Microfluidic chips and organoid capture, drug sensitivity testing, release and lysis methods

Abstract

The embodiments of the present disclosure provide a microfluidic chip and a method for organoid capture, drug sensitivity testing, release and lysis, wherein the chip comprises an air valve layer, a thin film layer and a drug sensitivity layer; the thin film layer and the air valve layer are sequentially stacked on the drug sensitivity layer; the air valve layer controls the on-off of the flow channel on the drug sensitivity layer through the thin film layer; the drug sensitivity layer is provided with a first liquid inlet, a drug incubation area, a release liquid inlet, a collection area and a first liquid outlet; the drug incubation area is connected to the first liquid inlet and the first liquid outlet through the flow channel respectively; the drug incubation area has an organoid capture sub-area; the collection area is connected to the drug incubation area through the flow channel; the release liquid inlet is connected to the drug incubation area through the flow channel; wherein the release liquid is introduced into the drug incubation area through the release liquid inlet; and based on the on-off control of the flow channel on the drug sensitivity layer by the air valve layer, the organoid capture sub-area releases the organoid; the organoids released from the organoid capture sub-area are gathered in the collection area through the flow channel.

Description

Technical Field

[0001] The embodiments of the present disclosure belong to the field of microfluidic biochip technology, and specifically relate to a microfluidic chip and an organoid capture, drug sensitivity testing, release and lysis method. Background Art

[0002] Organoids are self-assembled cell structures cultivated in vitro through three-dimensional culture technology, which have structures and functions that are very similar to organs in the body. Since organoids retain the heterogeneity of tumor cells in the body to a large extent, they can simulate the cellular composition, structure and physiological functions of organs in the body with high accuracy, which makes them have extremely good application prospects in the field of personalized medicine. Specifically, tumor organoids can be constructed from the patient's tumor tissue, and then high-throughput drug sensitivity testing can be performed through organoids. This method can help patients provide effective treatment reference drugs before taking the medicine, greatly improving the targetedness and effectiveness of the treatment.

[0003] Traditional plate-based organoid drug sensitivity testing is mainly done manually in the laboratory with the help of a pipette and a plate. Due to the errors in manual spotting, more parallel groups need to be set up to achieve balance, which places a greater demand on the base number of organoids.

[0004] However, for patients who are diagnosed early through puncture biopsy, the sample size is often very limited, and constructing organoids that meet the drug sensitivity requirements usually requires a long culture cycle, which makes it difficult to meet the timeliness requirements of drug sensitivity testing.

[0005] With the continuous advancement of science and technology, the emergence of organ chips has brought hope for solving this problem. Organ chips can achieve accurate capture of organoids and real-time observation in situ through micro-nano processing technology. This technology precisely controls the organoids in terms of quantity and environment, greatly reducing the error of manual spotting, further reducing the demand for sample volume base, and thus shortening the culture period before drug sensitivity testing.

[0006] However, the in situ analysis methods for organoids on a chip are relatively limited, and the dimensions provided by these methods can only reach the protein level at most, which is far from enough to gain a deep understanding of the characteristics of organoids and the mechanism of drug action. In order to better understand the mechanism of drug action on tumors, it is urgent to collect the on-chip organoids after achieving drug sensitivity for downstream molecular level analysis outside the chip. Summary of the invention

[0007] The embodiments of the present disclosure aim to solve at least one of the technical problems existing in the prior art and provide a microfluidic chip and organoid capture, drug sensitivity testing, release and lysis method.

[0008] In a first aspect of an embodiment of the present disclosure, a microfluidic chip is provided, the chip comprising an air valve layer, a thin film layer and a drug-sensitive layer; the thin film layer and the air valve layer are sequentially stacked on the drug-sensitive layer; the air valve layer controls the on-off of a flow channel on the drug-sensitive layer through the thin film layer;

[0009] The drug-sensitive layer is provided with a first liquid inlet, a drug incubation area, a release liquid inlet, a collection area and a first liquid outlet;

[0010] The drug incubation area is connected to the first liquid inlet and the first liquid outlet through flow channels respectively; the drug incubation area has an organoid capture sub-area for capturing organoids in the organoid matrix gel suspension introduced through the first liquid inlet;

[0011] The collection area is connected to the drug incubation area through a flow channel; the release liquid inlet is connected to the drug incubation area through a flow channel;

[0012] The releasing liquid is introduced into the drug incubation area through the releasing liquid inlet; and based on the on-off control of the flow channel on the drug sensitive layer by the air valve layer, the organoid capture sub-area releases the organoid; the organoids released from the organoid capture sub-area are collected in the collection area through the flow channel.

