Organoid chip and method for constructing and real-time monitoring of immune killing phenomena

Abstract

The present invention provides an organoid chip and method for constructing and monitoring immune killing phenomena in real time. The organoid chip includes a valve layer, a valve membrane layer and a culture detection layer; the valve layer is provided with a capture chamber inlet, a capture chamber outlet and a flow medium inlet; the side of the valve layer close to the valve membrane layer is provided with a capture flow channel and an isolation flow channel; the capture flow channel includes a capture area, and a plurality of baffles are staggered on the capture area; the valve membrane layer is provided with avoidance holes at positions corresponding to the capture area; the capture area, the avoidance holes and the microporous area together constitute a capture chamber; a seepage flow channel is provided near the microporous area, and a detection flow channel is provided on the side of the seepage flow channel away from the microporous area. One technical effect of the present invention is that it is reasonably designed and can construct and monitor the immune killing phenomenon of tumor organoids in real time.

Description

Technical Field

[0001] The present invention belongs to the technical field of microfluidics, and specifically relates to an organoid chip and method for constructing and monitoring immune killing phenomena in real time. Background Art

[0002] The development and treatment of cancer are closely linked to tumor immunity. Tumors can inhibit the immune system's ability to effectively recognize and kill tumor cells through abnormalities in various pathways, thereby creating immune tolerance and promoting tumor development and progression. Therefore, we need to construct a suitable tumor immune microenvironment in vitro to rapidly detect the recognition and killing of tumor cells by the patient's immune cells, thereby guiding subsequent treatment.

[0003] Currently, in vitro immune microenvironments are mostly constructed using cell lines and often within microplates. This results in a significant difference between the constructed immune environment and the in vivo environment, making it difficult to accurately predict responses. Patient-derived tumor organoids are miniature, self-organizing tumor cell clusters cultured in vitro from tumor samples removed by surgical resection or biopsy. Because they largely retain the histological, transcriptional, and genetic characteristics of the original tumor, tumor organoids are widely used in research on cancer mechanisms, cancer drug screening, and personalized medicine. Co-culturing patient-derived tumor organoids with the patient's lymphocytes can better establish an in vitro immune microenvironment. By monitoring the recognition and killing of cancer cells by immune cells, the in vivo immune response can be predicted. During the killing process, immune cells can produce cytokines that target tumor cells and destroy them by binding to T cell receptors (TCRs), thereby inducing tumor clearance. Therefore, the secretion of immune cytokines reflects immune cell activation parameters, and measuring cytokine levels can monitor immune cell activation and killing in real time.

[0004] The emergence of microfluidics has opened the door to the possibility of controlled co-culture of cells and real-time monitoring of their interactions. Microfluidics can control the co-culture of cells in a continuous perfusion chamber, controlling the flow rate to provide an in vivo-related flow environment. Most importantly, the integration of sensor devices into organoid chips enables accurate real-time monitoring of reactions.

[0005] Currently, it is impossible to monitor multiple cytokines in real time without affecting the immune microenvironment constructed by organoids. When it is necessary to construct and monitor the immune killing phenomenon of tumor organoids in real time, it is necessary to transfer samples between different chambers. This is not only complicated to operate, but also causes large errors in the monitoring results. Summary of the Invention

[0006] The present invention aims to solve at least one of the technical problems existing in the prior art and provide a new technical solution of an organoid chip and method for constructing and monitoring immune killing phenomena in real time.

[0007] According to a first aspect of the present application, there is provided an organoid chip for constructing and real-time monitoring of immune killing phenomena, comprising a valve sheet layer, a valve membrane layer, and a culture detection layer, wherein the valve membrane layer is disposed between the valve sheet layer and the culture detection layer;

[0008] The valve layer is provided with a capture chamber inlet, a capture chamber outlet, and a flow medium inlet; a capture flow channel and an isolation flow channel are provided on the side of the valve layer close to the valve membrane layer, one end of the capture flow channel is connected to the capture chamber inlet, and the other end is connected to the capture chamber outlet; the capture flow channel includes a capture area, and a plurality of baffles are staggered on the capture area; the isolation flow channel is connected to the flow medium inlet;

[0009] The valve membrane layer is provided with an avoidance hole at a position corresponding to the capture area;

[0010] The culture detection layer is provided with a microporous area corresponding to the capture area, and the microporous area is provided with a plurality of micropores corresponding one-to-one to the baffle. The capture area, the avoidance hole, and the microporous area together constitute a capture chamber; a seepage flow channel is provided near the microporous area, and a detection flow channel is provided on the side of the seepage flow channel away from the microporous area, one end of the detection flow channel is connected to a detection sample inlet, and the other end is connected to a detection sample outlet, and the detection sample inlet and the detection sample outlet respectively pass through the valve membrane layer and the valve sheet layer;

[0011] When tumor organoids and immune cells enter the capture chamber from the capture chamber inlet at a preset flow rate, the baffle captures the tumor organoid clusters, and the tumor organoids and immune cells are deposited together in the micropores to construct an immune microenvironment;

[0012] Filling the isolation flow channel with a flow medium through the flow medium inlet causes a portion of the valve membrane layer to move toward the culture detection layer, isolating the seepage flow channel and the detection flow channel; introducing capture microbeads and detection microbeads into the detection flow channel through the detection inlet;

[0013] The seepage channel is connected to the detection channel; the cytokines released from the capture chamber can penetrate into the detection channel along the seepage channel, and the cytokines are detected in the detection channel through the capture microbeads and the detection microbeads.

