Cell and organoid long-acting self-culture drug-excited micro-fluidic chip integrated system and detection method using same

Abstract

The invention relates to a cell and organ-like long-acting self-culture drug excitation micro-fluidic chip integrated system and a detection method using the system, and belongs to the technical field of micro-fluidic chips. The system comprises an air compressor, a pressure controller, a multi-path manifold, a liquid storage tank, a PTFE guide pipe, a liquid distribution valve, a Coriolis flowmeter, a flow resistance pipe, a two-path switching valve, a micro-fluidic chip, a three-path switching valve, a collection tank and the like, the air compressor feeds a reagent into the PTFE guide pipe from the liquid storage tank through pressure via the pressure controller and the multi-path manifold, then the reagent is distributed based on the liquid distribution valve, and the collection tank is used for collecting the reagent. And the distributed liquid enters the micro-fluidic chip through the Coriolis flowmeter, the flow resistance tube, the multi-path manifold and the two-path switching valve, and finally enters the collection pool or the waste liquid pool through the three-path switching valve. According to the system, through cooperative work of the components such as the air compressor, the pressure controller, the multi-path manifold and the liquid distribution valve, a reagent can be accurately fed and distributed from the liquid storage tank in a pressure mode. And the quantity and the flow velocity of the liquid entering the micro-fluidic chip are more accurate.

Description

Technical Field

[0001] The present invention belongs to the technical field of microfluidic chips, and in particular relates to an integrated system of long-acting self-stimulated microfluidic chips for cells and organoids and a detection method using the system. Background Art

[0002] Among the related technologies, in the field of biomedical research, cell and organoid culture and drug stimulation experiments play a key role in gaining a deeper understanding of life processes, disease mechanisms, and drug development. In recent years, microfluidics has gradually emerged as a cutting-edge technology.

[0003] Based on microfluidic technology, researchers hope to achieve accurate simulation and fine regulation of the growth environment of cells and organoids, and thus achieve long-term autonomous cultivation and precise drug stimulation at a microscale. Microfluidic chips provide a three-dimensional microenvironment close to the physiological environment for cell culture, which can theoretically highly restore the complex cell-to-cell interactions and material exchange processes in the body. For example, through the tiny channels designed in the chip, the supply of nutrients, oxygen, etc. to cells can be accurately controlled to maintain their long-term stable growth state.

[0004] However, in actual application, relying solely on microfluidic technology has caused many thorny problems. On the one hand, the accuracy of fluid manipulation is difficult to guarantee. Most traditional microfluidic systems use simple pressure drive or injection pump drive, lacking an accurate pressure feedback regulation mechanism, which causes large fluctuations in flow rate and pressure when reagents enter the microfluidic chip. Different types of cells and organoids are extremely sensitive to fluid mechanics parameters. Slight deviations in flow rate and pressure may change the growth morphology and proliferation rate of cells and even cause cell death, seriously affecting the repeatability and accuracy of the experiment.

[0005] On the other hand, liquid distribution and management are chaotic. Early microfluidic devices lacked flexibility in reagent distribution and were unable to accurately distribute multiple reagents to specific areas within the chip at different times according to complex experimental designs. Moreover, the waste liquid generated by the experiment and the samples that needed to be collected were difficult to effectively distinguish and properly handle, and the experimental process often needed to be interrupted and tedious manual operations performed, which not only reduced experimental efficiency, but also easily introduced human contamination and interfered with experimental results.

[0006] Furthermore, low system integration is also a major obstacle. Although microfluidic chips are powerful, when working with external power sources, liquid storage devices, monitoring equipment, etc., they are often fragmented and messy due to problems such as interface incompatibility and poor signal transmission. Professionals need to spend a lot of time debugging and installing, which makes it difficult to meet the urgent needs of scientific research frontline for efficient and convenient experimental equipment. Summary of the invention

[0007] In view of the above deficiencies in the prior art, the purpose of the invention is to provide an integrated system of long-term self-cultured drug-stimulated microfluidic chips for cells and organoids.

