Multi-drug combination screening chip, design method and application thereof

Abstract

The application discloses a multi-drug combination screening chip and a design method and application thereof, wherein the chip is a multi-layer structure and sequentially comprises a single drug concentration gradient generation plate, a cell culture plate and a plurality of drug mixing plates from top to bottom; the single drug concentration gradient generation plate comprises a drug adding hole and a concentration gradient drug generation hole, and the drug adding hole and the concentration gradient drug generation hole are communicated through a mixing flow channel; the cell culture plate is provided with a plurality of cell culture chambers, and each cell culture chamber is communicated with a drug mixing hole; each drug mixing plate is provided with a concentration gradient drug transition hole and a drug mixing hole; the concentration gradient drug transition hole is in one-to-one correspondence with and communicated with the concentration gradient drug generation hole; the concentration gradient drug transition hole and the drug mixing hole are communicated through a distribution flow channel; and the drug mixing hole is communicated with the cell culture chamber of the cell culture plate.

Description

Technical Field

[0001] This invention relates to the field of biomedical devices, and more specifically, to high-throughput organ-on-a-chip for screening multiple drug combinations, its preparation method, and its applications. Background Technology

[0002] To achieve better patient treatment, combination therapy has become increasingly popular in recent decades to increase patient benefit rates. Generally, combination therapy is more effective than single-drug therapy for cancer. Taking cancer treatment as an example, combination chemotherapy regimens are widely used clinically, such as the TP regimen (paclitaxel and cisplatin), and the Folfox regimen (oxaliplatin, leucovorin, and fluorouracil). Extensive clinical data has verified that multi-drug combination therapy is often more effective in cancer treatment.

[0003] Combination therapy with multiple drugs can address some drug resistance issues. Even if a drug can cure a certain disease, it may not be effective for everyone due to individual differences. Using two drugs in combination can reduce the probability of drug resistance to some extent. Furthermore, different drugs act on lesions through different mechanisms, potentially leading to synergistic effects. For example, different chemotherapy drugs target different cell cycles in tumor cells, and studies have shown that combination therapy within appropriate concentration ranges can achieve better results. However, combination therapy is not always beneficial. Clinical evidence suggests that the combined use of some drugs may lead to antagonistic effects and potentially cause severe toxic side effects. In conclusion, choosing appropriate drug combination regimens and concentrations can significantly increase patient benefit.

[0004] (1) Existing drug screening chips generally suffer from insufficient throughput. Many existing combination drug screening chips can only perform gradient concentration screening of a single drug (microfluidic chip integrating concentration gradient and bacterial detection, patent number: CN 113171807 A) and combined concentration gradient screening of two drugs (an integrated microfluidic chip for drug screening and its application, patent number: CN 109722387 A), which cannot meet the screening and evaluation of three-drug combination therapy commonly used in clinical practice.

[0005] (2) Existing combination drug screening chips generally suffer from insufficient simulation. Currently, most chips used for drug combination screening are still based on two-dimensional planar cultured cells for detection. (A microfluidic chip and its preparation method and application, patent number: CN 105713834 A) (A microfluidic chip for efficient drug screening, patent number: CN113751091 B) In recent years, although researchers have begun to use three-dimensional tumor spheres for combination drug screening and evaluation, they generally lack exomatrix components and are not biomimetic enough. (A microfluidic chip for realizing three-dimensional cell culture and drug screening and its application, patent number: CN 110373321 A)

[0006] (3) In addition, in terms of detection, the traditional detection method involves staining with a cell live / dead reagent kit and then observing and counting the cells under a fluorescence microscope, which cannot achieve online real-time detection. At the same time, the difficulty in sample recovery also adds to the difficulty of biochemical detection. Summary of the Invention

[0007] The purpose of this invention is to provide a multi-drug screening chip capable of simultaneously detecting different combinations of multiple concentration gradients of various drugs.

[0008] This invention provides a high-throughput organ-on-a-chip for screening multiple drug combinations. The chip has a multi-layer structure, comprising, from top to bottom, a single drug concentration gradient generation plate, a cell culture plate, and multiple drug mixing plates.

[0009] The single drug concentration gradient generating plate includes a drug inlet hole and a concentration gradient drug generating hole, and the drug inlet hole and the concentration gradient drug generating hole are connected by a mixing channel.