[0013] Optionally, the collection area includes a collection chamber, a functional liquid inlet and a functional liquid outlet; the functional liquid inlet and the functional liquid outlet are respectively connected to the collection chamber through flow channels; and the collection chamber is connected to the drug incubation area through the flow channel.

[0014] Optionally, the drug incubation area includes a plurality of main channels arranged side by side and connected in sequence; the organoid capture sub-area includes a capture structure arranged between two adjacent main channels; the main channel is used for the circulation of organoid matrix gel suspension; and the capture structure is used for capturing organoids in the organoid matrix gel suspension.

[0015] Optionally, there are a plurality of capture structures; along the length direction of the main flow channel, the plurality of capture structures are spaced apart between two adjacent main flow channels.

[0016] Optionally, the capture structure includes a capture cavity and a branch channel; wherein the capture cavity is arranged on the side wall of one of the two adjacent main channels; one end of the branch channel is connected to the capture cavity, and the other end is connected to the other main channel; the capture cavity is used to capture organoids in the organoid matrix gel suspension.

[0017] Optionally, the branch channel includes a narrow channel section and a transition section; the narrow channel section is arranged between the capture chamber and the transition section, and is respectively connected to the capture chamber and the transition section; one end of the transition section away from the narrow channel section is connected to the main channel; wherein the width dimension of the channel section is less than or equal to the width dimension of the transition section.

[0018] A second aspect of the embodiments of the present disclosure provides an organoid capture method, wherein the method is implemented based on the microfluidic chip described above.

[0019] Using the air valve layer to cut off the flow channel between the collection area and the drug incubation area, the flow channel between the collection area and the first liquid inlet, and the flow channel between the release liquid inlet and the drug incubation area;

[0020] Passing the organoid matrix gel suspension into the drug incubation area from the first liquid inlet, wherein the organoid capture sub-area captures the organoids in the organoid matrix gel suspension;

[0021] After standing for a preset time at a certain temperature, a flushing liquid is introduced into the drug incubation area from the first liquid inlet to flush the organoid matrix gel suspension in the main channel.

[0022] A third aspect of the embodiments of the present disclosure provides a method for testing drug sensitivity of organoids, wherein the method is implemented based on the microfluidic chip described above.

[0023] Using the air valve layer to cut off the flow channel between the collection area and the drug incubation area, the flow channel between the collection area and the first liquid inlet, and the flow channel between the release liquid inlet and the drug incubation area;

[0024] Passing the drug into the drug incubation area from the first liquid outlet, and incubating the organoids in the drug incubation area with the drug at a certain temperature;

[0025] The status of the organoids in the captured structure was observed under a microscope.

[0026] A fourth aspect of the embodiments of the present disclosure provides an organoid release method, wherein the method is implemented based on the microfluidic chip described above.

[0027] Using the air valve layer to cut off the flow channel between the drug incubation area and the first liquid outlet, the flow channel between the first liquid inlet and the drug incubation area, and the flow channel between the first liquid inlet and the collection area;

[0028] Passing a release liquid into the drug incubation area from the release liquid inlet, so that the organoid capture sub-area in the drug incubation area releases the organoid;

[0029] The collection area is used to collect the released fluid containing the organoids.

[0030] A fifth aspect of the embodiments of the present disclosure provides an organoid lysis method, wherein the method is implemented based on the microfluidic chip described above.

[0031] Using the air valve layer to cut off the flow channel between the drug incubation area and the collection area;

[0032] Passing the lysate into the collection chamber from the functional liquid inlet;

[0033] The air valve layer is used to cut off the flow channel between the functional liquid inlet and the collection chamber, and the air valve layer is used to agitate the thin film layer to mix the lysate and the organoid in the collection chamber.

