CN115466680A

CN115466680A - Micro-fluidic chip for monitoring immune killing of organoid in real time and method thereof

Info

Publication number: CN115466680A
Application number: CN202211309026.5A
Authority: CN
Inventors: 魏泽文; 丁文拥
Original assignee: Beijing Institute of Technology BIT
Current assignee: Beijing Institute of Technology BIT
Priority date: 2022-09-09
Filing date: 2022-10-25
Publication date: 2022-12-13

Abstract

The invention provides a micro-fluidic chip for monitoring immune killing of organoids in real time and a method thereof, wherein the micro-fluidic chip comprises a micro-structural channel layer and a glass substrate, wherein the micro-structural channel layer is arranged above the glass substrate; a cell culture region, a gel isolation region and a factor detection region are arranged on one side, close to the glass substrate, of the microstructure channel layer, and the gel isolation region is arranged between the cell culture region and the factor detection region; the gel isolation area is filled with a gel layer to prevent cells in the cell culture area from entering the factor detection area; organoids and immune cells enter a cell culture area, an immune microenvironment is constructed in the cell culture area, and cytokines in the cell culture area can pass through a gel layer in the gel isolation area to enter a factor detection area, and the cytokines are detected in the factor detection area. The invention has the technical effects of reasonable design, capability of realizing the implementation monitoring of the organoid immune killing and more accurate detection structure.

Description

Micro-fluidic chip for monitoring immune killing of organoid in real time and method thereof

Technical Field

The invention belongs to the technical field of microfluidic chips, and particularly relates to a microfluidic chip for monitoring immune killing of an organoid in real time and a method thereof.

Background

In recent years, the morbidity and mortality of cancers rapidly rise, the occurrence and treatment of the cancers are related to tumor immunity, different tumors can inhibit the effective recognition and killing of the immune system on tumor cells through different links, so that the immune tolerance is generated, and the occurrence and development of the tumors are promoted. Therefore, it is necessary to construct a suitable tumor immune microenvironment in vitro, and rapidly detect the recognition and killing of tumor cells by immune cells in a patient, so as to guide the subsequent treatment of the patient.

At present, the in vitro immune microenvironment is mostly constructed by using a cell line and is mostly constructed in a pore plate, so that the constructed immune environment is far from the in vivo immune environment, and accurate prediction reaction is difficult to achieve. The patient-derived organoid is used as a novel self-organizing three-dimensional structure, compared with a two-dimensional cell line, the structure and the function of a tumor can be better kept in vitro, and an immune microenvironment can be better constructed in vitro by co-culturing the patient-derived tumor organoid and immune cells of the patient. During the killing process, immune cells can be damaged by targeting tumor cells through relevant cytokines generated after connecting T Cell Receptors (TCR), thereby inducing tumor clearance. Therefore, the secretion of the immune cell factors can reflect the activation parameters of the immune cells, and the immune activation and killing of the immune cells can be monitored in real time by detecting the quantity of the immune cell factors. In the conventional detection, the limitation of the sample amount is that the supernatant is taken at the last stage and the related factors are detected by using a kit, so that the real-time detection cannot be realized, and the manual sample collection and detection method also has the problems of large workload and interference among holes.

Disclosure of Invention

The invention aims to at least solve one of the technical problems in the prior art and provides a novel technical scheme of a micro-fluidic chip for monitoring the immune killing of the organoid in real time and a method thereof.

According to a first aspect of the invention, a micro-fluidic chip for monitoring organoid immune killing in real time is provided, which comprises a micro-structural channel layer and a glass substrate, wherein the micro-structural channel layer is arranged above the glass substrate;

a cell culture region, a gel isolation region and a factor detection region are arranged on one side, close to the glass substrate, of the microstructure channel layer, and the gel isolation region is arranged between the cell culture region and the factor detection region; the gel isolation area is filled with a gel layer to prevent cells in the cell culture area from entering the factor detection area;

organoids and immune cells enter a cell culture area, an immune microenvironment is constructed in the cell culture area, and cytokines in the cell culture area can pass through a gel layer in the gel isolation area to enter a factor detection area, and the cytokines are detected in the factor detection area.