[0008] In the first aspect of the present invention, a long-acting self-cultured drug-stimulated microfluidic chip integrated system for cells and organoids is proposed, comprising: an air compressor, a pressure controller, a multi-way manifold, a liquid reservoir, a PTFE catheter, a liquid dispensing valve, a Coriolis flowmeter, a flow resistance tube, a two-way switching valve, a microfluidic chip, a three-way switching valve, a collection tank and a waste liquid tank, wherein the air compressor presses the reagent from the liquid reservoir into the PTFE catheter through the pressure controller and the multi-way manifold, and then distributes it based on the liquid dispensing valve, and the distributed liquid enters the microfluidic chip via the Coriolis flowmeter, the flow resistance tube, the multi-way manifold, the two-way switching valve, and finally enters the collection tank or the waste liquid tank through the three-way switching valve.

[0009] Furthermore, the microfluidic chip is arranged on the first component, the two-way switching valve is arranged on the second component, the three-way switching valve is arranged on the third component, the second component is connected to the support, the Coriolis flowmeter is arranged on the support, and the liquid dispensing valve is arranged at one end of the fourth component.

[0010] Furthermore, the first component, the second component, the third component, the fourth component, and the support member are all arranged at the bottom of the air compressor, and the collecting tank is arranged at the outer side of the air compressor.

[0011] Furthermore, a preset number of fixing parts are provided on the outer side of the air compressor, and the fixing parts are used to fix the collection tank.

[0012] Furthermore, the microfluidic chip is prepared on the PDMS material by using photolithography technology, wherein the silicon wafer is cleaned with piranha solution before photolithography, and then rinsed with deionized water, SU-8 photoresist is spin-coated on the surface of the silicon wafer to prepare the microfluidic chip with a thickness of 100 μm, wherein the SU-8 photoresist is irradiated with ultraviolet light to promote a cross-linking reaction, and the unexposed area will be dissolved. After exposure, it is placed on a hot stage for drying, and then the uncross-linked part is removed with a developer, and the silicon wafer is cleaned with isopropyl alcohol and deionized water, and finally the film is cured.

[0013] Furthermore, the microfluidic chip molding process includes silanization treatment of the mold surface, placing the silicon wafer mold into a dryer, using tin foil to make a container, adding a preset amount of perfluorooctyltrichlorosilane, vacuuming and silanization treatment for a preset time to ensure that the silicon wafer alkane vapor evenly covers the mold surface to achieve hydrophobic treatment.

[0014] Furthermore, the operation flow of the microfluidic chip reverse molding processing includes: weighing PDMS and coagulant with a mass ratio of 10:1 with a balance, stirring thoroughly with a glue homogenizer, casting the PDMS solution on the mold, placing it in a vacuum dryer, evacuating until there are no bubbles inside the solution, and then placing it in a heating box at 60 degrees for more than two hours, taking it out and cooling it to room temperature, cutting the chip along the silicon wafer with a scalpel, selecting a puncher of corresponding size to punch holes at the inlet and outlet of the microfluidic chip, and then placing the microfluidic chip with the channel facing up on a glass plate at the same time as the glass sheet for bonding, placing it in a plasma cleaning machine, processing it for two minutes, taking out the microfluidic chip and the glass sheet, fitting the upward sides of the two, placing them in a 60-degree heating box and heating them for more than 2 hours to complete the bonding process.

[0015] Furthermore, the airtightness of the microfluidic chip is detected based on an airtightness detection method, wherein the airtightness of the microfluidic chip is detected based on the airtightness detection method, including: filling the microfluidic chip to be tested with compressed air gas of a preset pressure; monitoring the pressure change of the compressed air gas based on a pressure sensor, and judging whether the sealing of the microfluidic chip to be tested is qualified based on the pressure change.

[0016] Furthermore, the flow loss of the microfluidic chip is detected based on the injection pump, wherein the flow loss of the microfluidic chip is detected based on the injection pump, including: using the injection pump to drive the reagent into the microfluidic chip, and using multiple flow rates to detect the flow loss of the microfluidic chip, wherein the multiple flow rates include high speed, medium speed and low speed; when the flow loss is lower than a preset threshold, it is determined that the flow loss of the microfluidic chip to be tested is qualified.