[0034] The beneficial effects of the embodiments of the present disclosure include:

[0035] The microfluidic chip of the present invention integrates organoid capture, drug sensitivity, and release functions to meet the high-throughput drug sensitivity and molecular detection needs of organoids, so as to be better used for the study of tumor drug sensitivity and mechanism of action. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 It is a schematic diagram of the exploded structure of a microfluidic chip according to an embodiment of the present disclosure;

[0037] Figure 2 It is a structural schematic diagram of a gas valve layer according to an embodiment of the present disclosure;

[0038] Figure 3 It is a schematic structural diagram of a drug-sensitive layer according to an embodiment of the present disclosure;

[0039] Figure 4 for Figure 3 Schematic diagram of the enlarged structure of the organoid capture subregion;

[0040] Figure 5 for Figure 3 A magnified schematic diagram of the structure of the collection area;

[0041] Figure 6 is a schematic structural diagram of a microfluidic chip according to another embodiment of the present disclosure;

[0042] Figure 7 A schematic diagram of a process for capturing an organoid according to another embodiment of the present disclosure.

[0043] In the figure, 100, air valve layer; 200, thin film layer; 300, drug sensitive layer; 301, first liquid inlet; 302, first liquid outlet; 303, release liquid inlet; 304, functional liquid inlet; 305, functional liquid outlet; 306, collection chamber; 310, collection area; 320, organoid capture sub-area; 330, drug incubation area; 321, capture structure; 331, main channel; 3211, capture cavity; 3212, branch channel; 32121, narrow channel section; 32122, transition section. DETAILED DESCRIPTION

[0044] In order to enable those skilled in the art to better understand the technical solution of the present disclosure, the present disclosure is further described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0045] The following is a further detailed description of the implementation methods of the present application in conjunction with the accompanying drawings and examples. The detailed descriptions and drawings of the following examples are used to illustrate the principles of the present application, but cannot be used to limit the scope of the present application, that is, the present application is not limited to the described embodiments. In the description of the present application, it should be noted that, unless otherwise specified, the meaning of "multiple" is more than two; the orientation or position relationship indicated by the terms "upper", "lower", "left", "right", "inside", "outside", etc. is only for the convenience of describing the present application and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present application. In addition, the terms "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or implying relative importance. "Vertical" is not vertical in the strict sense, but within the error tolerance range. "Parallel" is not parallel in the strict sense, but within the error tolerance range.

[0046] In the description of this application, it should also be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be directly connected or indirectly connected through an intermediate medium. For ordinary technicians in this field, the specific meanings of the above terms in this application can be understood according to the specific circumstances.

[0047] like Figure 1-6 As shown, a microfluidic chip includes an air valve layer 100, a thin film layer 200 and a drug-sensitive layer 300, wherein the thin film layer 200 and the air valve layer 100 are sequentially stacked on the drug-sensitive layer 300, and the air valve layer 100 controls the on-off of the flow channel on the drug-sensitive layer 300 through the thin film layer 200.

[0048] The drug sensitive layer 300 is provided with a first liquid inlet 301, a drug incubation area 330, a release liquid inlet 303, a collection area 310 and a first liquid outlet 302. The drug incubation area 330 is connected to the first liquid inlet 301 and the first liquid outlet 302 through a flow channel, respectively, and the drug incubation area 330 has an organoid capture sub-area 320 for capturing organoids in the organoid matrix gel suspension introduced by the first liquid inlet. The collection area 310 is connected to the drug incubation area 330 through a flow channel, and the release liquid inlet 303 is connected to the drug incubation area 330 through a flow channel.

[0049] The release liquid is introduced into the drug incubation area 330 through the release liquid inlet 303, and the organoid capture sub-area 320 releases the organoid based on the on-off control of the flow channel on the drug sensitive layer 300 by the air valve layer 100, and the organoid released from the organoid capture sub-area 320 is collected in the collection area 310 through the flow channel. It can be understood that the liquid introduced into the first liquid inlet 301 includes but is not limited to the organoid matrix gel suspension, which can be selected as needed.

[0050] In the present disclosure, the release liquid is introduced into the drug incubation area 330 through the release liquid inlet 303, and based on the control of the gas valve layer 100, the organoid capture sub-area 320 releases the organoids, and the organoids released from the organoid capture sub-area 320 are collected in the collection area 310 through the flow channel for subsequent molecular detection needs.

[0051] In some embodiments, a plurality of organoid capture sub-regions 320 are disposed in the drug incubation region 330 , and the plurality of organoid capture sub-regions 320 are arranged in an array in the drug incubation region 330 .