Optionally, a culture sample inlet, a culture sample outlet, an isolation sample inlet, an isolation sample outlet, a detection sample inlet and a detection sample outlet are arranged on the microstructure channel layer;

one end of the cell culture area is communicated with the culture sample inlet, and the other end of the cell culture area is communicated with the culture sample outlet; one end of the gel isolation area is communicated with the isolation sample inlet, and the other end of the gel isolation area is communicated with the isolation sample outlet; one end of the factor detection area is communicated with the detection sample inlet, and the other end of the factor detection area is communicated with the detection sample outlet.

Optionally, a plurality of rows of first positioning units are arranged at intervals along the flowing direction of the liquid in the cell culture area, the first positioning units in adjacent rows are staggered with each other, the first positioning units are shaped like V-shaped with openings facing the culture sample inlet, and the first positioning units are used for capturing and positioning cells.

Optionally, the first positioning unit includes a plurality of first microcolumns, the plurality of first microcolumns form a V-shape, and a gap is formed between adjacent first microcolumns.

Optionally, the height of the first positioning units is 20-500 microns, and the number of the first microcolumns of each first positioning unit is 10-30; the first microcolumn is a cylinder, and the diameter of the first microcolumn is 10 micrometers; the gap between adjacent first microcolumns was 5 μm.

Optionally, a plurality of rows of second positioning units are arranged at intervals along the flowing direction of the liquid in the factor detection area, the second positioning units in adjacent rows are staggered with each other, each second positioning unit is in a V shape formed by three second micro-columns, the opening of each second positioning unit faces the detection sample inlet, and a gap is formed between every two adjacent second micro-columns; the second positioning unit is used for capturing the capture positioning of the microbeads;

the capture micro-beads and the detection micro-beads enter the factor detection area from a detection sample inlet; the capture beads are polystyrene beads which are connected with cytokine antibodies and do not have fluorescence and have the diameter of 15 micrometers, and the detection beads are polystyrene beads which are connected with cytokine antibodies and have the fluorescence and the diameter of 0.5 micrometer; each of the second positioning units captures one of the capture microbeads, and the detection microbeads pass through a gap formed between adjacent second microcolumns.

Optionally, the height of the second positioning unit is 20-500 micrometers, and the second microcolumn is a cylinder with a diameter of 10 micrometers; the gap between adjacent second microcolumns was 10 μm.

Optionally, the gel isolation region is provided with two rows of third micropillar arrays, and the row spacing is 100-500 micrometers; wherein one row of the third micro-column array is located between the cell culture region and the gel isolation region, and the other row of the third micro-column array is located between the gel isolation region and the factor detection region;

the height of the third microcolumns is 20-500 micrometers, and the gap between the connected third microcolumns is 10-50 micrometers.

Optionally, two gel isolation regions are respectively disposed on two opposite sides of the cell culture region, and one side of each gel isolation region, which is far away from the cell culture region, is provided with the factor detection region.

According to a second aspect of the invention there is provided a method of monitoring organoid immune killing in real time comprising the steps of:

introducing the gel into a gel isolation area at a preset flow rate, and spontaneously solidifying under the irradiation of ultraviolet rays to form a gel layer; the cell culture area and the factor detection area are isolated by the gel isolation area, so that cell factors in the cell culture area can penetrate through a gel layer in the gel isolation area to enter the factor detection area, and cells in the cell culture area cannot enter the factor detection area under the action of the gel layer;

introducing tumor organoids and immune cells from a patient into a cell culture area at a preset flow rate, and capturing a group of cells at each first positioning unit in the cell culture area to construct an immune microenvironment; performing death and survival calibration on killing of immune cells in a cell culture area on tumor organoids in a fluorescence microscope in a fluorescence staining mode;

introducing the capture microbeads and the detection microbeads into the factor detection area at a preset flow rate, wherein each second positioning unit in the factor detection area captures one capture microbead, and the detection microbeads can pass through the second positioning units; when the cell factors released by the cell culture area permeate into the factor detection area through the gel isolation area, the capture microbeads and the detection microbeads form a connection structure of the capture microbeads-the antibodies-the cytokines-the antibodies-the detection microbeads and are captured by the second positioning unit.

One technical effect of the invention is that:

in the embodiment of the application, an immune microenvironment is constructed in a cell culture area, and death and survival calibration is carried out on killing of tumor organoids by immune cells in the cell culture area in a fluorescence microscope in a fluorescent staining mode, so that real-time monitoring of organoid immune killing is realized. In addition, the gel isolation region isolates cells in the cell culture region, and cytokines can be allowed to pass through the gel isolation region and enter the factor detection region for detection, so that the immune microenvironment of the fine culture region is not influenced in the detection process of the cytokines, the cells are prevented from being influenced, and the accuracy of detection results is favorably ensured.