[0014] Optionally, the detection channel is provided with a plurality of staggered capture units;

[0015] A groove is provided on one side of the capture unit, and a gap is provided in the middle of the groove extending to the other side of the capture unit;

[0016] The capture microbeads and detection microbeads enter the detection flow channel from the detection inlet; wherein, the capture microbeads are non-fluorescent polystyrene microbeads with a diameter of 15 microns and linked to cytokine antibodies, and the detection microbeads are fluorescent polystyrene microbeads with a diameter of 0.5 microns and linked to cytokine antibodies; the groove of each capture unit captures one capture microbead, and the detection microbeads pass through the gap.

[0017] Optionally, the baffle is a curved baffle, and a surface of the curved baffle facing the capture chamber inlet is a concave surface.

[0018] Optionally, the seepage channels are respectively provided on opposite sides of the microporous area; each of the seepage channels is correspondingly connected to one of the detection channels;

[0019] The seepage flow channel and the detection flow channel can be isolated by the isolation flow channel.

[0020] Optionally, when the isolation flow channel isolates the seepage flow channel and the detection flow channel, the isolation flow channel is close to the detection flow channel.

[0021] Optionally, the flow medium inlet is an air valve inlet, and the flow medium is air.

[0022] Optionally, the capture flow channel includes an inlet flow channel, a capture region, and an outlet flow channel; the capture chamber inlet, the inlet flow channel, the capture region, the outlet flow channel, and the capture chamber outlet are sequentially connected;

[0023] The inlet flow channel and the outlet flow channel are both long strip structures with a preset length.

[0024] Optionally, the thickness of the valve layer is 2 mm to 5 mm, and the depths of the capture flow channel and the isolation flow channel are both 100 μm to 500 μm;

[0025] The thickness of the culture detection layer is 2 mm to 5 mm, and the depths of the seepage flow channel and the detection flow channel are both 30 μm to 200 μm.

[0026] Optionally, the number of the baffles is 50-1000; the number of the capture units is 50-300.

[0027] According to a second aspect of the present application, a method for constructing and real-time monitoring of immune killing phenomena in an organoid chip is provided, using the organoid chip described in the first aspect, comprising the following steps:

[0028] Step S1: Patient-derived tumor organoids and immune cells are introduced into the capture flow channel through the capture chamber inlet at a preset flow rate. In the capture chamber, the tumor organoid clusters are captured by a baffle and co-precipitated into the microwells to establish an immune microenvironment.

[0029] Step S2, passing air into the isolation flow channel through the flow medium inlet, and the air squeezes part of the valve membrane layer to isolate the seepage flow channel from the detection flow channel;

[0030] Step S3: introducing capture microbeads and detection microbeads into the detection flow channel through the detection inlet; wherein the capture microbeads are non-fluorescent polystyrene microbeads with a diameter of 15 microns and linked to cytokine antibodies, and the detection microbeads are fluorescent polystyrene microbeads with a diameter of 0.5 microns and linked to cytokine antibodies; the capture microbeads are captured by capture units, each capture unit can capture one capture microbead, and the detection microbeads pass through the gaps in the capture units;

[0031] Step S4, stop introducing air into the isolation flow channel, and the seepage flow channel and the detection flow channel are connected; the cytokines released from the capture chamber can penetrate into the detection flow channel along the seepage flow channel, and the capture beads, detection beads, and cytokines together form a connection structure of capture beads-antibody-cytokine-antibody-detection beads, and the cytokine content in the capture chamber is detected by counting the proportion of fluorescent capture beads in the detection flow channel.

[0032] Optionally, the culture medium contains PI dye and a reagent for detecting caspase 3, and fluorescent staining is used to calibrate the killing of tumor organoids by immune cells under a fluorescence microscope.

[0033] A technical effect of the present invention is:

[0034] In an embodiment of the present application, the organoid chip and method for constructing and monitoring the immune killing phenomenon in real time realizes the construction of an immune microenvironment in vitro while being able to monitor the immune killing phenomenon in real time. Through the one-to-one corresponding baffles and micropores provided in the capture chamber, tumor organoids and immune cells can be captured into the micropores together to achieve the construction of an immune microenvironment and immune killing. The isolation flow channel isolates the cells in the capture chamber from the capture microbeads and detection microbeads in the detection flow channel. When the seepage flow channel is connected to the detection flow channel, the cytokines in the capture chamber penetrate along the seepage flow channel to the detection flow channel and are thereby detected. Compared with traditional methods, it is only necessary to continuously replace the capture microbeads and detection microbeads that replace the detection flow channel to achieve real-time and long-term detection of cytokines without any effect on the cells, and the detection effect is better.

[0035] Furthermore, this organoid chip and method for constructing and monitoring immune killing in real time achieves integrated and automated culture and detection. All operations are performed on a single chip, eliminating the unpredictable loss of samples caused by transferring them between chambers. From sample injection to detection, the entire process is automated, significantly reducing manual operations and saving significant manpower and time.