[0052] In some embodiments, the collection area 310 includes a collection chamber 306, a functional liquid inlet 304 and a functional liquid outlet 305, the functional liquid inlet 304 and the functional liquid outlet 305 are respectively connected to the collection chamber 306 through flow channels, and the collection chamber 306 is connected to the drug incubation area 330 through the flow channel.

[0053] In the present disclosure, by providing the collection chamber 306, the organoids released from the organoid capture sub-region 320 can be effectively received and temporarily stored, avoiding the loss of organoids due to excessive flow rate during the release process, thereby improving the collection efficiency of the organoids.

[0054] Through the functional liquid inlet 304 and the functional liquid outlet 305, the organoid can be further processed in the collection chamber 306, such as adding a lysis solution, so that molecular analysis can be performed after the organoid is lysed. It is understood that the liquid introduced into the functional liquid inlet 304 includes but is not limited to the lysis solution, which can be selected as needed.

[0055] In some embodiments, the drug incubation area 330 includes a plurality of main channels 331 arranged side by side and connected in sequence, and the organoid capture sub-area 320 includes a capture structure 321 arranged between two adjacent main channels 331, the main channel 331 is used for the circulation of the organoid matrix gel suspension, and the capture structure 321 is used for capturing organoids in the organoid matrix gel suspension.

[0056] In the present disclosure, by providing a capture structure 321 between a plurality of main channels 331 arranged side by side and connected in sequence, the surface area of ​​organoid capture can be significantly increased, thereby improving the capture efficiency of organoids. The design of the capture structure 321 can ensure that the organoids are effectively captured when flowing through the main channel 331, thereby reducing the loss of organoids.

[0057] The design of multiple main channels 331 arranged side by side and connected in sequence can optimize fluid dynamics performance, ensure that the organoid matrix gel suspension is evenly distributed and flows in the main channel 331, and reduce fluid resistance and pressure loss. This design helps to maintain a stable flow rate and pressure, ensuring a stable environment for organoids during culture.

[0058] In some embodiments, there are multiple capture structures 321 , and along the length direction of the main flow channel 331 , the multiple capture structures 321 are spaced apart between two adjacent main flow channels 331 .

[0059] In the present disclosure, by providing multiple capture structures 321 in the length direction of the main channel 331, the capture points of the organoids can be significantly increased, thereby improving the capture efficiency of the organoids. The provision of multiple capture structures 321 allows the organoids to have more opportunities to be captured when flowing through the main channel 331, thereby reducing the loss of the organoids.

[0060] Arranging multiple capture structures 321 along the length of the main channel 331 can better disperse the fluid dynamics and reduce the damage to the organoids caused by excessive local flow velocity. The evenly distributed capture structures 321 help maintain a stable flow rate and pressure, ensuring a stable environment for the organoids during the culture process.

[0061] In some embodiments, the capture structure 321 includes a capture chamber 3211 and a branch channel 3212, wherein the capture chamber 3211 is arranged on the side wall of one of the two adjacent main channels 331, one end of the branch channel 3212 is connected to the capture chamber 3211, and the other end is connected to the other main channel 331, and the capture chamber 3211 is used to capture organoids in the organoid matrix gel suspension.

[0062] In the present disclosure, the capture chamber 3211 is disposed on the side wall of the main channel 331 , so that the capture chamber 3211 can effectively capture the organoids in the main channel 331 .

[0063] The function of the branch channel 3212 is to limit the capture of organoids in the capture chamber through the narrow channel size of the branch channel during the capture process, while allowing liquid to flow through. During the drug sensitivity process and the release process, the flow resistance of the channel is adjusted by the wide and narrow channel size of the branch channel so that the organoids will not be released by mistake during the drug application process.

[0064] In some embodiments, the branch channel 3212 includes a narrow channel section 32121 and a transition section 32122, wherein the narrow channel section 32121 is disposed between the capture chamber 3211 and the transition section 32122, and is respectively connected to the capture chamber 3211 and the transition section 32122. One end of the transition section 32122 away from the narrow channel section 32121 is connected to the main channel 331, wherein the width of the channel section is less than or equal to the width of the transition section 32122.

[0065] In the present disclosure, the narrow channel section 32121 has a small width, which can generate a certain fluid resistance, making it easier for the organoid to be captured in the narrow channel by the capture chamber 3211. The transition section 32122 has a large width, which can smoothly guide the organoid matrix gel suspension from the narrow channel section 32121 to the main channel 331, reduce fluid resistance and pressure loss, and ensure that the organoid enters the capture chamber 3211 smoothly.