[0017] In the second aspect of the present invention, a microfluidic chip cell culture and activity detection method is proposed, using the above-mentioned cell and organoid long-acting self-culture drug to stimulate the microfluidic chip integrated system, including: passing 75% ethanol into the prepared microfluidic chip and irradiating it with ultraviolet light for 1 hour for disinfection; using a syringe pump to pass cell culture fluid into the microfluidic chip for 2 hours to eliminate residual ethanol and bubbles in the microfluidic chip, which is conducive to the cell growth in the culture chamber; using a live / dead assay kit to evaluate the viability of cells cultured in the microfluidic chip, incubating the cells in a culture medium solution containing 2 μL calcein AM solution and 5 μL ethidium homodimer-1 solution at 37 degrees Celsius for 30 minutes, then rinsing the culture chamber with PBS for 3-5 minutes to remove the reagents and observing under a fluorescence microscope, and calculating the percentage of cell viability based on dividing the number of live cells by the total number of cells.

[0018] The beneficial effects of the present invention are as follows: The cell and organoid long-acting self-cultivation drug-stimulated microfluidic chip integrated system described in the present invention can accurately pressurize and distribute reagents from the liquid reservoir through the coordinated work of components such as an air compressor, a pressure controller, a multi-way manifold, and a liquid distribution valve. The use of a Coriolis flowmeter further ensures the accurate measurement and control of the liquid flow, so that the amount and flow rate of the liquid entering the microfluidic chip are more accurate, which is conducive to the accurate supply of nutrients and drugs during the cell and organoid culture process. Microfluidic chips are prepared by photolithography technology, and a series of steps from silicon wafer cleaning, spin coating of photoresist to ultraviolet light irradiation, drying, development and hard film curing ensure the high precision of the chip structure. For example, a 100μm thick chip is prepared, and the pattern is stable, clear and residue-free, providing a good microenvironmental foundation for cell and organoid culture. The silanization treatment during the mold processing can make the mold surface hydrophobic and improve the quality of the chip. By strictly controlling the mass ratio of PDMS to coagulant, as well as operations such as casting, vacuuming, heating, cutting, punching, plasma cleaning and bonding, the physical and chemical properties of the microfluidic chip are ensured to be good, which is conducive to the long-term and stable use of the chip. By filling the microfluidic chip with compressed air, the pressure sensor is used to monitor the pressure change to detect the airtightness. The Elveflow microfluidic multi-channel microfluidic pressure controller can be used for testing at higher pressures, such as up to 2000mbar. By comparing the pressure sensor measurement value with the limit value to determine whether there is a leak, when the input pressure is 1200mbar, the pressure stability reaches 99.9% and is judged to be qualified. This detection method can effectively ensure the sealing of the chip in actual use. The reagent is driven into the chip by a syringe pump, and the flow loss is detected using multiple flow rates. By setting different flow rates (high speed 3ml / min, medium speed 1ml / min, low speed 0.1ml / min), comparing the input and output liquid volume ratios, calculating the mean and standard deviation to detect whether there are significant differences, and the flow loss rate is less than 5% to be judged as qualified, it can ensure that the flow performance of the chip at different working flow rates meets the requirements and ensure the normal flow of liquid in the chip. In addition, the layout of the various components of the system is reasonable. The microfluidic chip, switching valve, flow meter and other components are set at the bottom of the air compressor, and the collection pool is set on the outside and fixed with fixings. This compact layout facilitates the integration of the system, reduces space occupancy, and also facilitates operation, maintenance and management, improving the practicality of the entire system. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings are only used to illustrate specific embodiments and are not considered to limit the present invention. In the entire drawings, the same reference symbols represent the same components. Obviously, the drawings described below are only some embodiments recorded in the embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings.

[0020] Figure 1 2 is a schematic diagram of an integrated system of a microfluidic chip for long-term self-stimulation of cells and organoids according to an embodiment of the present invention; Figure 2 is a schematic diagram of an integrated system of a microfluidic chip for long-term self-stimulation of cells and organoids according to another embodiment of the present invention; Figure 3 This is a bright field image of mouse small intestinal organoids as the culture time increases according to one embodiment of the present invention, wherein the scale is 50 μm; Figure 4 This is an immunostaining image of a mouse small intestinal organoid according to an embodiment of the present invention, wherein the scale is 50 μm.