[0036] At the same time, the organoid chip and method for constructing and real-time monitoring of immune killing phenomena have a wide range of applications. The size of the baffle can be adjusted according to different cells, which is highly flexible and does not increase costs. The cytokines detected in the present invention can be arbitrarily replaced by changing the type of antibody on the detection microbeads, which will not affect the sensitivity of the detection and is conducive to cost savings. The types of cytokines detected in the present invention can also be increased by adding detection flow channels, and this can be done without affecting the sensitivity of the detection, and the operation is simple. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a schematic diagram of the decomposed structure of an organoid chip for constructing and real-time monitoring of immune killing phenomena according to one embodiment of the present invention;

[0038] Figure 2 This is a bottom view of a valve layer of an organoid chip for constructing and real-time monitoring of immune killing phenomena according to one embodiment of the present invention;

[0039] Figure 3 This is a schematic structural diagram of a valve membrane layer of an organoid chip for constructing and real-time monitoring of immune killing phenomena according to one embodiment of the present invention;

[0040] Figure 4 This is a top view of a culture detection layer of an organoid chip for constructing and real-time monitoring of immune killing phenomena according to one embodiment of the present invention;

[0041] Figure 5 This is a schematic structural diagram of a baffle for constructing an organoid chip for real-time monitoring of immune killing phenomena according to one embodiment of the present invention;

[0042] Figure 6 This is a schematic diagram of the structure of a microwell of an organoid chip for constructing and real-time monitoring of immune killing phenomena according to one embodiment of the present invention;

[0043] Figure 7 This is a schematic structural diagram of a capture unit of an organoid chip for constructing and real-time monitoring of immune killing phenomena according to one embodiment of the present invention;

[0044] Figure 8This is a schematic diagram of an organoid chip for constructing and real-time monitoring of immune killing phenomena according to one embodiment of the present invention, in which a baffle captures tumor organoid clusters and tumor organoids and immune cells are deposited together in microwells;

[0045] Figure 9 This is a flow chart of a method for constructing an organoid chip and monitoring immune killing phenomena in real time according to one embodiment of the present invention.

[0046] In the figure: 1. Valve plate layer; 2. Valve membrane layer; 3. Culture detection layer; 31. Micropore area; 311. Micropore; 4. Capture chamber inlet; 5. Capture chamber outlet; 6. Flow medium inlet; 71. Inlet flow channel; 72. Capture area; 73. Outlet flow channel; 8. Isolation flow channel; 9. Detection flow channel; 91. Detection inlet; 92. Detection outlet; 93. Capture unit; 931. Groove; 932. Gap; 10. Seepage flow channel; 11. Baffle; 12. Avoidance hole; 13. Capture chamber. DETAILED DESCRIPTION

[0047] Various exemplary embodiments of the present application will now be described in detail with reference to the accompanying drawings. It should be noted that unless otherwise specifically stated, the relative arrangements of components and steps, numerical expressions and numerical values set forth in these embodiments do not limit the scope of the present application.

[0048] The embodiments of the present application will be described in detail below. Examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present application and are not to be construed as limiting the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0049] The terms "first" and "second" in the specification and claims of this application may explicitly or implicitly refer to one or more of the features. Throughout the description of this application, unless otherwise specified, "plurality" means two or more. Furthermore, "and / or" in the specification and claims refers to at least one of the connected entities, and the character " / " generally indicates an "or" relationship between the connected entities.

[0050] In the description of the present application, it should be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", "clockwise", "counterclockwise", "axial", "radial", "circumferential" and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present application and simplifying the description, and do 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 should not be understood as a limitation on the present application.

[0051] In the description of this application, it should be noted that, unless otherwise expressly specified or limited, the terms "mounted," "connected," and "connected" should be understood in a broad sense. For example, they can refer to fixed connections, detachable connections, or integral connections; mechanical connections or electrical connections; direct connections or indirect connections through an intermediate medium; and internal connections between two components. Those skilled in the art will understand the specific meanings of the above terms in this application based on the specific circumstances.

[0052] See also Figures 1 to 8 According to the first aspect of the present application, an organoid chip for constructing and monitoring the immune killing phenomenon in real time is provided, which is used to construct and monitor the immune killing phenomenon of tumor organoids in real time.

[0053] Specifically, the organoid chip for constructing and real-time monitoring of immune killing phenomena includes a valve sheet layer 1, a valve membrane layer 2 and a culture detection layer 3, wherein the valve membrane layer 2 is arranged between the valve sheet layer 1 and the culture detection layer 3.

[0054] It should be noted that a sealed environment is formed between the valve sheet layer 1 and the valve membrane layer 2 so as to form a capture flow channel and an isolation flow channel 8 on the side of the valve sheet layer 1 close to the valve membrane layer 2; a sealed environment is formed between the culture and detection layer 3 and the valve membrane layer 2 so as to form a seepage flow channel 10 and a detection flow channel 9 on the side of the culture and detection layer 3 close to the valve membrane layer 2. Furthermore, the capture flow channel and the isolation flow channel 8 are formed by setting a recess on the surface of the valve sheet layer 1 close to the valve membrane layer 2, and covering the recess with the valve membrane layer 2 to form the capture flow channel and the isolation flow channel 8. Similarly, the seepage flow channel 10 and the detection flow channel 9 are also formed by setting a recess on the surface of the culture and detection layer 3 close to the valve membrane layer 2, and covering the recess with the valve membrane layer 2 to form the seepage flow channel 10 and the detection flow channel 9.