[0010] The cell culture plate is provided with multiple cell culture chambers, and each cell culture chamber is connected to a drug mixing well through a flow channel;

[0011] Each of the drug mixing plates is provided with a concentration gradient drug transition hole and a drug mixing hole; the concentration gradient drug transition hole corresponds one-to-one with the concentration gradient drug generation hole and is connected; the concentration gradient drug transition hole and the drug mixing hole are connected through a distribution channel; the drug mixing hole is connected to the cell culture chamber of the cell culture plate.

[0012] In a further embodiment of the present invention, when testing m concentration gradients of n drugs, the single drug concentration gradient generation plate has 2n drug inlet holes and m*n concentration gradient drug generation holes. Each pair of drug inlet holes is connected to the m concentration gradient drug generation holes via a mixing channel to obtain m different concentrations of one drug. For example, when n=3 and m=3, three sets of mixing channel combinations are arranged side-by-side within a circle to generate the respective concentration gradients of the three drugs. The first-stage mixing channel has three mixing channels, and the mixer in the mixing channel is a spiral mixer. If the number of concentration gradients needs to be increased, one more mixing channel can be added to each subsequent stage. The center position between any two mixing channels is the input port of that stage of the mixing channel.

[0013] As a further embodiment of the present invention, the mixing channel is an asymmetric channel.

[0014] In a further embodiment of the present invention, the drug mixing plate is an n*m layer, and each layer of the drug mixing plate is used to distribute n*m ​​different concentrations and types of drugs generated in the single concentration gradient generation section into a specific cell culture chamber.

[0015] In a further embodiment of the invention, the inlet of the distribution channel is connected to the concentration gradient drug generation orifice, and the distribution channel connects from the inlet to m^(n-1) culture chambers. Here, through a two-stage channel connection, m^(n-1) can be decomposed into two factors p*q. The first factor p is used as the number of channels in the first stage, the outlet of the first-stage channel is used as the inlet of the second-stage channel, the number of channels in the second stage is the second factor q, and p*q = m^(n-1).

[0016] In a further embodiment of the present invention, a mixer is provided between the mixing channel and the concentration gradient drug generation orifice; a damper is provided between the distribution channel and the drug mixing orifice; a mixer is provided between the drug mixing orifice and the cell culture chamber; the damper controls the flow rate to control the mixing ratio by balancing the flow resistance, and can be a serpentine microchannel or a spiral microchannel; the mixer uses repeatedly opposing curved channels to mix the liquid by the action of Dean flow, and can be a serpentine microchannel or a spiral microchannel.

[0017] In a further embodiment of the present invention, the depth of each of the aforementioned flow channels is 50 μm-300 μm, preferably 200 μm; the flow channel width of the single drug concentration gradient generation section is 200 μm, the flow channel width of the damper in the drug combination concentration generation section is 200 μm, the flow channel width of the asymmetric structure at the front end of the damper is 1000 μm, the flow channel width of the drug mixing section is 200 μm, and the flow channel width of the drug mixing section in the cell culture layer is 400 μm. The cell channel width of the cell culture section is 1000-1500 μm, preferably 1200 μm. The overall chip diameter is 100 mm and the height is 11 mm.

[0018] In a further embodiment of the present invention, the cell culture chamber is composed of a three-channel structure, with the middle channel being the cell channel and the two side channels being drug channels. The spacing between the three channels is formed by trapezoidal micropillars or half-height walls. The distance between the micropillars is 50μm-100μm, preferably 100μm. The width of the trapezoid is 100μm, and the length of the long side is 150μm-300μm, preferably 150μm. The angle between the long side and the hypotenuse of the trapezoid is 70°-80°, preferably 79.4°.

[0019] The single drug concentration gradient generating plate has cell loading wells at positions corresponding to both ends of the cell flow channel. At the same positions corresponding to both ends of the drug flow channel, one end of the single drug concentration gradient generating plate has a sampling well, and the other end is connected to the drug mixing well.

[0020] The sampling well, serving as the detection section, is located at the end of the cell culture chamber and has a circular structure. Within the sampling well, the carboxyl groups on the activated surface are first treated with PLASMA, followed by chemical grafting of amino groups using a silane coupling agent. Quantum dots are then grafted onto the chip using EDC solution, and subsequently, nucleic acid aptamers for the target protein are linked to the chip using both EDC and NHs solutions. Simultaneously, antibodies targeting the protein are grafted onto gold nanoparticles using PEG.