[0021] Figure numerals: 11, air compressor; 12, pressure controller; 13, multi-way manifold; 14, liquid storage tank; 15, PTFE catheter; 16, liquid distribution valve; 17, Coriolis flowmeter; 18, flow resistance tube; 19, two-way switching valve; 20, microfluidic chip; 21, three-way switching valve; 22, collection tank; 23, waste liquid tank; 1, first component; 2, second component; 3, third component; 4, support member; 5, fourth component; 6, fixing member. DETAILED DESCRIPTION

[0022] In order to enable those skilled in the art to better understand the technical solutions in the embodiments of the present invention, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. It should be understood that these descriptions are only exemplary and are not intended to limit the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work should fall within the scope of protection of the present invention.

[0023] Furthermore, in the following description, descriptions of well-known structures and technologies are omitted to avoid unnecessarily obscuring the concepts disclosed in the present invention.

[0024] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the orientation or position relationship indicated by the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inside", "outside", etc. is based on the orientation or position relationship shown in the drawings, which is only for the convenience of describing the present invention and simplifying the description, and does not indicate or imply 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 a limitation of the present invention. In addition, the terms "first", "second", and "third" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance. The terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be a connection between the two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0025] Exemplary embodiments will be described in detail herein, examples of which are shown in the accompanying drawings. When the following description refers to the drawings, unless otherwise indicated, the same numbers in different drawings represent the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present invention. Instead, they are merely examples of methods and systems consistent with some aspects of the present invention as detailed in the appended claims.

[0026] The present invention proposes an integrated system of a microfluidic chip for long-acting self-cultured drug stimulation of cells and organoids. Specifically, the following describes an integrated system of a microfluidic chip for long-acting self-cultured drug stimulation of cells and organoids according to an embodiment of the present invention with reference to the accompanying drawings.

[0027] Figure 1 FIG. 1 is a schematic diagram of an integrated system of a microfluidic chip for long-term self-stimulation of cells and organoids according to an embodiment of the present invention. Figure 1 As shown, the integrated system of long-acting self-cultured drug-stimulated microfluidic chip for cells and organoids includes: an air compressor 11, a pressure controller 12, a multi-way manifold 13, a liquid reservoir 14, a PTFE catheter 15, a liquid dispensing valve 16, a Coriolis flowmeter 17, a flow resistance tube 18, a two-way switching valve 19, a microfluidic chip 20, a three-way switching valve 21, a collection tank 22 and a waste liquid tank 23, wherein the air compressor 11 presses the reagent from the liquid reservoir 14 into the PTFE catheter 15 through the pressure controller 12 and the multi-way manifold 13, and then distributes it based on the liquid dispensing valve 16, and the distributed liquid enters the microfluidic chip 20 via the Coriolis flowmeter 17, the flow resistance tube 18, the multi-way manifold 13, the two-way switching valve 19, and finally enters the collection tank 22 or the waste liquid tank 23 through the three-way switching valve 21.

[0028] like Figure 2 As shown, in an embodiment of the present invention, the microfluidic chip 20 is arranged on the first component 1, the two-way switching valve 19 is arranged on the second component 2, the three-way switching valve 21 is arranged on the third component 3, the second component 2 is connected to the support 4, the Coriolis flowmeter 17 is arranged on the support 4, and the liquid dispensing valve 16 is arranged at one end of the fourth component 5.

[0029] In the embodiment of the present invention, the first component 1 , the second component 2 , the third component 3 , the fourth component 5 , and the support member 4 are all arranged at the bottom of the air compressor 11 , and the collecting tank 22 is arranged at the outer side of the air compressor 11 .

[0030] In the embodiment of the present invention, a preset number of fixing members 6 are provided on the outer side of the air compressor 11 , and the fixing members 6 are used to fix the collection tank 22 .