[0055] Furthermore, to accommodate the size of cells, materials compatible with existing microfabrication techniques must be selected. Exemplarily, PDMS (polydimethylsiloxane) is chosen as the material for the entire organoid chip because it is well compatible with microfabrication processes based on photolithography and etching, and its machining precision is easily controlled, making it easy to obtain a capture array that matches the cell size. Those skilled in the art can also select machinable materials such as glass and silicon as needed.

[0056] For example, oxygen plasma-assisted bonding can be used to achieve good sealing between PDMS.

[0057] In other implementation methods, a suitable sealing method can be selected according to different materials.

[0058] More specifically, the valve layer 1 is provided with a capture chamber inlet 4, a capture chamber outlet 5 and a flow medium inlet 6; the side of the valve layer 1 close to the valve membrane layer 2 is provided with a capture flow channel and an isolation flow channel 8, one end of the capture flow channel is connected to the capture chamber inlet 4, and the other end is connected to the capture chamber outlet 5; the capture flow channel includes a capture area 72, and a plurality of baffles 11 are staggered on the capture area 72; the isolation flow channel 8 is connected to the flow medium inlet 6.

[0059] For example, a pump can be used to introduce patient-derived tumor organoids and immune cells into capture chamber 13 at a specific flow rate through capture chamber inlet 4 for culture and capture. These cells can then be discharged through capture chamber outlet 5, forming a circulation loop. Air or liquid can be introduced into isolation channel 8 through flow medium inlet 6, forcing valve membrane layer 2 toward culture and detection layer 3, thereby isolating certain areas of culture and detection layer 3.

[0060] The valve membrane layer 2 is provided with a bypass hole 12 at a position corresponding to the capture area 72. The bypass hole 12 allows patient-derived tumor organoids and immune cells to pass through the capture chamber inlet 4 into the capture flow channel and then into the capture chamber 13, thereby creating a microenvironment for the immune killing of tumor organoids.

[0061] The culture detection layer 3 is provided with a microporous area 31 corresponding to the capture area 72. The microporous area 31 is provided with a plurality of micropores 311 corresponding one-to-one with the baffle 11. For example, the micropores 311 are cylindrical micropores 311. The capture area 72, the avoidance hole 12, and the microporous area 31 together constitute a capture chamber 13. A seepage channel 10 is provided near the microporous area 31. A detection channel 9 is provided on the side of the seepage channel 10 away from the microporous area 31. One end of the detection channel 9 is connected to a detection sample inlet 91, and the other end is connected to a detection sample outlet 92. The detection sample inlet 91 and the detection sample outlet 92 respectively penetrate the valve membrane layer 2 and the valve sheet layer 1.

[0062] When tumor organoids and immune cells enter the capture chamber 13 from the capture chamber inlet 4 at a preset flow rate, the baffle 11 captures the tumor organoid clusters, and the tumor organoids and immune cells settle together into the micropores 311 to construct an immune microenvironment.

[0063] The flow medium is filled into the isolation channel 8 through the flow medium inlet 6, so that part of the valve membrane layer 2 moves toward the culture detection layer 3, isolating the seepage channel 10 and the detection channel 9; the capture microbeads and detection microbeads are introduced into the detection channel 9 through the detection injection port 91.

[0064] The seepage channel 10 is connected to the detection channel 9; the cytokines released from the capture chamber 13 can penetrate along the seepage channel 10 to the detection channel 9, and the cytokines are detected in the detection channel 9 by capturing microbeads and detecting microbeads.

[0065] In an embodiment of the present application, the organoid chip for constructing and monitoring the immune killing phenomenon in real time realizes the construction of an immune microenvironment in vitro while being able to monitor the immune killing phenomenon in real time. Through the one-to-one corresponding baffle 11 and micropore 311 set in the capture chamber 13, the tumor organoids and immune cells can be captured into the micropore 311 together to achieve the construction of an immune microenvironment and immune killing. The isolation channel 8 will isolate the cells in the capture chamber 13 from the capture microbeads and detection microbeads in the detection channel 9. When the seepage channel 10 is connected to the detection channel 9, the cytokines in the capture chamber 13 penetrate into the detection channel 9 along the seepage channel and are thereby detected. Compared with traditional methods, it is only necessary to continuously replace the capture microbeads and detection microbeads of the detection channel 9 to achieve real-time and long-term detection of cytokines without any effect on the cells, and the detection effect is better.

[0066] Furthermore, this organoid-on-a-chip, used to construct and monitor immune killing in real time, integrates and automates both culture and detection. All operations are performed on a single chip, eliminating the unpredictable loss of samples caused by transferring them between chambers. From sample injection to detection, the entire process is automated, significantly reducing manual labor and saving significant time.

[0067] At the same time, the organoid chip used to construct and monitor immune killing phenomena in real time has a wide range of applications. The size of the baffle 11 can be adjusted according to different cells, which is highly flexible and does not increase costs. The cytokines detected in the present invention can be arbitrarily replaced by changing the type of antibody on the detection microbeads without affecting the sensitivity of the detection, which is conducive to cost savings. The types of cytokines detected in the present invention can also be increased by adding detection channels 9, without affecting the sensitivity of the detection, and the operation is simple.