[0021] This invention also provides a design method for the above-mentioned high-throughput organ-on-a-chip for screening multiple drug combinations, comprising the following steps:

[0022] 1) Set the flow rate of the target solution input and the length, width, spacing, and types of drugs, as well as the number of concentration gradients for each drug in the organ-on-a-chip;

[0023] 2) Model using SolidWorks software;

[0024] 3) Import the solution into the COMSOL simulation software and input parameters such as the initial solution concentration and viscosity;

[0025] 4) Compare the concentration of each drug in each cell culture chamber obtained from the simulation in step 3) with the preset concentration to obtain the solution concentration difference.

[0026] 5) If the solution concentration differences are all less than or equal to the error threshold, then the model is used as the final model; if the solution concentration error is greater than the error threshold, then modify the channel size parameters and repeat steps 2)-4) until the solution concentrations are all less than or equal to the error threshold. The above modification of the channel size parameters is to increase the channel width or length.

[0027] This invention also provides a high-throughput screening method for multi-drug combination therapy, using the above-mentioned high-throughput organ-on-a-chip for multi-drug combination therapy screening, comprising the following steps:

[0028] 1) Add hydrogel containing cell clusters into the cell culture chamber through the cell injection well;

[0029] 2) Cross-linked hydrogels;

[0030] 3) Simultaneously introduce culture medium without drugs and culture medium with different drugs into two drug inlets.

[0031] 4) Continuously add the culture medium at a constant drug loading rate until the flow rate of the culture medium flowing out of the sampling well stabilizes;

[0032] 5) Place the chip in a cell culture incubator, change the culture medium daily, repeat steps 3)-4), and culture and detect the cells.

[0033] The culture conditions involve irradiation with a laser emitter, and the detection involves taking pictures with a camera and analyzing the fluorescence intensity using image processing software.

[0034] Furthermore, step 1) is preceded by a step of sterilizing and drying the chip under high temperature and high pressure.

[0035] The above-mentioned chip should also be within the scope of protection of this invention in any of the following applications:

[0036] A1) Application in screening for combined drug use;

[0037] A2) Application in drug sensitivity testing.

[0038] The beneficial effects of this invention are as follows:

[0039] 1. Increased throughput of multi-drug co-screening chips. Traditional concentration gradient generation modules or drug mixing modules utilize symmetrical flow channel structures to control flow resistance and achieve on-demand drug mixing. However, due to spatial limitations, symmetrical flow channel structures cannot achieve screening of three or more combined concentration gradients. This invention can meet the need for generating multiple concentration gradient combinations of various drugs, and can realize the combined concentration gradient testing of three or more drugs.

[0040] 2. Improved accuracy of drug sensitivity testing. By adding extracellular matrix components to the cell culture chamber and selecting different cell units for detection, the accuracy of drug sensitivity testing is improved through enhanced biomimicry of cell culture. The cell culture chamber, with its biomimetic extracellular matrix environment, allows for the evaluation of various three-dimensional cluster structural units. It can support the evaluation of various structural units such as tumor cell clusters and organoid models.

[0041] 3. Improved the convenience of organ-on-a-chip detection. Based on DNA probe technology, nucleic acid aptamers are immobilized in the chip via chemical bonds, enabling real-time monitoring using a simple SLR camera and laser generator module, eliminating the need for traditional staining, incubation, fluorescence microscopy, and other procedures. Detection modules can be developed at specific locations within the chip to achieve real-time drug efficacy detection after drug action. Attached Figure Description

[0042] Figure 1 This is a perspective view of the high-throughput organ-on-a-chip for multidrug combination screening in Example 1.

[0043] Figure 2 This is a schematic diagram of the single drug concentration gradient generation part in Example 1.

[0044] Figure 3 This is an exploded view of the multilayer chip structure in Example 1, with the layers corresponding from left to right as the top to bottom of the chip.

[0045] Figure 4 This is a schematic diagram of the single-layer drug distribution channel in Example 1.

[0046] Figure 5 This is a schematic diagram of the cell culture layer in Example 1.

[0047] Figure 6 This is a perspective view of a high-throughput organ-on-a-chip for screening multiple drug combinations in the embodiments.