[0066] refer to Figure 7 According to a second aspect of the embodiments of the present disclosure, a method for capturing an organoid is provided. The method is implemented based on the above-mentioned microfluidic chip.

[0067] S101, using the air valve layer to cut off the flow channel between the collection area and the drug incubation area, the flow channel between the collection area and the first liquid inlet, and the flow channel between the release liquid inlet and the drug incubation area.

[0068] S102, passing the organoid matrix gel suspension into the drug incubation area from the first liquid inlet, wherein the organoid capture sub-area captures the organoids in the organoid matrix gel suspension.

[0069] S103, after standing for a preset time at a certain temperature, a flushing liquid is introduced into the drug incubation area from the first liquid inlet to flush the organoid matrix gel suspension in the main channel.

[0070] A third aspect of the embodiments of the present disclosure provides a method for testing drug sensitivity of organoids, which is implemented based on the above-mentioned microfluidic chip.

[0071] S201, using the air valve layer to cut off the flow channel between the collection area and the drug incubation area, the flow channel between the collection area and the first liquid inlet, and the flow channel between the release liquid inlet and the drug incubation area.

[0072] S202, introducing the drug into the drug incubation area from the first liquid outlet, and incubating the organoids in the drug incubation area at a certain temperature.

[0073] S203. Observe the state of the organoid in the captured structure through a microscope.

[0074] A fourth aspect of the embodiments of the present disclosure provides an organoid release method, which is implemented based on the above-mentioned microfluidic chip.

[0075] S301, using the air valve layer to cut off the flow channel between the drug incubation area and the first liquid outlet, the flow channel between the first liquid inlet and the drug incubation area, and the flow channel between the first liquid inlet and the collection area.

[0076] S302, passing the releasing liquid into the drug incubation area from the releasing liquid inlet, so that the organoid capture sub-area in the drug incubation area releases the organoid.

[0077] S303. Use the collection area to collect the release liquid containing the organoids.

[0078] A fifth aspect of the embodiments of the present disclosure provides an organoid lysis method, which is implemented based on the above-mentioned microfluidic chip.

[0079] S401, using the air valve layer to cut off the flow channel between the drug incubation area and the collection area.

[0080] S402, passing the lysate into the collection chamber from the functional liquid inlet.

[0081] S403, using the air valve layer to cut off the flow channel between the functional liquid inlet and the collection chamber, and using the air valve layer to agitate the thin film layer to mix the lysis solution and the organoid in the collection chamber.

[0082] The present disclosure provides a specific example, including:

[0083] The present disclosure provides a microfluidic chip, comprising: a valve layer 100, a film layer 200 and a drug-sensitive layer 300 stacked in sequence from top to bottom. Four drug incubation areas 330 are arranged on the drug-sensitive layer 300, each drug incubation area 330 is correspondingly provided with a collection area 310, and each drug incubation area 330 is correspondingly provided with a first liquid outlet 302, and the first liquid outlet 302 is connected to the drug incubation area 330 through a flow channel.

[0084] The drug-sensitive layer 300 includes two first liquid inlets 301 and one release liquid inlet 303. One first liquid inlet 301 is connected to two drug incubation areas 330 through flow channels, and the other first liquid inlet 301 is connected to the other two drug incubation areas 330 through flow channels. The release liquid inlet 303 is connected to the four drug incubation areas 330 through flow channels.

[0085] Wherein, one or more organoid capture sub-areas 320 are arranged in each drug incubation area 330 .

[0086] The air valve layer 100 is used to cooperate with the film layer 200 to control the flow and closing of the flow channel on the drug-sensitive layer 300.

[0087] The drug-sensitive layer 300 has five flow channel inlets and outlets, including:

[0088] The first liquid inlet 301 is used as the first liquid inlet / culture medium inlet / drug outlet. The first liquid outlet 302 is used as the drug inlet / first liquid inlet / culture medium outlet. The release liquid inlet 303 is used as the organoid release liquid, such as culture medium / PBS. The functional liquid inlet 304 is used as the cell lysis liquid / other functional liquid inlet. The functional liquid outlet 305 is used as the cell lysis liquid / other functional liquid outlet.