In addition, the micro-fluidic chip realizes the integration and automation of culture and detection. All operations are performed on one chip, avoiding the unpredictable losses caused by the transfer of samples between different chambers. Moreover, the whole process from the injection of the sample into the microfluidic chip to the completion of the detection is automatic, so that the manual operation is greatly reduced, and a large amount of labor and time are saved.

Drawings

Fig. 1 is a schematic exploded view of a microfluidic chip for monitoring organoid immune killing in real time according to an embodiment of the present invention;

fig. 2 is a schematic structural diagram of a microstructure channel layer of a microfluidic chip for monitoring organoid immune killing in real time according to an embodiment of the present invention;

FIG. 3 is an enlarged detail view taken at A in FIG. 2;

fig. 4 is a schematic structural diagram of a third micropillar array of the microfluidic chip for monitoring organoid immune killing in real time according to an embodiment of the present invention.

In the figure: 1. a microstructure channel layer; 2. a glass substrate; 3. culturing a sample inlet; 4. isolating the sample inlet; 5. detecting a sample inlet; 6. detecting a sample outlet; 7. isolating the sample outlet; 8. culturing a sample outlet; 9. a factor detection zone; 10. a cell culture zone; 11. a gel isolation region; 12. a first positioning unit; 13. a second positioning unit; 14. a third micropillar array.

Detailed Description

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

Reference will now be made in detail to embodiments of the present application, examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are exemplary only for the purpose of explaining the present application and are not to be construed as limiting the present application. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

The features of the terms first and second in the description and in the claims of the present application may explicitly or implicitly include one or more of such features. In the description of the present application, "a plurality" means two or more unless otherwise specified. In addition, "and/or" in the specification and claims means at least one of connected objects, a character "/" generally means that a preceding and succeeding related objects are in an "or" relationship.

In the description of the present application, it is to be understood that the terms "central," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," "counterclockwise," "axial," "radial," "circumferential," and the like are used in the orientations and positional relationships indicated in the drawings for convenience in describing the present application and to simplify the description, and are not intended to indicate or imply that the referenced devices or elements must have a particular orientation, be constructed and operated in a particular orientation, and are therefore not to be considered limiting of the present application.

In the description of the present application, it should be noted that, unless otherwise explicitly stated or limited, the terms "mounted," "connected," and "connected" are to be construed broadly, and may be, for example, a fixed connection, a detachable connection, or an integral connection; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meaning of the above terms in the present application can be understood in a specific case by those of ordinary skill in the art.

Referring to fig. 1 to 4, according to a first aspect of the present invention, there is provided a microfluidic chip for monitoring organoid immune killing in real time. The immune killing condition of the organoid can be monitored in real time through the microfluidic chip.

Specifically, the device comprises a microstructure channel layer 1 and a glass substrate 2, wherein the microstructure channel layer 1 is arranged above the glass substrate 2. And the microstructure channel layer 1 and the glass substrate 2 are sealed with each other. Preferably, the thickness of the glass substrate 2 is 0.15mm to 2mm. The glass substrate 2 can well realize the sealing of the microstructure channel layer 1, and is convenient for real-time monitoring of organoid immune killing under a fluorescence microscope.

For example, the side of the microstructure channel layer 1 close to the glass substrate 2 is provided with microstructure grooves, and the microstructure grooves are closed by the glass substrate 2 to form microstructure channels of the microfluidic chip.

In one embodiment, the thickness of the microstructure channel layer 1 is 2 to 5mm, and the depth of the microstructure channel is 30 to 500 μm, which can better realize the function of the microstructure channel layer 1, and can also ensure the structural stability of the microstructure channel layer 1.

Further specifically, a cell culture region 10, a gel isolation region 11 and a factor detection region 9 are arranged on one side of the microstructure channel layer 1 close to the glass substrate 2, and the gel isolation region 11 is arranged between the cell culture region 10 and the factor detection region 9; the gel isolation zone 11 is filled with a gel layer to prevent the cells in the cell culture zone 10 from entering the factor detection zone 9. The cell culture region 10, the gel isolation region 11 and the factor detection region 9 are all part of a micro-structure channel, so as to realize the culture and isolation of cells and the detection of cytokines.