[0068] Optionally, the detection channel 9 is provided with a plurality of staggered capture units 93;

[0069] A groove 931 is provided on one side of the capture unit 93, and a gap 932 is provided in the middle of the groove 931, extending to the other side of the capture unit 93. The capture unit 93 has a height of 30 to 200 microns, a length of 50 to 200 microns, and a width of 50 to 200 microns. The width of the gap 932 is 2 to 10 microns.

[0070] The capture microbeads and detection microbeads enter the detection flow channel 9 through the detection injection port 91; wherein, the capture microbeads are non-fluorescent polystyrene microbeads with a diameter of 15 microns and attached to cytokine antibodies, and the detection microbeads are fluorescent polystyrene microbeads with a diameter of 0.5 microns and attached to cytokine antibodies; the groove 931 of each capture unit 93 captures one capture microbead, and the detection microbeads pass through the gap 932.

[0071] That is, the cytokines released from the chamber penetrate into the detection channel 9 along the seepage channel 10, and the capture beads, detection beads, and cytokines together form a connection structure of capture beads-antibody-cytokine-antibody-detection beads. The proportion of fluorescent capture beads in the detection channel 9 is counted to detect the cytokine content in the capture chamber 13.

[0072] In the above embodiment, when the cytokines released from the capture chamber 13 penetrate into the detection channel 9 along the seepage channel 10 , the cytokines can be better detected, the detection result is more accurate, and the operation is simple.

[0073] Optionally, the baffle 11 is curved, with the surface facing the capture chamber inlet 4 being concave. This allows the baffle 11 to quickly and accurately capture tumor organoid clusters. Furthermore, the size of the baffle 11 can be adjusted to suit different tumor cell types, providing high flexibility without increasing costs and a wide range of applicability.

[0074] In a specific embodiment, the baffle 11 is a semicircular ring-shaped baffle 11. The baffle 11 has a height of 80 to 400 microns, an outer diameter of 100 to 1000 microns, and an inner diameter of 80 to 900 microns, thereby enabling better capture of tumor organoid clusters.

[0075] Optionally, the seepage channels 10 are respectively provided on two opposite sides of the microporous area 31; each of the seepage channels 10 is correspondingly connected to one of the detection channels 9;

[0076] The seepage channel 10 and the detection channel 9 can be isolated by the isolation channel 8 .

[0077] In the above embodiment, the types of antibodies on the detection microbeads in the two detection channels 9 are different, so different cytokines can be detected, which is simple to operate, has high detection efficiency, and also helps to save costs.

[0078] Of course, by increasing the number of detection channels 9, that is, by having different types of antibodies on the detection microbeads in multiple detection channels 9, it is possible to detect a variety of different cytokines.

[0079] Optionally, when the isolation channel 8 isolates the seepage channel 10 and the detection channel 9, the isolation channel 8 is close to the detection channel 9. This helps to ensure the accuracy of the cytokine detection results.

[0080] Optionally, the flow medium inlet 6 is an air valve inlet, and the flow medium is air. This makes the use of the organoid chip simpler and more convenient, and also helps to ensure the safety of the organoid chip.

[0081] Optionally, the capture flow channel includes an inlet flow channel 71, a capture area 72, and an outlet flow channel 73; the capture chamber inlet 4, the inlet flow channel 71, the capture area 72, the outlet flow channel 73, and the capture chamber outlet 5 are connected in sequence;

[0082] The inlet flow channel 71 and the outlet flow channel 73 are both long strip structures with a preset length.

[0083] In the above embodiment, it helps to pass the patient-derived tumor organoids and immune cells into the capture flow channel through the capture chamber inlet 4 at a preset flow rate, enter the capture chamber 13 through the inlet flow channel 71, and flow out of the capture chamber 13 through the outlet flow channel 73, which helps to build a continuous and stable immune microenvironment and is conducive to ensuring the accuracy of the test results.

[0084] Optionally, the thickness of the valve layer 1 is 2 mm to 5 mm, and the depths of the capture flow channel and the isolation flow channel 8 are both 100 μm to 500 μm;

[0085] The thickness of the valve membrane layer 2 is 10 micrometers to 100 micrometers; for example, the thickness of the valve membrane layer 2 is 50 micrometers.

[0086] The thickness of the culture detection layer 3 is 2 mm to 5 mm, and the depths of the seepage flow channel 10 and the detection flow channel 9 are both 30 μm to 200 μm.

[0087] Optionally, the number of the baffles 11 is 50-1000, the number of the micropores 311 is also 50-1000; and the number of the capture units 93 is 50-300.

[0088] For example, the culture detection layer 3 has a thickness of 3 mm, and the depths of the seepage channel 10, the detection channel 9, and the micropores 311 are all 50 μm. The detection channel 9 contains 150 capture cells 93, each 50 μm in height, 100 μm in length, and 80 μm in width. The width of the gaps 932 is 8 μm.

[0089] In the above embodiment, the design of each structural layer of the organoid chip is relatively reasonable, which not only helps to construct a microenvironment for the immune killing phenomenon of tumor organoids, but also helps to monitor the immune killing phenomenon of tumor organoids in real time, and is very convenient to use.