[0031] In an embodiment of the present invention, a microfluidic chip 20 is prepared on a PDMS material by using photolithography technology, wherein the silicon wafer is cleaned with piranha solution before photolithography, and then rinsed with deionized water, and SU-8 photoresist is spin-coated on the surface of the silicon wafer to prepare a 100 μm thick microfluidic chip 20, wherein the SU-8 photoresist is irradiated with ultraviolet light to promote a cross-linking reaction, and the unexposed area will be dissolved. After exposure, it is placed on a hot stage for drying to further promote the polymerization of the photoresist, increase the interlayer bonding force, and ensure the stability of the pattern. Thereafter, the uncross-linked part is removed with a developer, and the silicon wafer is cleaned with isopropyl alcohol and deionized water to ensure that the pattern is clear and free of residue, and finally the film is cured.

[0032] In an embodiment of the present invention, the molding process of the microfluidic chip 20 includes silanization treatment of the mold surface, placing the silicon wafer mold into a dryer, using tin foil to make a container, adding a preset amount of perfluorooctyltrichlorosilane, vacuuming and silanization treatment, and continuing for a preset time to ensure that the silicon wafer silane vapor evenly covers the mold surface to achieve hydrophobic treatment.

[0033] In an embodiment of the present invention, the operation process of the microfluidic chip 20 molding process includes: weighing PDMS and coagulant with a mass ratio of 10:1 with a balance, stirring thoroughly with a glue homogenizer, casting the PDMS solution on the mold, placing it in a vacuum dryer, evacuating until there are no bubbles inside the solution, and then placing it in a heating box and heating it at 60 degrees for more than two hours, taking it out and cooling it to room temperature, cutting the chip along the silicon wafer with a scalpel, selecting a puncher of corresponding size to punch holes at the inlet and outlet of the microfluidic chip 20, and then placing the microfluidic chip 20 with the channel facing up on the glass plate at the same time as the glass sheet for bonding, placing it in a plasma cleaning machine, processing for two minutes, taking out the microfluidic chip 20 and the glass sheet, fitting the upward sides of the two, and placing them in a 60-degree heating box and heating them for more than 2 hours to complete the bonding process.

[0034] In an embodiment of the present invention, the air tightness of the microfluidic chip 20 can be detected based on an air tightness detection method, wherein compressed air gas of a preset pressure can be filled into the microfluidic chip 20 to be tested; the pressure change of the compressed air gas is monitored based on a pressure sensor, and based on the pressure change, it is determined whether the sealing of the microfluidic chip 20 to be tested is qualified.

[0035] Among them, the Elveflow microfluidic multi-channel microfluidic pressure controller 12 is used to test the air tightness of the microfluidic chip 20, with the highest test pressure of 2000mbar. The channels of the microfluidic chip 20 are connected to the ports of the test instrument, all channel outlets are blocked, the channels are pressurized to the preset pressure, the pressure decay is observed, the data of the pressure sensor is recorded, the pressure sensor measurement value is compared with the preset minimum / maximum limit, and whether the channel is leaking. Among them, in the air tightness test, when the input pressure is 1200mbar, the output pressure is 1198mbar, and the pressure stability is 99.9%, which determines that the air tightness of the microfluidic chip 20 is qualified.

[0036] In an embodiment of the present invention, the flow loss of the microfluidic chip can be detected based on an injection pump, wherein the injection pump can be used to drive the reagent into the microfluidic chip 20, and a variety of flow rates can be used to detect the flow loss of the microfluidic chip 20, wherein the multiple flow rates include high speed, medium speed and low speed; when the flow loss is lower than a preset threshold, it is determined that the flow loss of the microfluidic chip 20 to be tested is qualified.

[0037] That is to say, a syringe pump is used to drive the cell culture fluid and drugs into the microfluidic chip 20 to achieve multiple gears of liquid flow rate control; multiple flow rates are used to detect the flow loss of the microfluidic chip 20, and the final flow value is recorded and compared with the minimum / maximum flow limit to determine whether there is a certain degree of leakage; the volume ratio between the input liquid and the outflow liquid at different flow rates is examined, and three liquid flow rates of high speed (3ml / min), medium speed (1ml / min), and low speed (0.1ml / min) are set, and the mean and standard deviation between each group of data are calculated to detect whether there are significant differences between the groups. Among them, in the flow loss test, the flow loss rate is less than 5% and is judged to be qualified.