Organoids and immune cells enter the cell culture area 10, an immune microenvironment is constructed in the cell culture area 10, cytokines in the cell culture area 10 can pass through the gel layer in the gel isolation area 11 to enter the factor detection area 9, and the cytokines are detected in the factor detection area 9.

It should be noted that the microfluidic chip is manufactured based on the microfluidic technology. The advent of microfluidic technology has laid the possibility for controlled co-culture of cells and real-time monitoring of interactions. Microfluidic technology can facilitate co-culture of tumor organoids with immune cells in a continuous perfusion chamber, and sensors and actuators can be integrated with the microfluidic device for precise monitoring and control. The micro-fluidic chip for monitoring the immune killing of the organoid in real time can efficiently capture the organoid by designing a micro structure in the cell culture area 10, and form the gel isolation area 11 based on a gel isolation method, so that various factors can be monitored in real time under the condition of not influencing the organoid to construct an immune microenvironment, and the aim of monitoring the immune killing of the organoid in real time is better achieved.

In the embodiment of the application, an immune microenvironment is constructed in the cell culture zone 10, and killing of tumor organoids by immune cells in the cell culture zone 10 is calibrated by death and survival in a fluorescent microscope in a fluorescent staining manner, so that real-time monitoring of organoid immune killing is realized. In addition, the gel isolation region 11 isolates cells in the cell culture region 10, and can allow cytokines to pass through the gel isolation region 11 and enter the factor detection region 9 for detection, so that the immune microenvironment of the fine culture region is not influenced in the detection process of the cytokines, the cells are prevented from being influenced, and the accuracy of detection results is ensured.

In addition, the micro-fluidic chip realizes the integration and automation of culture and detection. All operations are performed on one chip, avoiding the unpredictable losses caused by the transfer of samples between different chambers. Moreover, the whole process is automatic from the time when the sample is injected into the microfluidic chip to the time when the detection is finished, so that the manual operation is greatly reduced, and a large amount of labor and time are saved.

Illustratively, the cell culture area 10 includes an inlet channel, a culture channel and an outlet channel, both ends of the culture channel are respectively connected to the inlet channel and the outlet channel, and the inlet channel is communicated with the culture inlet 3, and the outlet channel is communicated with the culture outlet 8, and the width of the culture channel is greater than the widths of the inlet channel and the outlet channel, so as to construct a stable immune microenvironment in the cell culture area 10.

Optionally, a culture sample inlet 3, a culture sample outlet 8, an isolation sample inlet 4, an isolation sample outlet 7, a detection sample inlet 5 and a detection sample outlet 6 are arranged on the microstructure channel layer 1;

one end of the cell culture area 10 is communicated with the culture sample inlet 3, and the other end is communicated with the culture sample outlet 8; one end of the gel isolation region 11 is communicated with the isolation sample inlet 4, and the other end is communicated with the isolation sample outlet 7; one end of the factor detection area 9 is communicated with the detection sample inlet 5, and the other end is communicated with the detection sample outlet 6.

In the above embodiment, it is helpful to introduce the culture medium containing organoids and immune cells into the cell culture region 10 through the culture sample inlet 3, and the culture medium can flow out from the culture sample inlet 3, so as to realize the construction of immune microenvironment; the gel is also facilitated to be introduced into the gel isolation area 11 through the isolation sample inlet 4 so as to form a gel layer to prevent the cells and the like from permeating into the factor detection area 9; the device is also favorable for introducing detection beads and capturing the beads through the detection sample inlet 5 so as to realize the detection of the cell factors, has reasonable structural design, does not influence cells, and ensures the accuracy of detection results.

Optionally, a plurality of rows of first positioning units 12 are arranged at intervals along the flowing direction of the liquid in the cell culture region 10, the first positioning units 12 of adjacent rows are staggered with each other, the first positioning units 12 are shaped as V-shaped openings facing the culture sample inlet 3, and the first positioning units 12 are used for capturing and positioning cells.

In the above embodiment, the first positioning unit 12 can perform precise capture and positioning on a group of cells, so as to form a stable immune microenvironment in the cell culture zone 10.