[0048] Figure 7 This is a schematic diagram of the single drug concentration gradient generation part in Example 3.

[0049] Figure 8 This is an exploded view of the multilayer chip structure in Example 3, with the top to bottom structural layers of the chip arranged from left to right.

[0050] Figure 9 This is a schematic diagram of the single-layer drug distribution channel in Example 3.

[0051] Figure 10 This is a schematic diagram of the cell culture layer in Example 3;

[0052] Figure 11 This is a partial structural diagram of the cell culture chamber in the embodiment.

[0053] Explanation of reference numerals in the attached figures:

[0054] 1 Single drug concentration gradient generation plate, 11 Drug addition well, 12 Concentration gradient drug generation well, 13 Mixing channel; 2 Drug mixing plate, 21 Concentration gradient drug transition well, 22 Drug mixing well, 23 Distribution channel, 231 Primary channel, 232 Secondary channel, 233 Damper; 3 Cell culture plate, 31 Cell culture chamber, 32 Cell channel, 321 Cell sample port, 33 Drug channel, 34 Spacing, 35 Sampling well. Detailed Implementation

[0055] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0056] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0057] Example 1

[0058] like Figure 1 As shown, this embodiment provides a high-throughput organ-on-a-chip for screening multiple drug combinations. Functionally, it includes a single drug concentration gradient generation section, a drug combination concentration generation section, a drug mixing section, a cell culture section, and a detection section. Structurally, the chip has a multi-layer structure, which includes a single drug concentration gradient generation plate 1, a cell culture plate 3, and multiple drug mixing plates 2 from top to bottom.

[0059] The single concentration gradient generation section is located on the single drug concentration gradient generation plate 1 (cover layer) of the chip, adopting a "Christmas tree" structure to generate m concentration gradients for n drugs; in this example, n=3, m=3. Figure 2As shown, three sets of "Christmas tree" structures are arranged side-by-side within the circumference to generate the respective concentration gradients of the three drugs. The first-stage mixing channel has three mixing channels, and the mixer within these channels uses a spiral-shaped channel structure. To increase the number of concentration gradients, one more channel can be added to each subsequent stage. The inlet of any stage is located at the center of any two mixing channels. This refers to the spiral mixer. Spiral mixers and dampers have similar structures, but their applications and principles differ. Spiral mixers utilize repeatedly curved channels to mix liquids through Dean's flow. The principle of a damper is to increase local resistance through a relatively small channel size and repeatedly curved channels, ignoring the resistance of other parts of the channel, thus achieving a balance of flow resistance for each drug entering the cell culture chamber.

[0060] The drug combination concentration generation section employs a multi-layer asymmetric flow channel structure design, requiring a total of n*m layers. Each layer distributes the n*m ​​different concentrations and types of drugs generated in the single concentration gradient generation section into a specific cell culture chamber. In this example, n=3, m=3, so the drug combination concentration generation section requires a total of 9 flow channel layers, as shown below. Figure 3 As shown. To ensure that the mixing ratio of the drugs is controllable, as... Figure 4 As shown, in each layer of the flow channel structure, the distribution channel is divided into three channels from the inlet, and then the outlet of each of the three channels serves as the inlet, each connecting to the other three channels. At the end of the channels, dampers are designed to balance the flow resistance, control the flow velocity, and control the mixing ratio.

[0061] The drug mixing section is located in the cell culture layer of the chip, and a serpentine mixer is used to uniformly mix three drugs in different proportions that flow in from the drug combination concentration generation section, such as... Figure 5 As shown, the serpentine mixer directly connects the drug combination generated in the drug combination concentration generation section to the cell culture chamber after mixing.

[0062] The damper is located in the combined concentration generation section of the chip. To ensure that the drug mixing ratio is controllable, the damper increases the fluid resistance through continuous flow channel bends. The flow channel at the front end of the damper is negligible compared to the resistance of the damper, so as to ensure that the flow resistance of each liquid entering different chambers is almost the same, making the drug mixing ratio controllable and stable.