[0038] like Figure 3 As shown, mouse small intestinal organoids were successfully cultured in this system for one week. The organoids grew well and their diameter increased over time, indicating the feasibility of long-term culture of the system.

[0039] like Figure 4As shown, the mouse small intestinal organoids were removed from the system and immunostained. The staining of E-Cadherin indicated good cell-to-cell connections, and the staining of SOX-9 indicated stem cells in the organoids, indicating that the organoids in the system had good proliferation and differentiation capabilities. Hochest marked the position of the cell nucleus, indicating that the system can be used for automated culture of organoids.

[0040] According to the embodiment of the present invention, the integrated system of long-acting self-cultivation drug-stimulated microfluidic chip for cells and organoids can accurately pressurize and distribute reagents from the liquid reservoir through the coordinated work of components such as air compressors, pressure controllers, multi-way manifolds, and liquid distribution valves. The use of a Coriolis flowmeter further ensures the accurate measurement and control of the liquid flow, making the amount and flow rate of the liquid entering the microfluidic chip more accurate, which is conducive to the accurate supply of nutrients and drugs during the culture of cells and organoids. Microfluidic chips are prepared by photolithography technology, from silicon wafer cleaning, spin coating of photoresist to ultraviolet light irradiation, drying, development and hard film curing, etc. A series of steps ensure the high precision of the chip structure. For example, a 100μm thick chip is prepared, and the pattern is stable, clear and residue-free, providing a good microenvironmental foundation for cell and organoid culture. The silanization treatment during the mold processing can make the mold surface hydrophobic and improve the quality of the chip. By strictly controlling the mass ratio of PDMS to coagulant, as well as operations such as casting, vacuuming, heating, cutting, punching, plasma cleaning and bonding, the physical and chemical properties of the microfluidic chip are ensured to be good, which is conducive to the long-term and stable use of the chip. By filling the microfluidic chip with compressed air, the pressure sensor is used to monitor the pressure change to detect the airtightness. The Elveflow microfluidic multi-channel microfluidic pressure controller can be used for testing at higher pressures, such as up to 2000mbar. By comparing the pressure sensor measurement value with the limit value to determine whether there is a leak, when the input pressure is 1200mbar, the pressure stability reaches 99.9% and is judged to be qualified. This detection method can effectively ensure the sealing of the chip in actual use. The reagent is driven into the chip by a syringe pump, and the flow loss is detected using multiple flow rates. By setting different flow rates (high speed 3ml / min, medium speed 1ml / min, low speed 0.1ml / min), comparing the input and output liquid volume ratios, calculating the mean and standard deviation to detect whether there are significant differences, and the flow loss rate is less than 5% to be judged as qualified, it can ensure that the flow performance of the chip at different working flow rates meets the requirements and ensure the normal flow of liquid in the chip. In addition, the layout of the various components of the system is reasonable. The microfluidic chip, switching valve, flow meter and other components are set at the bottom of the air compressor, and the collection pool is set on the outside and fixed with fixings. This compact layout facilitates the integration of the system, reduces space occupancy, and also facilitates operation, maintenance and management, improving the practicality of the entire system.

[0041] The present invention also proposes a microfluidic chip cell culture and activity detection method, comprising: The prepared microfluidic chip was passed through 75% ethanol and irradiated with ultraviolet light for 1 h for disinfection; Use a syringe pump to pass cell culture fluid into the microfluidic chip for 2 hours to remove residual ethanol and bubbles in the microfluidic chip and facilitate the growth of cells adhering to the wall in the culture chamber; The viability of cells cultured in the microfluidic chip was assessed using a live / dead assay kit. The cells were incubated in a culture medium solution containing 2 μL of calcein AM solution and 5 μL of ethidium homodimer-1 solution at 37 degrees Celsius for 30 minutes. The culture chamber was then rinsed with PBS for 3-5 minutes to remove the reagents and observed under a fluorescence microscope. The percentage of cell viability was calculated based on the number of live cells divided by the total number of cells.