Optionally, the first positioning unit 12 includes a plurality of first micro-pillars, the plurality of first micro-pillars form a V shape, and a gap is formed between adjacent first micro-pillars. The first positioning unit 12 can accurately capture organoids and immune cells, and the gap between the adjacent first microcolumns is helpful for reducing the flow resistance of the culture medium containing organoids and immune cells in the cell culture region 10 and maintaining the stability of the immune microenvironment.

Optionally, the height of the first positioning units 12 is 20-500 micrometers, and the number of the first microcolumns of each first positioning unit 12 is 10-30; the first microcolumn is a cylinder, and the diameter of the first microcolumn is 10 micrometers; the gap between adjacent first microcolumns was 5 μm. This allows the first positioning unit 12 to be designed reasonably to capture a mass of cells, and further reduces the flow resistance of the culture medium containing organoids and immune cells in the cell culture region 10.

Optionally, a plurality of rows of second positioning units 13 are arranged at intervals along the flowing direction of the liquid in the factor detection area 9, the second positioning units 13 in adjacent rows are staggered with each other, the second positioning units 13 are V-shaped and are formed by three second micro-columns, the openings of the second positioning units 13 face the detection sample inlet 5, and a gap is formed between the adjacent second micro-columns; the second positioning unit 13 is used for capturing and positioning the microbeads;

the capture micro-beads and the detection micro-beads enter the factor detection area 9 from the detection sample inlet 5; the capture beads are polystyrene beads which are connected with cytokine antibodies and do not have fluorescence and have the diameter of 15 micrometers, and the detection beads are polystyrene beads which are connected with cytokine antibodies and have the fluorescence and the diameter of 0.5 micrometer; each of the second positioning units 13 captures one of the capture microbeads, and the detection microbead passes through a gap formed between the adjacent second microcolumns.

In the above embodiment, the second positioning unit 13 is reasonably designed to effectively capture the capture beads, and the detection beads can pass through the gaps formed between the adjacent second microcolumns.

After the cell factors released by the cell culture area 10 permeate into the factor detection area 9 through the gel isolation area 11, the capture beads and the detection beads form a connection structure of capture beads-antibody-cytokine-antibody-detection beads, so that the capture beads captured by the second positioning unit 13 gradually have fluorescence, and the proportion of the capture beads with fluorescence in the factor detection area 9 is counted, so that the content of the cell factors in the cell culture area 10 is detected, the detection mode is simple, and the detection result is accurate.

Optionally, the height of the second positioning unit 13 is 20-500 micrometers, and the second microcolumn is a cylinder with a diameter of 10 micrometers; the gap between adjacent second microcolumns was 10 μm. This makes the structural design of the second positioning unit 13 reasonable, and can better realize the function of the second positioning unit 13.

In a specific embodiment, the microstructure channel layer 1 is made of a transparent material, and the transparent material may be glass or PDMS, so that the observation of the microfluidic chip is facilitated. Since the microstructure channel layer 1 needs to be provided with each sample inlet and sample outlet, holes need to be formed.

Optionally, the gel isolation region 11 is provided with two rows of third micropillar arrays 14, and the row spacing is 100-500 micrometers; one row of the third micro-column array 14 is located between the cell culture region 10 and the gel isolation region 11, and the other row of the third micro-column array 14 is located between the gel isolation region 11 and the factor detection region 9;

In the above embodiment, the third micro-column array 14 of the gel isolation region 11 can fix the gel well when the gel isolation region 11 is filled with the gel to form a gel layer, thereby isolating the cells and allowing the cytokine to pass through.

In the embodiment of the application, the application range of the microfluidic chip is wide. The sizes of the first positioning unit 12, the second positioning unit 13 and the third micro-column array 14 can be adjusted according to different cells, the flexibility is high, and the cost is not increased. Furthermore, the cytokine detected by the microfluidic chip can be changed at will by changing the types of the antibodies on the detection microbeads, so that the detection sensitivity is not influenced, and the cost is not increased. Meanwhile, the number of the detection factors can be increased by increasing the number of the gel isolation area 11 and the factor detection area 9, so that the detection sensitivity is not influenced, and the operation is very convenient.

Optionally, two gel isolation regions 11 are respectively disposed on two opposite sides of the cell culture region 10, and one side of each gel isolation region 11 away from the cell culture region 10 is provided with the factor detection region 9.