[0063] The cell culture section is located in the cell culture layer of the chip and consists of n^m cell culture chambers. In this example, n=3 and m=3, so the cell culture section requires 27 cell culture chambers. Each cell culture chamber has a three-channel structure, with the middle channel being the cell channel and the two side channels being drug channels. The three channels are connected by trapezoidal micropillars, with a spacing of 50μm-100μm, preferably 100μm. The width of the trapezoid is 100μm, and the length of the long side is 150μm-300μm, preferably 150μm. The angle between the long side and the hypotenuse of the trapezoid is 70°-80°, preferably 79.4°.

[0064] The detection section is located in the cell culture layer of the chip, at the end of the cell culture chamber, and has a circular structure. Within the circular detection section, the carboxyl groups on the surface are first activated by PLASMA treatment, followed by chemical grafting of amino groups using a silane coupling agent. Quantum dots are then grafted onto the chip using EDC solution, and subsequently, the nucleic acid aptamer of the target protein is linked to the chip using EDC solution and NHs solution. Simultaneously, the antibody for the target protein is grafted onto gold nanoparticles using PEG. In this example, liver cancer was selected as the target disease for evaluating the combination therapy regimen; therefore, liver cancer-specific nucleic acid aptamers and specific antibodies were chosen.

[0065] The channel depth is 50μm-300μm, preferably 200μm; the channel width of the single drug concentration gradient generation section is 200μm, the channel width of the damper in the drug combination concentration generation section is 200μm, the channel width of the asymmetric structure at the front end of the damper is 1000μm, the channel width of the drug mixing section is 200μm, and the channel width of the drug mixing section in the cell culture layer is 400μm. The cell channel width of the cell culture section is 1000-1500μm, preferably 1200μm. The overall chip diameter is 100mm and the height is 11mm.

[0066] In this example, the micropillar structure in the cell culture section can prevent the gel structure from intruding into the culture medium channels during cell loading due to surface tension, and it is also convenient to process by soft lithography or DLP photopolymerization printing.

[0067] The high-throughput organ-on-a-chip method for screening multi-drug combinations in this embodiment is as follows:

[0068] 1) Add hydrogel containing cell clusters to 27 cell culture chambers through the cell sample introduction channel.

[0069] 2) Cross-linked hydrogel.

[0070] 3) Simultaneously introduce culture medium without drugs and culture medium with different drugs from the culture medium inlet and the drug inlet respectively. Gold nanoparticles modified with antibodies are mixed in the culture medium.

[0071] 4) Continuously input at a flow rate of 50 μl / min for 10 min until the outlet flow rate stabilizes.

[0072] 5) Place the chip in a cell culture incubator, change the medium daily, irradiate it with a laser emitter, take pictures with a camera, and analyze the fluorescence intensity using image processing software.

[0073] Example 2

[0074] Based on the structure of Example 1, this embodiment replaces the trapezoidal microcolumns with a half-height wall structure in the middle of the three-channel structure of the cell culture section. The height of the half-height wall is 50μm-100μm, preferably 100μm.

[0075] In this embodiment, the semi-high wall design can prevent the gel structure from invading the culture medium channel during cell loading through surface tension, and also facilitates CNC milling.

[0076] Example 3

[0077] This embodiment provides a high-throughput organ-on-a-chip for detecting two drugs at four gradients, oriented towards multi-drug combination screening. The cell culture layer of the chip has eight (2x4) cell culture chambers; the chip includes:

[0078] The drug combination concentration generation section employs a multi-layer asymmetric flow channel structure design, requiring a total of n*m layers. Each layer distributes the n*m ​​different concentrations and types of drugs generated in the single concentration gradient generation section into a specific cell culture chamber. In this example, n=2, m=4, so the drug combination concentration generation section requires a total of 8 flow channel layers, as shown below. Figure 3 As shown. To ensure that the mixing ratio of the drugs is controllable, as... Figure 4 As shown, in each layer of the flow channel structure, the distribution channel is divided into three channels from the inlet, and then the outlet of each of the three channels serves as the inlet, each connecting to the other three channels. At the end of the channels, dampers are designed to balance the flow resistance, control the flow velocity, and control the mixing ratio.

[0079] The drug mixing section is located in the cell culture layer of the chip, and a serpentine mixer is used to uniformly mix three drugs in different proportions that flow in from the drug combination concentration generation section, such as... Figure 5 As shown, the serpentine mixer directly connects the drug combination generated in the drug combination concentration generation section to the cell culture chamber after mixing.