[0090] In one embodiment, the valve layer 1 and the culture and detection layer 3 are made of transparent materials, such as glass and PDMS, to facilitate observation of the organoid chip. The valve membrane layer 2 is made of PDMS and has a certain degree of elasticity. The valve layer 1 needs to have interfaces for fluid inlet and outlet, so it needs to be able to open holes.

[0091] Furthermore, when constructing and monitoring the immune killing phenomenon of tumor organoids in real time, the organoid chip requires the use of a fluorescence microscope (fluorescence imaging system), a pump, etc. to accurately construct and monitor the immune killing phenomenon of tumor organoids in real time. Fluorescence microscopes (fluorescence imaging systems), pumps, etc. are commonly used in this field and are not described in detail in this application.

[0092] That is, in addition to the organoid chip, a complete system consisting of tumor organoids, immune cells, capture beads, detection beads, a fluorescence microscope (fluorescence imaging system), and a pump is needed to complete the construction and monitor the immune killing phenomenon of tumor organoids in real time.

[0093] Tumor organoids are 3D cell clusters cultured in gel from tumor cells lysed from the patient's tumor. Immune cells are PBMC cells extracted from the patient's blood and then induced to become tumor-reactive immune cells. The tumor organoids and immune cells are passed into the capture chamber 13 together and captured by the baffle 11 and micropores 311.

[0094] The capture microbeads are 15-micron-diameter non-fluorescent polystyrene microbeads linked to immune cytokine antibodies, while the detection microbeads are 0.5-micron-diameter fluorescent polystyrene microbeads linked to immune cytokine antibodies. The capture microbeads are captured by the capture units 93 of the detection channel 9. Each capture unit 93 can capture one capture microbead, while the detection microbeads pass through the gaps 932 without being captured. When the cytokines in the capture chamber 13 permeate into the detection channel 9 along the permeation channel, the capture microbeads and the detection microbeads form a connection structure of capture microbeads-antibody-cytokine-antibody-detection microbeads. Therefore, the capture microbeads captured by the capture units 93 gradually become fluorescent. By counting the proportion of fluorescent capture microbeads in the detection channel 9, the cytokine content in the cell capture culture chamber is detected.

[0095] A fluorescence microscope is used to observe the microbeads in the capture unit 93 in the detection channel 9 and the cells in the microwells 311 in the capture chamber 13, staining and imaging the death and apoptosis of the organoid. A pump is used to drive cell samples and related reagents into the organoid chip.

[0096] In a specific embodiment, the method for preparing the organoid chip is as follows:

[0097] The valve layer 1 is fabricated using a 4-inch N-type silicon wafer. After photolithography to create the planar shapes of the isolation channel 8 and baffle 11, ICP dry etching (inductively coupled plasma etching, which uses high-energy plasma of sulfur hexafluoride and carbon tetrafluoride to etch silicon) is then used to create a mold for forming the 80-micron-high baffle 11 and the 100-micron-deep capture channel and isolation channel 8. Liquid PDMS is poured into the mold and baked in an 80-degree oven for 2 hours to solidify the PDMS. The mold is then removed from the mold, the excess PDMS removed, and the wafer is cut into rectangular pieces 2 cm long and 1 cm wide. Holes are then punched at the desired locations using a hole punch. This completes the valve layer 1.

[0098] The method for making the culture detection layer 3 is as follows: Using an N-type 4-inch silicon wafer, after photolithography to form the planar shapes of the micropores 311, capture units 93, and other structures, ICP dry etching (inductively coupled plasma etching, i.e., using high-energy plasma of sulfur hexafluoride and carbon tetrafluoride to etch silicon) is then used to obtain a mold for forming the 30-micron-high capture units 93, the 30-micron-deep seepage channels 10, the detection channels 9, and the micropores 311. Liquid PDMS is poured into the mold, and the mold is baked in an 80-degree oven for 2 hours to solidify the PDMS. After demolding, the mold is removed, the excess PDMS is trimmed, and the mold is cut into rectangular pieces with dimensions of 2 cm long and 1 cm wide. This completes the culture detection layer 3.

[0099] The manufacturing method of the valve membrane layer 2 is as follows: liquid PDMS is poured onto an N-type 4-inch silicon wafer, centrifuged at 500 rpm using a glue spreader, and then baked in an oven at 80 degrees for 2 hours to solidify the PDMS to obtain a valve membrane layer 2 with a thickness of about 50 microns. The middle area of the valve membrane layer 2 is then removed with a blade to form an avoidance hole 12.

[0100] Sealing process: Use oxygen plasma to treat the valve sheet layer 1, valve membrane layer 2 and culture detection layer 3 separately, and bond them together in sequence to finally obtain a complete organoid chip.

[0101] It should be noted that on the basis of the organoid chip, polytetrafluoroethylene tubes are used to connect the fluid inlet and outlet of the pump and the valve layer 1. When the results need to be analyzed, the chip is placed under an inverted fluorescence microscope that can automatically perform fluorescence imaging, and the fluorescence image is automatically analyzed and processed in the computer's image processing software to complete the construction of the entire system.