In the above embodiment, each gel isolation region 11 is connected with the isolation sample inlet 4 and the isolation sample outlet 8, each factor detection region 9 is connected with the detection sample inlet 5 and the detection sample outlet 6, as shown in fig. 1 and fig. 2, different detection microbeads can be introduced into different factor detection regions 9, and different cytokine detection regions 9 can detect different cytokines due to different types of antibodies on different detection microbeads, so that the operation is very simple and efficient.

In one embodiment, the glass substrate 2 has a thickness of 2mm. The thickness of the glass sheet has an influence on the imaging magnification of the microscope, so if high-power microscope imaging is required for cells, detection beads and the like, a thinner glass substrate 2 can be selected as required.

PDMS is selected as the material of the microstructure channel layer 1, because PDMS can be well compatible with the micro-processing technology based on photoetching and corrosion, and the processing precision is easy to control, so that the capture array which is consistent with the cell size can be easily obtained. The skilled person can also select processable materials such as glass, silicon etc. according to the requirements.

For example, the thickness of the microstructure channel layer 1 is 3 mm, and the depth of the microstructure channel is 30 μm. The height of the first positioning units 12 is 25 micrometers, the first positioning units are formed by arranging 18 first micro-columns, each first micro-column is a cylinder with the diameter of 10 micrometers, the gap between every two adjacent first micro-columns is 5 micrometers, and the cell culture area 10 is totally provided with 77 first positioning units 12.

The height of the second positioning unit 13 of the factor detection area 9 is 25 micrometers, the factor detection area is formed by arranging 3 second microcolumns, each second microcolumn is a cylinder with the diameter of 10 micrometers, the gap between every two adjacent second microcolumns is 10 micrometers, and the factor detection area 9 has 200 second positioning units 13 in total.

The gel isolation region 11 is provided with two rows of third micropillar arrays 14, the row spacing is 100 micrometers, the height of the third micropillars is 25 micrometers, and the gap between adjacent third micropillars is 37 micrometers. When other cells need to be processed, the appropriate size and number of microstructures can be selected by one skilled in the art.

In another embodiment, the microstructure channel layer 1 has a thickness of 2mm and the microstructure channels have a depth of 35 μm. The height of the first positioning units 12 is 30 micrometers, the first positioning units are formed by arranging 36 first micro-columns, each first micro-column is a cylinder with the diameter of 10 micrometers, the gap between every two adjacent first micro-columns is 5 micrometers, and the total number of the first positioning units 12 in the cell culture area 10 is 150.

The height of the second positioning unit 13 of the factor detection area 9 is 30 micrometers, the factor detection area is formed by arranging 3 second microcolumns, each second microcolumn is a cylinder with the diameter of 10 micrometers, the gap between every two adjacent second microcolumns is 10 micrometers, and the factor detection area 9 has 200 second positioning units 13 in total.

The gel isolation region 11 is provided with two rows of third microcolumn arrays 14, the row spacing is 200 micrometers, the height of the third microcolumns is 30 micrometers, and the gap between adjacent third microcolumns is 45 micrometers.

In the application, the micro-structure channel layer 1 and the glass substrate 2 are sealed and connected by adopting an oxygen plasma auxiliary bonding method.

The manufacturing method of the micro-fluidic chip comprises the following steps:

firstly, an N-type 4-inch silicon wafer is adopted, after the plane shape of a microstructure groove is photoetched, each positioning unit with the height of 25 micrometers and a flow channel with the depth of 30 micrometers are obtained by adopting an ICP (inductive plasma etching, namely, silicon is etched by high-energy plasma of sulfur hexafluoride and carbon tetrafluoride) dry etching method. Pouring liquid PDMS into a tank, baking for 2h in an oven at 80 ℃ to solidify the PDMS, demolding and taking out the solidified PDMS, cutting off the redundant part of the PDMS, cutting the PDMS into rectangular small pieces according to the overall dimensions of 2 cm in length and 1 cm in width, and punching holes at the required positions by using a puncher, thereby obtaining the microstructure channel layer 1.

Then, the bottom surface of the transparent microstructure channel layer 1 and the top surface of the glass substrate 2 are treated with oxygen plasma and bonded together, and finally, a complete chip is fabricated.