[0042] According to the microfluidic chip cell culture and activity detection method of the embodiment of the present invention, by passing 75% ethanol into the microfluidic chip and irradiating it with ultraviolet light for 1 hour for disinfection, the microorganisms that may exist in the chip can be effectively killed, providing a sterile environment for cell culture. Then, the cell culture solution is passed through the syringe pump for 2 hours, which can not only eliminate the residual ethanol in the chip and avoid the toxic effects of ethanol on cells, but also eliminate bubbles. The removal of bubbles is conducive to the growth of cells adhering to the wall in the culture chamber, because bubbles may interfere with the contact between cells and the wall of the culture chamber, thereby providing cells with more suitable conditions for adhering to the wall. The live / dead assay kit was used to evaluate cell viability, and the dosage of the reagents (2 μL calcein AM solution and 5 μL ethidium homodimer-1 solution) and incubation conditions (incubation at 37 degrees Celsius for 30 minutes) were clarified. This standardized operating procedure makes cell viability detection more accurate. After incubation, rinse the culture chamber with PBS for 3-5 minutes to remove the reagent, then observe under a fluorescence microscope, and finally determine the cell viability by calculating the percentage of live cells to the total number of cells. This can accurately understand the survival status of cells in the microfluidic chip culture environment and provide reliable data support for subsequent cell research and applications.

[0043] The applicant of the present invention has made a detailed explanation and description of the implementation examples of the present invention in conjunction with the drawings in the specification. However, those skilled in the art should understand that the above implementation examples are only preferred implementation schemes of the present invention, and the detailed description is only to help readers better understand the spirit of the present invention, but not to limit the scope of protection of the present invention. On the contrary, any improvements or modifications based on the inventive spirit of the present invention should fall within the scope of protection of the present invention.

[0044] In addition, each functional unit in each embodiment of the present invention may be integrated into a processing module, or each unit may exist physically separately, or two or more units may be integrated into one module. The above-mentioned integrated module may be implemented in the form of hardware or in the form of a software functional module. If the integrated module is implemented in the form of a software functional module and sold or used as an independent product, it may also be stored in a computer-readable storage medium.

[0045] The storage medium mentioned above may be a read-only memory, a magnetic disk or an optical disk, etc. Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limiting the present invention. A person of ordinary skill in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

[0046] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the embodiments of the present invention, rather than to limit them. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions recorded in the above embodiments, or replace some of the technical features therein by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention. Any changes or replacements that can be easily thought of by those skilled in the art within the technical scope disclosed in the present invention should be covered within the protection scope of the present invention.

Claims

1. A cell and organoid long-term self-cultured drug-stimulated microfluidic chip integrated system, characterized in that: include: It includes an air compressor, a pressure controller, a multi-way manifold, a liquid storage tank, a PTFE catheter, a liquid distribution valve, a Coriolis flowmeter, a flow resistance tube, a two-way switching valve, a microfluidic chip, a three-way switching valve, a collection tank and a waste liquid tank, wherein the air compressor presses the reagent from the liquid storage tank into the PTFE catheter through the pressure controller and the multi-way manifold, and then distributes it based on the liquid distribution valve, and the distributed liquid enters the microfluidic chip through the Coriolis flowmeter, the flow resistance tube, the multi-way manifold, and the two-way switching valve, and finally enters the collection tank or the waste liquid tank through the three-way switching valve.

2. The integrated system of long-acting self-stimulated microfluidic chip for cells and organoids according to claim 1, characterized in that: The microfluidic chip is arranged on the first component, the two-way switching valve is arranged on the second component, the three-way switching valve is arranged on the third component, the second component is connected to the support, the Coriolis flowmeter is arranged on the support, and the liquid dispensing valve is arranged at one end of the fourth component.

3. The cell and organoid long-acting self-stimulated microfluidic chip integrated system according to claim 2, characterized in that: The first component, the second component, the third component, the fourth component, and the support member are all arranged at the bottom of the air compressor, and the collecting tank is arranged at the outer side of the air compressor.

4. The cell and organoid long-acting self-stimulated microfluidic chip integrated system according to claim 3, characterized in that: A preset number of fixing parts are provided on the outer side of the air compressor, and the fixing parts are used to fix the collection tank.