[0102] See also Figure 9 According to the second aspect of the present application, a method for constructing and real-time monitoring of immune killing phenomena in an organoid chip is provided, using the organoid chip as described in the first aspect, comprising the following steps:

[0103] Step S1: Patient-derived tumor organoids and immune cells are introduced into the capture flow channel through the capture chamber inlet 4 at a preset flow rate. In the capture chamber 13, the tumor organoid clusters are captured by the baffle 11 and are deposited together into the micropores 311 to establish an immune microenvironment.

[0104] Step S2: air is introduced into the isolation flow channel 8 through the flow medium inlet 6, and the air squeezes part of the valve membrane layer 2 to isolate the seepage flow channel 10 from the detection flow channel 9;

[0105] Step S3: The capture microbeads and the detection microbeads are introduced into the detection flow channel 9 through the detection inlet 91; the capture microbeads are 15-micron-diameter non-fluorescent polystyrene microbeads linked to cytokine antibodies, and the detection microbeads are 0.5-micron-diameter fluorescent polystyrene microbeads linked to cytokine antibodies; the capture microbeads are captured by the capture units 93, each of which can capture one capture microbead, and the detection microbeads pass through the gaps 932 of the capture units 93;

[0106] In step S4, the air supply to the isolation channel 8 is stopped, and the seepage channel 10 and the detection channel 9 are connected; the cytokines released from the capture chamber 13 can penetrate into the detection channel 9 along the seepage channel 10, and the capture beads, detection beads, and cytokines together form a connection structure of capture beads-antibody-cytokine-antibody-detection beads. The proportion of fluorescent capture beads in the detection channel 9 is counted to detect the cytokine content in the capture chamber 13.

[0107] In the above-mentioned embodiment, in the embodiment of the present application, the method for constructing and monitoring the immune killing phenomenon on an organoid chip in real time realizes the construction of an immune microenvironment in vitro while being able to monitor the immune killing phenomenon in real time. Through the one-to-one corresponding baffles 11 and micropores 311 provided in the capture chamber 13, tumor organoids and immune cells can be captured into the micropores 311 together to realize the construction of an immune microenvironment and immune killing. The isolation channel 8 will isolate the cells in the capture chamber 13 from the capture microbeads and detection microbeads in the detection channel 9. When the seepage channel 10 is connected to the detection channel 9, the cytokines in the capture chamber 13 penetrate into the detection channel 9 along the seepage channel and are thereby detected. Compared with the traditional method, it is only necessary to continuously replace the capture microbeads and detection microbeads of the detection channel 9 to achieve real-time and long-term detection of cytokines without any effect on the cells, and the detection effect is better.

[0108] Furthermore, this method for constructing and monitoring immune killing in real time in an organoid chip integrates and automates both culture and detection. All operations are performed on a single chip, eliminating the unpredictable loss of samples caused by transferring them between chambers. From sample injection to detection, the entire process is automated, significantly reducing manual labor and saving significant time.

[0109] At the same time, the method for constructing and real-time monitoring of immune killing phenomena in an organoid chip has a wide range of applications. The size of the baffle 11 can be adjusted according to different cells, which is highly flexible and does not increase costs. The cytokines detected in the present invention can be arbitrarily replaced by changing the type of antibody on the detection microbeads, without affecting the sensitivity of the detection, which is conducive to cost savings. The types of cytokines detected in the present invention can also be increased by adding a detection channel 9, without affecting the sensitivity of the detection, and the operation is simple.

[0110] Optionally, the culture medium contains PI dye and a reagent for detecting caspase 3, and fluorescent staining is used to determine the live / dead nature of immune cell killing of tumor organoids under a fluorescence microscope. This allows for accurate detection of immune cell killing of tumor organoids, helping to ensure the accuracy of the detection of immune cell killing of tumor organoids.

[0111] It's important to note that PI (propidium iodide) dye is a DNA-staining nuclear staining reagent and an analog of ethidium bromide. It emits red fluorescence after intercalating into double-stranded DNA. Although PI cannot pass through living cell membranes, it can penetrate damaged cell membranes and stain nuclei. This allows for live-death calibration of immune cell killing of tumor organoids, improving the accuracy of test results.

[0112] Therefore, the organoid chip and method for constructing and real-time monitoring of immune killing phenomena achieves efficient capture of organoids by setting a baffle 11 in the capture chamber 13, and uses an isolation flow channel 8 and detection microbeads to monitor multiple factors in real time without affecting the construction of the immune microenvironment of the organoid, thereby monitoring the immune killing phenomenon of the organoid in real time.

[0113] In one specific application, patient-derived tumor organoids and immune cells are introduced into capture chamber 13. Baffle 11 captures the tumor organoid clusters, which then settle together into micropores 311 to establish an immune microenvironment that enables immune cell killing of the tumor organoids. Fluorescence microscopy images the cells in the culture zone, providing organoid staining for both live and dead tissue and apoptosis. Capture microbeads and detection microbeads linked to cytokine antibodies are introduced into detection channel 9, with each capture unit 93 capturing one capture microbead. After cytokines from capture chamber 13 permeate detection channel 9, the capture microbeads and detection microbeads connect via a capture microbead-antibody-cytokine-antibody-detection microbead structure, causing the capture microbeads captured by capture unit 93 to gradually become fluorescent. By counting the proportion of fluorescent capture microbeads in detection channel 9, the cytokine content in the cell capture culture chamber is detected. Fluid is then passed through detection channel 9 in the reverse direction to flush out the microbeads in capture unit 93, and the next batch of microbeads is introduced through detection inlet 91 to complete the next test.