It should be noted that, on the basis of the microfluidic chip, a polytetrafluoroethylene tube is used to connect the pump and each sample inlet, when the analysis result is needed, the chip is placed under an inverted fluorescence microscope capable of performing fluorescence imaging, and the automatic analysis processing of the fluorescence image is performed in the image processing software of the computer, so that the construction of the whole set of system can be completed.

introducing the gel into the gel isolation area 11 at a preset flow rate, and spontaneously solidifying under the irradiation of ultraviolet rays to form a gel layer; the gel isolation region 11 isolates the cell culture region 10 from the factor detection region 9, so that the cytokine in the cell culture region 10 can pass through the gel layer in the gel isolation region 11 to enter the factor detection region 9, and the cell in the cell culture region 10 cannot enter the factor detection region 9 under the action of the gel layer.

For example, the gel is pumped into the gel isolation region 11 at a predetermined flow rate, and the gel is bound between two rows of the micro-column array due to viscosity. Then, the gel spontaneously solidifies under ultraviolet irradiation to form a gel layer. The gel layer allows small molecule proteins such as cytokines to permeate therethrough, but does not allow large diameter structures such as cells to pass therethrough, thereby effectively isolating the cell culture zone 10 from the factor detection zone 9.

Introducing tumor organoids and immune cells from a patient into the cell culture region 10 at a predetermined flow rate, and capturing a mass of cells by each first positioning unit 12 in the cell culture region 10 to construct an immune microenvironment; and (3) performing death and survival calibration on killing of the tumor organoids by the immune cells in the cell culture area 10 in a fluorescence microscope in a fluorescence staining mode.

For example, a culture medium containing the organoid and the immune cells is pumped into the cell culture area 10, the culture medium contains PI and a reagent for detecting caspase9, and death and survival calibration can be performed on killing of tumor organoids by the immune cells under a fluorescence microscope through fluorescent staining, so that real-time monitoring on organoid immune killing is realized, and the monitoring effect is good.

It should be noted that the tumor organoid can be a 3D cell mass cultured in a gel from tumor cells lysed from a tumor of the patient, and the immune cells can be PBMC cells extracted from blood of the patient and then induced tumor-reactive immune cells, and the tumor organoid and the immune cells are passed into the cell culture region 10 and captured by the first positioning unit 12. The fluorescence microscope is used for observing the capture beads-antibody-cytokine-antibody-detection beads in the factor detection zone 9 and observing the cells in the cell culture zone 10, and the death and apoptosis staining of the organoids are imaged.

The method comprises the following steps of (1) introducing capture microbeads and detection microbeads into a factor detection area 9 at a preset flow rate, capturing one capture microbead by each second positioning unit 13 in the factor detection area 9, and enabling the detection microbead to pass through the second positioning unit 13; when the cytokine released from the cell culture region 10 permeates into the factor detection region 9 through the gel isolation region 11, the capture bead and the detection bead form a connection structure of the capture bead-antibody-cytokine-antibody-detection bead and are captured by the second positioning unit 13.

For example, capture beads and detection beads are pumped into the factor detection zone 9 at a predetermined flow rate. After the cytokine released by the cell culture area 10 permeates into the factor detection area 9 through the gel isolation area 11, the capture beads and the detection beads form a connection structure of capture beads-antibody-cytokine-antibody-detection beads, so that the capture beads captured by the second positioning unit 13 gradually have fluorescence, and the proportion of the capture beads with fluorescence in the factor detection area 9 is counted, so that the content of the cytokine in the cell culture area 10 is detected, the detection mode is simple, and the detection result is accurate.

And finally, washing off the microbeads captured by the capture unit by reversely feeding liquid to the factor detection area 9, and then feeding the next batch of microbeads from the detection sample inlet 5 to finish the next detection, so that the operation is simple.

In the above embodiment, the method for monitoring organoid immune killing in real time is very simple, and not only can death and viability calibration be performed on killing of tumor organoids by immune cells in the cell culture area 10 in a fluorescent microscope in a fluorescent staining mode, so that real-time monitoring of organoid immune killing is realized, but also the whole process is automatic from the time when a sample is injected into the microfluidic chip to the time when detection is completed, so that manual operation is greatly reduced, and a large amount of manpower and time are saved.

It will be understood that the above embodiments are merely exemplary embodiments taken to illustrate the principles of the present invention, which is not limited thereto. It will be apparent to those skilled in the art that various modifications and improvements can be made without departing from the spirit and scope of the invention, and such modifications and improvements are also considered to be within the scope of the invention.

