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

Abstract

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

Description

Technical Field

[0001] The present invention relates to the field of biomedical equipment, and in particular to a high-throughput organ chip for multi-drug combination screening and a preparation method and application thereof. Background Art

[0002] In order to achieve better treatment for patients, drug combination has gradually emerged in recent decades to increase the benefit rate of patients. Generally, drug combination has better therapeutic effect than single anticancer drug treatment. Taking cancer treatment as an example, combination chemotherapy regimens have been widely used in clinical practice, such as the TP regimen of paclitaxel and cisplatin, the folfox regimen of oxaliplatin, calcium folinate, and fluorouracil, etc. A large amount of clinical data has verified that multi-drug combination therapy is often more effective in cancer treatment.

[0003] The combination of multiple drugs can solve some of the drug resistance problems. Even if a drug can cure a certain human disease, it may not be effective for everyone due to individual differences. The combination of two drugs can reduce the probability of drug resistance to a certain extent. On the other hand, the different mechanisms of action of multiple drugs on lesions may produce synergistic effects between drugs. For example, different chemotherapy drugs act on different cell cycles of tumor cells. Studies have shown that the combination of multiple drugs within the appropriate concentration range can have a better effect. However, the combination of multiple drugs is not necessarily beneficial. Clinical evidence shows that the combined use of some drugs may lead to antagonism between drugs and may also cause strong toxic side effects. In summary, choosing the right drug combination regimen and choosing the right combination concentration can significantly increase the patient's benefit rate.

[0004] (1) Existing drug screening chips generally have the problem of insufficient throughput. A large number of 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: CN113171807A) and combined concentration gradient screening of two drugs (an integrated microfluidic chip for drug screening and its application, patent number: CN109722387A), which cannot meet the screening and evaluation of three-drug combination commonly used in clinical practice.

[0005] (2) The existing combination drug screening chips generally have the problem of insufficient simulation. At present, most chips used for drug combination screening are still based on two-dimensional plane cultured cells for detection. (A microfluidic chip and its preparation method and application, patent number: CN105713834A) (Microfluidic chip for efficient drug screening, patent number: CN113751091B) In recent years, although researchers have begun to use three-dimensional tumor spheres for combination drug screening and evaluation, they generally lack extracellular matrix components and are not biomimetic enough (A microfluidic chip and application for realizing three-dimensional cell culture and drug screening, patent number: CN110373321A).

[0006] (3) In addition, in terms of detection, the traditional detection scheme is to use a cell live / dead kit to stain and then observe and count under a fluorescence microscope, which cannot achieve online real-time detection. At the same time, the difficulty in sample recovery also adds difficulties to biochemical detection. Summary of the invention

[0007] The object of the present invention is to provide a multi-drug joint screening chip capable of simultaneously detecting different combinations of multiple concentration gradients of multiple drugs.

[0008] The present invention provides a high-throughput organ chip for multi-drug combination screening. The chip is a multi-layer structure, which includes, 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 comprises a drug adding hole and a concentration gradient drug generating hole, and the drug adding hole and the concentration gradient drug generating hole are connected through a mixing channel;

[0010] The cell culture plate is provided with a plurality of cell culture chambers, each of which is connected to a drug mixing hole 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 holes correspond to and are connected with the concentration gradient drug generation holes one by one; the concentration gradient drug transition holes and the drug mixing holes are connected through a distribution flow channel; the drug mixing holes are connected with the cell culture chamber of the cell culture plate.

[0012] As a further solution of the present invention, when testing m concentration gradients of n drugs; the number of drug addition holes on the single drug concentration gradient generating plate is 2n, and the number of concentration gradient drug generating holes is m*n, wherein every two drug addition holes are connected to the m concentration gradient drug generating holes through a mixing channel to obtain m different concentrations of a drug. For example, when n=3 and m=3, three groups of mixing channel combination structures are arranged in parallel within the circumference to generate respective concentration gradients of the three drugs, wherein the first-stage mixing channel is provided with three mixing channels, and the mixer in the mixing channel adopts a spiral mixer, and if the number of concentration gradients needs to be increased, one more mixing channel can be added to each subsequent stage compared to the previous stage. The center position of any two mixing channels is the input port of the mixing channel of that stage.

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

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

[0015] As a further solution of the present invention, the inlet of the distribution channel is connected to the concentration gradient drug generation hole, and the distribution channel is connected to m^(n-1) culture chambers through the channel from the inlet. Here, through the 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 first-stage channels, the outlet of the first-stage channel is used as the inlet of the second-stage channel, the number of the second-stage channels is the second factor q, and p*q=m^(n-1).