[0089] The depth of the flow channel on the drug-sensitive layer 300 is between 100 and 200 microns.

[0090] The film layer 200 is used to cooperate with the air valve layer 100 to control the flow and closing of the flow channel of the drug-sensitive layer 300.

[0091] The thickness of the film layer 200 is between 20 and 50 micrometers.

[0092] The air valve layer 100 is provided with air valves 101-115, each of which includes an air inlet, an air inlet channel, and a control node provided on the air inlet channel, wherein the control node and the thin film layer act together on the channel of the drug sensitive layer 300 to control the flow and closing of the channel. The depth of the air inlet channel on the air valve layer 100 is between 50-200 microns. The working principle of the air valve layer 100 includes:

[0093] Organoid Capture:

[0094] Valve state: Valve 114 is open, valve 101 is open, the flow of fluid in the flow channel is blocked, and the other valves are closed and do not work. That is, the flow channel between the collection area and the drug incubation area, the flow channel between the collection area and the first liquid inlet, and the flow channel between the release liquid inlet and the drug incubation area are cut off by the valve layer.

[0095] The organoid matrix gel suspension enters the drug incubation area of ​​the drug sensitive layer 300 from the first liquid inlet 301, and is captured by the organoid capture sub-area in the drug incubation area. Other liquids flow out through the first liquid outlet 302. The microfluidic chip is left to stand in a 37°C incubator for 20 minutes, and the organoid matrix gel suspension changes from liquid to solid.

[0096] The culture medium is introduced from the first liquid inlet 301 . As the organoids are captured by the organoid capture sub-region, the flow resistance in the main channel becomes larger. The liquid washes away the residual matrix gel in the flow channel through the main channel and flows out from the first liquid outlet 302 .

[0097] Organoid drug sensitivity:

[0098] Valve state: Valve 114 is open, valve 101 is open, the flow of fluid in the flow channel is blocked, and the other valves are closed and do not work. That is, the flow channel between the collection area and the drug incubation area, the flow channel between the collection area and the first liquid inlet, and the flow channel between the release liquid inlet and the drug incubation area are cut off by the valve layer.

[0099] The drug enters the drug incubation area from the first liquid outlet 302 . Since the organoids are captured at the capture structure, the flow resistance of the main channel increases, and the liquid flows through the main channel and flows out from the first liquid inlet 301 .

[0100] The microfluidic chip was placed in a 37° C. incubator for drug incubation, and oil was dripped onto the first liquid outlet 302 and the first liquid inlet 301 to seal the first liquid outlet 302 and the first liquid inlet 301 to prevent liquid evaporation.

[0101] During the drug sensitivity test, the state of individual organoids in the captured structure can be observed in real time by microscopy. It is understandable that the type of drug can be selected as needed.

[0102] Organoid Release:

[0103] The release process is layer by layer. The air valve changes and release process are as follows:

[0104] The air valve 102 is opened, the air valve 111 is opened, and the air valve 103 is opened to prevent the flow of the flow channel, and the other valves are closed and do not work. That is, the air valve layer is used to cut off the flow channel between the drug incubation area and the first liquid outlet, the flow channel between the first liquid inlet and the drug incubation area, and the flow channel between the first liquid inlet and the collection area.

[0105] The organoid releasing liquid enters from the releasing liquid inlet 303. Due to the blockage of the main channel, the liquid flushes the first row of organoids (the organoids in the main channel corresponding to the valve 103) through the branch channel and releases them into the main channel.

[0106] The air valve 102 is opened, the air valve 111 is opened, and the air valve 104 is opened to prevent the flow of the flow channel, and the remaining valves are closed and do not work.

[0107] The organoid releasing liquid enters from the releasing liquid inlet 303. Due to the blockage of the main channel, the liquid flushes the second row of organoids (the organoids in the main channel corresponding to the gas valve 104) through the branch channel and releases them into the main channel.

[0108] The air valve 102 is opened, the air valve 111 is opened, and the air valve 105 is opened to prevent the flow of the flow channel, and the other valves are closed and do not work.

[0109] The organoid releasing liquid enters from the releasing liquid inlet 303. Due to the blockage of the main channel, the liquid flushes the third row of organoids (the organoids in the main channel corresponding to the gas valve 105) through the branch channel and releases them into the main channel.

[0110] In this way, the organoids in the main flow channels corresponding to valves 106 to 111 are released one by one.