Claims

1. The micro-fluidic chip for monitoring the immune killing of the organoid in real time is characterized by comprising a micro-structural channel layer and a glass substrate, wherein the micro-structural channel layer is arranged above the glass substrate;

2. The microfluidic chip for monitoring organoid immune killing in real time according to claim 1, wherein the microstructure channel layer is provided with a culture sample inlet, a culture sample outlet, an isolation sample inlet, an isolation sample outlet, a detection sample inlet and a detection sample outlet;

3. The microfluidic chip for real-time monitoring organoid immune killing according to claim 2, wherein a plurality of rows of first positioning units are arranged at intervals along the flowing direction of the liquid in the cell culture region, the first positioning units of adjacent rows are staggered with each other, the first positioning units are in a V shape with an opening facing the culture sample inlet, and the first positioning units are used for capturing and positioning cells.

4. The microfluidic chip for real-time monitoring of organoid immune killing according to claim 3, wherein the first positioning unit comprises a plurality of first microcolumns, the plurality of first microcolumns form a V-shape, and a gap is formed between adjacent first microcolumns.

5. The microfluidic chip for real-time monitoring organoid immune killing according to claim 4, wherein the height of the first positioning units is 20-500 μm, and the number of the first microcolumns of each first positioning unit is 10-30; the first microcolumn is a cylinder, and the diameter of the first microcolumn is 10 micrometers; the gap between adjacent first microcolumns was 5 μm.

6. The microfluidic chip for real-time monitoring of organoid immune killing according to claim 2, wherein a plurality of rows of second positioning units are arranged at intervals along the flow direction of the liquid in the factor detection zone, the second positioning units of adjacent rows are staggered with each other, the second positioning units are V-shaped and are formed by three second microcolumns, the openings of the second positioning units face the detection sample inlet, and a gap is formed between the adjacent second microcolumns; the second positioning unit is used for capturing the capture positioning of the microbeads;

the capture micro-beads and the detection micro-beads enter the factor detection area from the detection sample inlet; wherein, the capture micro-beads are polystyrene micro-beads which are connected with cytokine antibodies and have no fluorescence and have the diameter of 15 micrometers, and the detection micro-beads are polystyrene micro-beads which are connected with cytokine antibodies and have fluorescence and have the diameter of 0.5 micrometer; each of the second positioning units captures one of the capture microbeads, and the detection microbeads pass through a gap formed between adjacent second microcolumns.

7. The microfluidic chip for real-time monitoring organoid immune killing according to claim 6, wherein the height of the second positioning unit is 20-500 microns, the second microcolumn is a cylinder with a diameter of 10 microns; the gap between adjacent second microcolumns was 10 μm.

8. The microfluidic chip for real-time monitoring organoid immune killing according to claim 2, wherein the gel isolation region is provided with two rows of the third micropillar arrays with a row spacing of 100-500 μm; wherein one row of the third micro-column array is positioned between the cell culture region and the gel isolation region, and the other row of the third micro-column array is positioned between the gel isolation region and the factor detection region;

9. The microfluidic chip for real-time monitoring organoid immune killing according to claim 1, wherein two of the gel isolation regions are disposed on opposite sides of the cell culture region, and the factor detection region is disposed on a side of each of the gel isolation regions away from the cell culture region.

10. A method for monitoring organoid immune killing in real time, which is characterized by comprising the following steps:

introducing the gel into the gel isolation area at a preset flow rate, and spontaneously solidifying under the irradiation of ultraviolet rays to form a gel layer; the gel isolation area isolates the cell culture area from the factor detection area, so that the cell factors in the cell culture area can pass through the gel layer in the gel isolation area to enter the factor detection area, and the cells in the cell culture area cannot enter the factor detection area under the action of the gel layer;

introducing the capture microbeads and the detection microbeads into the factor detection area at a preset flow rate, wherein each second positioning unit in the factor detection area captures one capture microbead, and the detection microbeads can pass through the second positioning units; when the cell factors released by the cell culture area permeate into the factor detection area through the gel isolation area, the capture microbeads and the detection microbeads form a connection structure of the capture microbeads-the antibodies-the cytokines-the antibodies-the detection microbeads, and the connection structure is captured by the second positioning unit.