[0080] The damper is located in the combined concentration generation section of the chip. To ensure that the drug mixing ratio is controllable, the damper increases the fluid resistance through continuous flow channel bends. The flow channel at the front end of the damper is negligible compared to the resistance of the damper, so as to ensure that the flow resistance of each liquid entering different chambers is almost the same, making the drug mixing ratio controllable and stable.

[0081] The cell culture section is located in the cell culture layer of the chip and consists of m^n cell culture chambers. In this example, n=3 and m=3, so the cell culture section requires 27 cell culture chambers. Each cell culture chamber has a three-channel structure, with the middle channel being the cell channel and the two side channels being drug channels. The three channels are connected by trapezoidal micropillars, with a spacing of 50μm-100μm, preferably 100μm. The width of the trapezoid is 100μm, and the length of the long side is 150μm-300μm, preferably 150μm. The angle between the long side and the hypotenuse of the trapezoid is 70°-80°, preferably 79.4°.

[0082] The detection section is located in the cell culture layer of the chip, at the end of the cell culture chamber, and has a circular structure. Within the circular detection section, the carboxyl groups on the surface are first activated by PLASMA treatment, followed by chemical grafting of amino groups using a silane coupling agent. Quantum dots are then grafted onto the chip using EDC solution, and subsequently, the nucleic acid aptamer of the target protein is linked to the chip using EDC solution and NHs solution. Simultaneously, the antibody for the target protein is grafted onto gold nanoparticles using PEG. In this example, liver cancer was selected as the target disease for evaluating the combination therapy regimen; therefore, liver cancer-specific nucleic acid aptamers and specific antibodies were chosen.

[0083] The channel depth is 50μm-300μm, preferably 200μm; the channel width of the single drug concentration gradient generation section is 200μm, the channel width of the damper in the drug combination concentration generation section is 200μm, the channel width of the asymmetric structure at the front end of the damper is 1000μm, the channel width of the drug mixing section is 200μm, and the channel width of the drug mixing section in the cell culture layer is 400μm. The cell channel width of the cell culture section is 1000-1500μm, preferably 1200μm. The overall chip diameter is 100mm and the height is 11mm.

[0084] In this example, the micropillar structure in the cell culture section can, on the one hand, prevent the gel structure from intruding into the culture medium channels during cell loading due to surface tension, and on the other hand, facilitate the processing of cells by soft lithography or DLP photopolymerization printing.

[0085] In the above embodiments, the chip design method is as follows:

[0086] 1) Set the flow rate of the target solution input and the length, width, spacing, and types of drugs, as well as the number of concentration gradients for each drug in the organ-on-a-chip;

[0087] 2) Model using SolidWorks software;

[0088] 3) Import the solution into the COMSOL simulation software and input parameters such as the initial solution concentration and viscosity;

[0089] 4) Compare the concentration of each drug in each cell culture chamber in step 3) with the preset concentration, where the concentration of each drug in each cell culture chamber refers to the drug concentration in each cell culture chamber in the COMSOL fluid dynamics simulation of step 3); the preset concentration refers to the concentration obtained through theoretical calculation.

[0090] 5) If the solution concentration is less than or equal to the error threshold, the model is used as the final model; if the solution concentration error is greater than the error threshold, the channel width or length needs to be increased (i.e., the channel size parameters are modified, mainly referring to the size of the damper, so as to adjust the solution concentration), and then steps 2)-4) are repeated until the solution concentration is less than or equal to the error threshold.

[0091] In the above embodiments, the distribution mechanism of the culture chambers is as follows: the culture chambers are arranged radially outward from the center, with m rings, each ring containing m^(n-1) culture chambers. For the first drug, the culture chambers in each ring from the inside out receive progressively increasing concentrations of the drug; that is, the first ring receives the lowest concentration of the first drug, the second ring receives the second concentration, the third ring receives the third concentration, and so on. For the remaining drugs, each column of culture chambers radially distributed from the inner ring to the outer ring receives the same concentration of the same drug. The allocation rule for each drug is as follows: for the y-th concentration of the x-th drug, starting from the (y-1)*m^(nx)+1th column of culture chambers, the same concentration of the drug is allocated to m^(nx) consecutive columns of culture chambers, and then every (m-1)*m^(nx) consecutive columns of culture chambers, the same concentration of the drug is allocated to m^(nx) consecutive columns of culture chambers.