[0111] The released organoids follow the fluid and flow backward to the collection chamber 321 in the center of the collection area 310. The volume of the collection chamber 321 is much larger than the total volume of the organoids in the capture structure. The functional liquid inlet 304 and the functional liquid outlet 305 are both provided with anti-blocking ports, which on the one hand prevent cell debris from clogging the chip, and on the other hand limit the outflow of organoids from the port by the size of the flow channel.

[0112] Organoid lysis for molecular analysis:

[0113] Valve status:

[0114] The air valve 114 is opened to prevent the flow of the flow channel fluid, and the other valves are closed and do not work. That is, the air valve layer is used to cut off the flow channel between the drug incubation area and the collection area.

[0115] The organoid lysate enters through the functional liquid inlet 304 and flows out through the functional liquid outlet 305. The functional liquid outlet and the functional liquid inlet are provided with an annular narrow flow channel, and the cross-sectional size of the annular narrow flow channel is smaller than the size of the organoid and the lysed cells, so as to limit the organoid and the lysed cells from passing through the annular narrow flow channel, so that the narrow flow channel only supports the outflow of the lysate.

[0116] The air valve 114 is opened, the air valve 113 is opened, and the flow of the fluid in the flow channel is blocked. The air valve 115 is opened and closed for 20 to 30 times continuously, and the other valves are closed and do not work.

[0117] The air valve agitates the thin film layer to fully mix the lysate with the organoids in the collection chamber 321 .

[0118] The air valve 114 is opened to prevent the flow of fluid in the flow channel, and the other valves are closed and do not work.

[0119] The organoid lysis solution flows in through the functional solution inlet 304 , and the organoid lysis solution, the lysed organoids and the molecules released therefrom flow out from the functional solution outlet 305 .

[0120] The outlet of the functional liquid outlet 305 is connected to a conduit and collected into an EP tube for subsequent testing.

[0121] In the drug-sensitive layer 300, the width of the main flow channel is 200-500 microns.

[0122] The capture cavity of the capture structure has a circular cross section with a diameter of 150-300 microns, and a width of an opening connecting the capture cavity and the main channel is 100-200 microns.

[0123] The width of the narrow channel section is 30-50 microns, and the width of the transition section is 50-100 microns.

[0124] The collection chamber is circular in shape, with a diameter of 2000-3000 microns.

[0125] The width of the flow channels corresponding to the functional liquid inlet 304 and the functional liquid outlet 305 are both 10-20 microns.

[0126] The present invention proposes a chip for organoid drug sensitivity testing and its application. By using micro-nano processing technology, the organoid capture, drug sensitivity and release functions are integrated to achieve high-throughput drug sensitivity of organoids and downstream molecular detection needs, so as to be better used for the study of tumor drug sensitivity and mechanism of action.

[0127] 1. The organoid capture sub-area can accurately capture and fix organoids, provide a uniform number and size of organoids for drug sensitivity testing, and facilitate real-time observation in situ without overlapping of organoids in the well plate.

[0128] 2. The drug sensitivity function can efficiently test the drug sensitivity of organoids. The chip can array multiple units, which can easily meet the needs of high-throughput drug testing and quickly screen out drugs that are effective for specific tumors.

[0129] 3. The release function is even more critical. It can release the organoids safely and completely, so that all test organoids can be lysed in the organoid lysis functional area of ​​the chip and downstream molecular detection can be performed.

[0130] The above-mentioned integrated design of the present disclosure realizes the high-throughput drug sensitivity of organoids and the downstream molecular detection needs, and provides a powerful tool and method for the study of tumor drug sensitivity and mechanism of action. Compared with traditional drug sensitivity testing methods, the present invention has higher efficiency, more accurate results and wider application prospects, which can better help scientific researchers to explore the sensitivity and mechanism of action of tumor drugs in depth, and make important contributions to conquering the medical problem of tumors.

[0131] It is to be understood that the above embodiments are merely exemplary embodiments used to illustrate the principles of the present disclosure, but the present disclosure is not limited thereto. For those of ordinary skill in the art, various modifications and improvements can be made without departing from the spirit and substance of the present disclosure, and these modifications and improvements are also considered to be within the scope of protection of the present disclosure.