5. The integrated system of long-acting self-stimulated microfluidic chip for cells and organoids according to claim 1, characterized in that: The microfluidic chip is prepared on the PDMS material by using photolithography technology, wherein the silicon wafer is cleaned with piranha solution before photolithography, and then rinsed with deionized water, SU-8 photoresist is spin-coated on the surface of the silicon wafer to prepare the microfluidic chip with a thickness of 100 μm, wherein the SU-8 photoresist is irradiated with ultraviolet light to promote a cross-linking reaction, and the unexposed area will be dissolved, and after exposure, it is placed on a hot stage for drying, and then the uncross-linked part is removed with a developer, and the silicon wafer is cleaned with isopropyl alcohol and deionized water, and finally the film is cured.

6. The cell and organoid long-acting self-stimulated microfluidic chip integrated system according to claim 5, characterized in that: The microfluidic chip molding process includes silanization treatment of the mold surface, placing the silicon chip mold into a dryer, using tin foil to make a container, adding a preset amount of perfluorooctyltrichlorosilane, vacuuming and silanization treatment for a preset time to ensure that the silicon chip silane vapor evenly covers the mold surface to achieve hydrophobic treatment.

7. The cell and organoid long-acting self-stimulated microfluidic chip integrated system according to claim 6, characterized in that: The operation flow of the microfluidic chip reverse molding processing includes: weighing PDMS and coagulant with a mass ratio of 10:1 with a balance, stirring thoroughly with a glue homogenizer, casting the PDMS solution on the mold, placing it in a vacuum dryer, evacuating until there are no bubbles inside the solution, then placing it in a heating box at 60 degrees for more than two hours, taking it out and cooling it to room temperature, cutting the chip along the silicon wafer with a scalpel, selecting a puncher of corresponding size to punch holes at the inlet and outlet of the microfluidic chip, and then placing the microfluidic chip with the channel facing up on a glass plate together with a glass sheet for bonding, placing it in a plasma cleaning machine, processing it for two minutes, taking out the microfluidic chip and the glass sheet, fitting the upward sides of the two, placing them in a 60-degree heating box and heating them for more than 2 hours to complete the bonding process.

8. The integrated system of long-acting self-stimulated microfluidic chip for cells and organoids according to claim 1, characterized in that: The air tightness of the microfluidic chip is detected based on an air tightness detection method, wherein the air tightness of the microfluidic chip is detected based on the air tightness detection method includes: Filling the microfluidic chip to be tested with compressed air gas at a preset pressure; The pressure change of the compressed air gas is monitored based on a pressure sensor, and whether the sealing of the microfluidic chip to be tested is qualified is determined based on the pressure change.

9. The cell and organoid long-acting self-stimulated microfluidic chip integrated system according to claim 8, characterized in that: Detecting the flow loss of the microfluidic chip based on a syringe pump, wherein detecting the flow loss of the microfluidic chip based on the syringe pump includes: The syringe pump is used to drive the reagent into the microfluidic chip, and multiple flow rates are used to detect the flow loss of the microfluidic chip, wherein the multiple flow rates include high speed, medium speed and low speed; When the flow loss is lower than a preset threshold, it is determined that the flow loss of the microfluidic chip to be tested is qualified.

10. A cell culture and activity detection method, characterized in that: include: Using the cell and organoid long-acting self-stimulated microfluidic chip integrated system described in any one of claims 1-9, The prepared microfluidic chip was passed through 75% ethanol and irradiated with ultraviolet light for 1 h for disinfection; A cell culture solution is introduced into the microfluidic chip using a syringe pump for 2 hours to remove residual ethanol and bubbles in the microfluidic chip and facilitate the cell growth in the culture chamber; The viability of cells cultured in the microfluidic chip was evaluated using a live / dead assay kit. The cells were incubated in a culture medium solution containing 2 μL of calcein AM solution and 5 μL of ethidium homodimer-1 solution at 37 degrees Celsius for 30 minutes. The culture chamber was then rinsed with PBS for 3-5 minutes to remove the reagents and observed under a fluorescence microscope. The percentage of cell viability was calculated based on the number of live cells divided by the total number of cells.

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