[0114] It will be understood that the above embodiments are merely exemplary embodiments for illustrating the principles of the present invention, and the present invention is not limited thereto. Those skilled in the art will appreciate that various modifications and improvements can be made without departing from the spirit and substance of the present invention, and such modifications and improvements are also considered to be within the scope of protection of the present invention.

Claims

1. An organoid chip for constructing and real-time monitoring of immune killing phenomena, characterized in that: It comprises a valve sheet layer, a valve membrane layer and a culture detection layer, wherein the valve membrane layer is arranged between the valve sheet layer and the culture detection layer; The valve layer is provided with a capture chamber inlet, a capture chamber outlet, and a flow medium inlet; a capture flow channel and an isolation flow channel are provided on the side of the valve layer close to the valve membrane layer, one end of the capture flow channel is connected to the capture chamber inlet, and the other end is connected to the capture chamber outlet; the capture flow channel includes a capture area, and a plurality of baffles are staggered on the capture area; the isolation flow channel is connected to the flow medium inlet; The valve membrane layer is provided with an avoidance hole at a position corresponding to the capture area; The culture detection layer is provided with a microporous area corresponding to the capture area, and the microporous area is provided with a plurality of micropores corresponding one-to-one to the baffle. The capture area, the avoidance hole, and the microporous area together constitute a capture chamber; a seepage flow channel is provided near the microporous area, and a detection flow channel is provided on the side of the seepage flow channel away from the microporous area, one end of the detection flow channel is connected to a detection sample inlet, and the other end is connected to a detection sample outlet, and the detection sample inlet and the detection sample outlet respectively pass through the valve membrane layer and the valve sheet layer; When tumor organoids and immune cells enter the capture chamber from the capture chamber inlet at a preset flow rate, the baffle captures the tumor organoid clusters, and the tumor organoids and immune cells are deposited together in the micropores to construct an immune microenvironment; Filling the isolation flow channel with a flow medium through the flow medium inlet causes a portion of the valve membrane layer to move toward the culture detection layer, isolating the seepage flow channel and the detection flow channel; introducing capture microbeads and detection microbeads into the detection flow channel through the detection inlet; The seepage channel is connected to the detection channel; the cytokines released from the capture chamber can penetrate into the detection channel along the seepage channel, and the cytokines are detected in the detection channel through the capture microbeads and the detection microbeads.

2. The organoid chip for constructing and real-time monitoring of immune killing phenomena according to claim 1, characterized in that: The detection channel is provided with a plurality of staggered capture units; A groove is provided on one side of the capture unit, and a gap is provided in the middle of the groove extending to the other side of the capture unit; The capture microbeads and detection microbeads enter the detection flow channel from the detection inlet; wherein, the capture microbeads are non-fluorescent polystyrene microbeads with a diameter of 15 microns and linked to cytokine antibodies, and the detection microbeads are fluorescent polystyrene microbeads with a diameter of 0.5 microns and linked to cytokine antibodies; the groove of each capture unit captures one capture microbead, and the detection microbeads pass through the gap.

3. The organoid chip for constructing and real-time monitoring of immune killing phenomena according to claim 1, characterized in that: The baffle is an arc-shaped baffle, and a surface of the arc-shaped baffle facing the capture chamber inlet is a concave surface.

4. The organoid chip for constructing and real-time monitoring of immune killing phenomena according to claim 1, characterized in that: The seepage channels are respectively provided on opposite sides of the microporous area; each of the seepage channels is correspondingly connected to one of the detection channels; The seepage flow channel and the detection flow channel can be isolated by the isolation flow channel.

5. The organoid chip for constructing and real-time monitoring of immune killing phenomena according to claim 4, characterized in that: When the isolation flow channel isolates the seepage flow channel and the detection flow channel, the isolation flow channel is close to the detection flow channel.

6. The organoid chip for constructing and real-time monitoring of immune killing phenomena according to claim 1, characterized in that: The flow medium inlet is an air valve air inlet, and the flow medium is air.

7. The organoid chip for constructing and real-time monitoring of immune killing phenomena according to claim 1, characterized in that: The capture flow channel includes an inlet flow channel, a capture area, and an outlet flow channel; the capture chamber inlet, the inlet flow channel, the capture area, the outlet flow channel, and the capture chamber outlet are connected in sequence; The inlet flow channel and the outlet flow channel are both long strip structures with a preset length.

8. The organoid chip for constructing and real-time monitoring of immune killing phenomena according to claim 1, characterized in that: The thickness of the valve layer is 2 mm to 5 mm, and the depths of the capture flow channel and the isolation flow channel are both 100 μm to 500 μm; The thickness of the culture detection layer is 2 mm to 5 mm, and the depths of the seepage flow channel and the detection flow channel are both 30 μm to 200 μm.

9. The organoid chip for constructing and real-time monitoring of immune killing phenomena according to claim 2, characterized in that: The number of the baffles is 50-1000; the number of the capture units is 50-300.

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