[0092] For example, consider a culture chamber design for high-throughput testing of two drugs, each with four concentration gradients. Let A represent the first drug, B represent the second drug, A1 be the lowest concentration of drug A, and B4 be the highest concentration of drug B. The design rules are as follows:

[0093]

[0094] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A high-throughput organ-on-a-chip for screening multiple drug combinations, characterized in that, The chip has a multi-layer structure, comprising, from top to bottom, a single drug concentration gradient generation plate, a cell culture plate, and multiple drug mixing plates: The single drug concentration gradient generating plate includes a drug inlet hole and a concentration gradient drug generating hole, and the drug inlet hole and the concentration gradient drug generating hole are connected by a mixing channel. The cell culture plate is provided with multiple cell culture chambers, and the cell culture chambers are connected to the drug mixing wells on the drug mixing plate. Each of the drug mixing plates is provided with a concentration gradient drug transition hole and a drug mixing hole; the concentration gradient drug transition hole corresponds one-to-one with the concentration gradient drug generation hole and is connected; the concentration gradient drug transition hole and the drug mixing hole are connected through a distribution channel; the drug mixing hole is connected to the cell culture chamber of the cell culture plate; When testing m concentration gradients of n drugs, the single drug concentration gradient generation plate has 2n drug addition wells and m concentration gradient drug generation wells. There are n orifices, where each pair of drug inlet holes forms a group, which are connected to m concentration gradient drug generation holes through a mixing channel to obtain m different concentrations of a drug; The mixing channel is an asymmetric channel; The drug mixing plate is n m layers, each layer of the drug mixing plate is used to distribute the n generated in the single concentration gradient generation section. m different concentrations and types of drugs were placed in the cell culture chamber; Each of the concentration gradient drug generation wells is connected to m^(n-1) cell culture chambers via a distribution channel; A mixer is provided between the mixing channel and the concentration gradient drug generation orifice on the single drug concentration gradient generation plate; a damper is provided between the distribution channel and the drug mixing orifice on the drug mixing plate; and a mixer is provided between the drug mixing orifice and the cell culture chamber on the cell culture plate.

2. The high-throughput organ-on-a-chip for screening multiple drug combinations according to claim 1, characterized in that, The cell culture chamber includes a three-channel structure, with the middle channel being the cell channel and the two side channels being the drug channels. The intervals between the three channels are composed of trapezoidal micropillars or half-height walls. Cell sample wells are provided at positions corresponding to both ends of the cell channels on the single drug concentration gradient generation plate. At positions corresponding to both ends of the drug channels on the single drug concentration gradient generation plate, one end is provided with a sampling well, and the other end is connected to a drug mixing well.

3. A method for preparing a high-throughput organ-on-a-chip for screening multiple drug combinations as described in any one of claims 1-2, characterized in that, Includes the following steps: 1) Set the flow rate of the target solution input, the length, width, spacing of each mixing channel of the organ-on-a-chip, the type of drug, and the number of concentration gradients for each drug; 2) Model using SolidWorks software; 3) Import the solution into the COMSOL simulation software and input the initial solution concentration and viscosity parameters; 4) Compare the concentration of each drug in each cell culture chamber obtained from the simulation in step 3) with the preset concentration to obtain the solution concentration difference. 5) If the solution concentration difference is less than or equal to the error threshold, the model is used as the final model; if the solution concentration error is greater than the error threshold, the flow channel size parameters are modified and steps 2)-4) are repeated until the solution concentration is less than or equal to the error threshold.

4. A high-throughput screening method for multi-drug combination therapy, characterized in that, Using the high-throughput organ-on-a-chip for multi-drug combination screening as described in any one of claims 1-2, the method includes the following steps: 1) Add hydrogel containing cell clusters into the cell culture chamber through the cell injection well; 2) Cross-linked hydrogels; 3) Simultaneously introduce culture medium without drugs and culture medium with different drugs into two drug inlets; 4) Continuously add the culture medium at a constant drug loading rate until the flow rate of the culture medium flowing out of the sampling well stabilizes; 5) Place the chip in a cell culture incubator, change the culture medium daily, repeat steps 3)-4), and culture and detect the cells.

5. The application of the chip according to any one of claims 1-2 in any of the following: A1) Application in screening for combined drug use; A2) Application in drug sensitivity testing.

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