Claims

1. A microfluidic chip, characterized in that: The chip comprises an air valve layer, a thin film layer and a drug-sensitive layer; the thin film layer and the air valve layer are sequentially stacked on the drug-sensitive layer; the air valve layer controls the flow channel on the drug-sensitive layer through the thin film layer; The drug-sensitive layer is provided with a first liquid inlet, a drug incubation area, a release liquid inlet, a collection area and a first liquid outlet; The drug incubation area is connected to the first liquid inlet and the first liquid outlet through flow channels respectively; the drug incubation area has an organoid capture sub-area for capturing organoids in the organoid matrix gel suspension introduced through the first liquid inlet; The collection area is connected to the drug incubation area through a flow channel; the release liquid inlet is connected to the drug incubation area through a flow channel; The release liquid is introduced into the drug incubation area through the release liquid inlet; and the organoid capture sub-area releases the organoid based on the on-off control of the flow channel on the drug sensitive layer by the air valve layer; the organoids released from the organoid capture sub-area are collected in the collection area through the flow channel; The collection area includes a collection chamber, a functional liquid inlet and a functional liquid outlet; the functional liquid inlet and the functional liquid outlet are respectively connected to the collection chamber through a flow channel; the collection chamber is connected to the drug incubation area through the flow channel; The drug incubation area includes a plurality of main channels arranged side by side and connected in sequence; the organoid capture sub-area includes a capture structure arranged between two adjacent main channels; the main channel is used for the circulation of the organoid matrix gel suspension; the capture structure is used for capturing the organoids in the organoid matrix gel suspension; The capture structure comprises a capture cavity and a branch channel; wherein the capture cavity is arranged on the side wall of one of the two adjacent main channels; one end of the branch channel is connected to the capture cavity, and the other end is connected to the other main channel; the capture cavity is used to capture the organoids in the organoid matrix gel suspension; The branch channel includes a narrow channel section and a transition section; the narrow channel section is arranged between the capture chamber and the transition section, and is respectively connected to the capture chamber and the transition section; one end of the transition section away from the narrow channel section is connected to the main channel; wherein the width dimension of the channel section is smaller than the width dimension of the transition section.

2. A microfluidic chip according to claim 1, characterized in that: There are multiple capture structures; along the length direction of the main flow channel, the multiple capture structures are arranged between two adjacent main flow channels at intervals.

3. A method for capturing organoids, the method being implemented based on the microfluidic chip according to claim 1, characterized in that: Using the air valve layer to cut off the flow channel between the collection area and the drug incubation area, the flow channel between the collection area and the first liquid inlet, and the flow channel between the release liquid inlet and the drug incubation area; Passing the organoid matrix gel suspension into the drug incubation area from the first liquid inlet, wherein the organoid capture sub-area captures the organoids in the organoid matrix gel suspension; After standing for a preset time at a certain temperature, the organoid matrix gel suspension changes from a liquid state to a gel state, and a flushing liquid is introduced into the drug incubation area from the first liquid inlet to flush the organoid matrix gel suspension remaining in the main channel.

4. A method for testing drug sensitivity of organoids, the method being implemented based on the microfluidic chip according to claim 1, characterized in that: Using the air valve layer to cut off the flow channel between the collection area and the drug incubation area, the flow channel between the collection area and the first liquid inlet, and the flow channel between the release liquid inlet and the drug incubation area; Passing the drug into the drug incubation area from the first liquid outlet, and incubating the organoids in the drug incubation area with the drug at a certain temperature; The status of the organoids in the captured structure was observed under a microscope.

5. A method for releasing organoids, the method being implemented based on the microfluidic chip according to claim 1, characterized in that: Using the air valve layer to cut off the flow channel between the drug incubation area and the first liquid outlet, the flow channel between the first liquid inlet and the drug incubation area, and the flow channel between the first liquid inlet and the collection area; Passing a release liquid into the drug incubation area from the release liquid inlet, so that the organoid capture sub-area in the drug incubation area releases the organoid; The collection area is used to collect the released fluid containing the organoids.

6. A method for lysing an organoid, the method being implemented based on the microfluidic chip according to claim 1, characterized in that: Using the air valve layer to cut off the flow channel between the drug incubation area and the collection area; Passing the lysate into the collection chamber from the functional liquid inlet; The air valve layer is used to cut off the flow channel between the functional liquid inlet and the collection chamber, and the air valve layer is used to agitate the thin film layer to mix the lysate and the organoid in the collection chamber.

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