide slide cylinder (33) is opened, compressed air flows out, and the cylinder piston moves to drive the pneumatic slider in the linear guide slide cylinder (33) to achieve linear motion, thereby pushing the loading platform (32) with groove positioning to move horizontally on the linear guide until it moves to the starting point reset state of the slide rail and fixes it.

[0093] After the mechanical reset action is completely completed, the experimenter rotates a pair of carrier clamps (31) with clamp handles clockwise to remove the cell culture experiment base (4) from the positioning groove of the carrier platform (32) with groove positioning, and can continue to carry out other subsequent processing.

[0094] At this point, the operation process of the device is completely completed.

[0095] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above specific embodiments. The above specific embodiments and the description in the specification are only for further illustrating the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of the present invention to be protected is defined by the claims and their equivalents.

Claims

1. A Transwell chamber invasion counting analysis device, characterized in that: It comprises a pneumatic test fixture body (1), a power interface and a data interface; The pneumatic test fixture table body (1) comprises, from top to bottom, a pneumatic downward pressure test device (2), a pneumatic sliding table clamp (3), a pneumatic test fixture table upper cover plate (5), and a pneumatic test fixture table bakelite box (8), and the frame structure is constructed of bakelite boards; the pneumatic downward pressure test device (2) and the pneumatic sliding table clamp (3) are installed above the pneumatic test fixture table upper cover plate (5), and the pneumatic sliding table clamp clamps a cell culture experiment base (4); The front face of the pneumatic test fixture bakelite box (8) is equipped with an emergency stop button (6) and a pair of test key switches (9). The emergency stop button (6) is used to shut down the device in an emergency when the device is out of control or in other emergency situations, and the test key switches (9) are used to start the device to start the test process. The pneumatic test fixture bakelite box (8) integrates all electrical function modules, including a power supply module, a main controller module, an impedance spectrum acquisition module and an electrode switching module; the power supply module supplies power to the main controller module, the impedance spectrum acquisition module and the electrode switching module, and the main controller module is responsible for communicating with the computer host computer to control the switching of the electrode switching module and the acquisition of the impedance spectrum acquisition module; The rear surface of the pneumatic test fixture bakelite box (8) is provided with a power interface and a data interface for external power supply and external communication of the device.

2. A Transwell chamber invasion counting analysis device according to claim 1, characterized in that: The two side surfaces of the pneumatic test fixture bakelite box (8) are symmetrically equipped with folding handles (7), which are convenient for experimenters to move the device and can be kept in a folded state when there is no need to move it, thereby reducing the occupied space.

3. A Transwell chamber invasion counting analysis device according to claim 1, characterized in that: The bottom of the pneumatic test fixture bakelite box (8) is provided with four rubber foot pads (10) for reducing vibration.

4. A Transwell chamber invasion counting analysis device according to claim 1, characterized in that: The pneumatic downward pressure test device (2) comprises a pneumatic test bench test bracket (21), a double-axis slide cylinder (22), a linear optical axis (23), a sliding bearing (24), a pressure plate (25), a test probe (26), and a spring pressure rod (27); A double-axis slide cylinder (22) and a pair of linear optical axes (23) are installed on the crossbeam of the pneumatic test bench test bracket (21). Through the coordinated control of an external air source and an air valve switch, the pneumatic push rod in the double-axis slide cylinder (22) realizes telescopic movement, thereby driving the lower end pressure plate (25) to move in the vertical direction. The pressure plate (25) is inlaid with a sliding bearing (24), and the function of the linear optical axis (23) is to guide the sliding bearing (24) to ensure that the pressure plate (25) realizes vertical linear movement along the guidance of the linear optical axis (23); A test probe (26) and a spring pressure rod (27) are mounted on the pressure plate, wherein the test probe (26) is used to form a second test current loop outside the interdigital electrode pair to detect the overall electrochemical impedance of the sample, and the spring pressure rod (27) is used to press the cell culture experimental base (4) to minimize the vibration of the device during the test process, which may cause the cell culture experimental base (4) to deviate, cause the electrode to shift, and cause measurement errors.

5. A Transwell chamber invasion counting analysis device according to claim 4, characterized in that: The spring pressure rod (27) comprises an annular buckle (261), a rubber gasket (262), a pressure sheet (263), a spring pressure rod housing (264), a pressure rod core (265), a spring (266), a limiting boss (267), a pressure rod body (268), and a conical rubber pressure head (269); The spring pressure rod housing (264) is provided with a pressure sheet (263), a pressure rod core (265), a spring (266), and a limiting boss (267); the pressure sheet (263) is arranged between the annular buckle (261) and the spring (266); one end of the pressure rod core (265) is movably fitted in the spring pressure rod housing (264) and matched with the spring (266); the spring pressure rod housing (264) is provided with a limiting boss (267) matched with one end of the pressure rod body (268) The setting of the limiting boss (267) limits the position of the pressure rod body (268) to prevent the pressure rod body (268) from being separated from the spring pressure rod housing (264). A rubber gasket (262) and a conical rubber pressure head (269) are also installed outside the spring pressure rod housing (264) to provide a buffering effect, thereby reducing the impact caused by the spring pressure rod (27) contacting the cell culture experimental base (4) when the pressure plate (25) is pressed down during the test process, thereby better protecting the cell culture experimental base (4); The spring pressure rod (27) is easy to install, and the position or model of the spring pressure rod (27) can be changed by disengaging and fastening the annular buckle (261) on the spring pressure rod (27) without disassembling the machine module.

6. A Transwell chamber invasion counting analysis device according to claim 1, characterized in that: The pneumatic slide fixture (3) comprises a pair of object clamps (31) with clamp handles, an object platform (32) with groove positioning, and a pair of linear guide slide cylinders (33); The loading platform (32) with groove positioning is installed on the slide of a pair of linear guide slide cylinders (33). Through the coordinated control of an external air source and an air valve switch, the pneumatic slider in the linear guide slide cylinder (33) realizes linear motion, thereby driving the loading platform (32) with groove positioning to move horizontally on the linear guide. At the same time, a pair of loading clamps (31) with clamp handles are installed on the groove edge of the loading platform (32) with groove positioning, which are used to clamp and tighten the cell culture experiment base (4) after placing the cell culture experiment base (4) in the groove of the loading platform (32), so as to minimize the vibration of the device caused by mechanical action during the test, which causes the cell culture experiment base (4) to be offset, resulting in electrode displacement and measurement errors.

7. A Transwell chamber invasion counting analysis device according to claim 1, characterized in that: The cell culture experimental base (4) comprises a PDMS base body (41), a Transwell invasion chamber body (42), a Transwell plug-in hole (43), a glass substrate (44), and interdigital electrodes (45); The main structure of the cell culture experimental base (4) is composed of a glass substrate (44) and a PDMS base body (41) bonded together. A Transwell plug-in hole (43) structure is cast in the PDMS base body (41). The shape and size of the Transwell plug-in hole (43) are designed according to the selected Transwell invasion chamber body (42) model and are adapted to the outer contour and size of the selected Transwell invasion chamber body (42). The Transwell invasion chamber body (42) can be inserted into the Transwell plug-in hole (43) and then fixed therein. The Transwell invasion chamber body (42) can be directly purchased from Tra Transwell standard parts can also be customized according to needs; the Transwell invasion chamber body (42) usually has a polycarbonate semipermeable membrane with micropores (423), a cup body (422) and a neck (421) connected in sequence from bottom to top; wherein, the cup body (422) is mainly used for culturing cells; the cup body (422) is usually a cylindrical structure, the side wall of which is made of polypropylene, and the bottom is a polycarbonate semipermeable membrane with micropores (423), the pore size of the semipermeable membrane is about 0.4μm, and the polycarbonate semipermeable membrane with micropores (423) is used to realize the material exchange between the cup body (422) and the cell culture fluid perfusion channel below the semipermeable membrane, as well as the invasion and migration behavior of the cells in the cup.

8. A Transwell chamber invasion counting analysis device according to claim 7, characterized in that: The bottom of the PDMS base body (41) is a forked electrode (45), including a pad (451) and a forked finger (452). The pad (451) is used for connecting the circuit of the test circuit, and the forked finger (452) is used as a sensor of cell impedance. The forked electrode (45) is a low-cost electrode film finished product produced in batches by depositing a metal thin layer of gold, platinum or the like in an electrode shape on a glass substrate through a sputtering process, or using PET material as a substrate. When in use, it is directly bonded to the glass substrate (44) using a simple process.

9. An analysis method for a Transwell chamber invasion counting analysis device, characterized in that: The Transwell chamber invasion counting analysis device with electrode pair switching function according to any one of claims 1 to 8, further comprising the following method: In a Transwell chamber cell invasion experiment, the Transwell chamber was placed vertically in a cell culture base. The cells were migrating and invading from top to bottom. The observer could use the polycarbonate semipermeable membrane at the bottom of the Transwell chamber as a boundary to distinguish between cells that had invaded and those that had not. The interdigital electrodes are placed directly below the Transwell chamber to detect the impedance response of cells below the polycarbonate semipermeable membrane during their attachment to the electrodes. The external probe electrode is vertically fixed above the Transwell chamber, and the conductive needle at the end of the probe electrode is immersed in the tissue fluid to form a loop with any interdigital electrode at the bottom to detect the impedance response of the entire tissue fluid to be tested. During the experiment, the device can select different electrodes to connect to the test circuit through electrical switching, and the total number of cells in the tissue fluid sample to be tested in the experimental device is recorded as N total , the number of cells above the polycarbonate semipermeable membrane that did not invade was recorded as N up , below the polycarbonate semipermeable membrane, the number of cells that have invaded is recorded as N down , the results of the electrochemical impedance test are recorded as Z1 and Z2 respectively, then the number of cells in the experimental device can be expressed as formula (1): N total =N up +N down (1) At this time, the tissue fluid sample to be tested is directly tested through a pair of interdigital electrodes at the bottom. During the test, the device applies weak AC excitation to the sample through the interdigital electrodes. Since the electric field is concentrated on the electrode surface, the current circulation path at this time is: one side of the interdigital electrode-tissue fluid near the bottom interdigital electrode-the other side of the interdigital electrode. When the area of ​​the interdigital electrode is appropriate, the electrochemical impedance test results can be deduced according to the theoretical model to approximately reflect the number of cells in the area below the polycarbonate semipermeable membrane, that is, the number of cells that have invaded; The function f(N) is used to represent the specific algebraic formula of the impedance model, where N refers to the number of cells. The result of the electrochemical impedance test can be simply expressed in the form of formula (2): Z1=f(N down ) (2) The electrode pair is electrically switched so that the external test probe electrode and any one of the two interdigitated electrodes are electrically connected to the test circuit. At this time, the two electrodes are far apart, and the current circulation path is: any interdigitated electrode-part of the tissue fluid below the polycarbonate semipermeable membrane-polycarbonate membrane-part of the tissue fluid above the polycarbonate semipermeable membrane-probe electrode. Therefore, the electrochemical impedance test result at this time can be deduced based on the theoretical model to approximately reflect the sum of the number of cells in the area below the polycarbonate semipermeable membrane and the number of cells in the area above the polycarbonate semipermeable membrane, that is, the number of cells that have invaded; the function g(N) is used to represent the specific algebraic formula of the impedance model, where N refers to the number of cells. The result of the electrochemical impedance test at this time can be simply expressed in the form of formula (3); Z2=g(N total ) (3) Through this theoretical analysis method of Transwell chamber cell counting analysis, experimenters can simultaneously test the number of cells that have undergone invasive behavior and the total number of cells in one test, and deduce the invasive ability of the cells.

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