[0016] As a further solution of the present invention, a mixer is provided between the mixing channel and the concentration gradient drug generating hole; a damper is provided between the distribution channel and the drug mixing hole; a mixer is provided between the drug mixing hole and the cell culture chamber; the damper controls the flow rate and the mixing ratio by balancing the flow resistance, and can be a serpentine microfluidic channel or a spiral microfluidic channel; the mixer utilizes repeatedly oppositely curved channels to mix liquids through the action of Dean flow, and can be a serpentine microfluidic channel or a spiral microfluidic channel.

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

[0018] As a further solution of the present invention, the cell culture chamber is composed of a three-channel structure, the middle channel is a cell channel, the two sides are drug channels, and the intervals between the three channels are composed of trapezoidal micro-columns or half-height walls, the micro-column minus distance is 50μm-100μm, preferably 100μm, the width of the trapezoid is 100μm, the length of the long side is 150μm-300μm, preferably 150μm. The angle between the long side of the trapezoid and the hypotenuse is 70°-80°, preferably 79.4°.

[0019] A cell loading hole is arranged at the position corresponding to both ends of the cell flow channel on the single drug concentration gradient generating plate, and a sampling hole is arranged at one end at the position corresponding to both ends of the drug flow channel on the single drug concentration gradient generating plate, and the other end is connected to the drug mixing hole.

[0020] The sampling hole, as the detection part, is located at the end of the cell culture chamber and has a circular structure. In the sampling hole, the carboxyl groups on the surface are first activated by PLASMA treatment, and then the amino groups are chemically grafted by a silane coupling agent. Then, the quantum dots are grafted onto the chip by an EDC solution, and then the nucleic acid aptamer of the target detection protein is connected to the chip by an EDC solution and an NHs solution. At the same time, the antibody of the target protein is grafted onto the gold nanoparticles by PEG. (This should refer to one of the tests, and it should be stated that it is the step for that test)

[0021] The present invention also provides a design method for the high-throughput organ chip for multi-drug combination screening, comprising the following steps:

[0022] 1) Setting the flow rate of the target solution input and the length, width, spacing of each mixing channel of the organ chip, the type of drug, the number of concentration gradients of each drug, etc.;

[0023] 2) Modeling using solidworks software;

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

[0025] 4) comparing the concentration of each drug in each cell culture chamber obtained by simulation in step 3) with the preset concentration to obtain a solution concentration difference,

[0026] 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 size parameters of the flow channel are modified and steps 2-4 are repeated until the solution concentration is less than or equal to the error threshold. The above modification of the flow channel size parameters is to increase the flow channel width or length.

[0027] The present invention also provides a high-throughput screening method for multi-drug combination, using the high-throughput organ chip for multi-drug combination screening, comprising the following steps:

[0028] 1) adding the hydrogel with cell clusters into the cell culture chamber through the cell injection hole;

[0029] 2) Cross-linked hydrogel;

[0030] 3) The culture medium without drugs and the culture medium with different drugs are respectively input into two drug inlets at the same time;.

[0031] 4) continuously inputting the culture medium at a constant drug addition flow rate until the flow rate of the culture medium flowing out of the sampling hole stabilizes;

[0032] 5) Place the chip in a cell culture incubator, change the culture medium every day, repeat steps 3-4 and perform culture and testing;

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

[0034] Furthermore, before step 1), the method further includes sterilizing and drying the chip under high temperature and high pressure.

[0035] The above chip may also be used in any of the following applications within the scope of protection of the present invention:

[0036] A1) Application in drug combination screening;

[0037] A2) Application in drug sensitivity testing.

[0038] The beneficial effects of the present invention are:

[0039] 1. Improved the throughput of multi-drug combined screening chips. Traditional concentration gradient generation modules or drug mixing modules use symmetrical flow channel structures to control flow resistance to achieve on-demand mixing of drugs. However, due to the limitations of the spatial structure, the symmetrical flow channel structure cannot achieve three or more combined concentration gradient screening. The present invention can meet the needs of generating multiple concentration gradient combinations of multiple drugs, and can achieve combined concentration gradient testing of three or more drugs.

[0040] 2. Improved the accuracy of drug sensitivity testing. By adding extracellular matrix components to the cell culture chamber and being able to select different cell units for testing, the accuracy of drug sensitivity testing is improved by improving the biomimetic nature of cell culture. A cell culture chamber with a biomimetic extracellular matrix environment that allows the evaluation of multiple three-dimensional cluster structure units. It can support the evaluation of multiple structural units such as tumor cell clusters and organoid models.

[0041] 3. Improved the convenience of organ chip detection. Based on DNA probe technology, by fixing nucleic acid aptamers in the chip through chemical bonds, real-time monitoring can be achieved through a simple SLR camera and laser generation module, without the need for traditional staining, incubation, fluorescence microscope observation and other operations. Develop a detection module at a specific location in the chip to achieve real-time efficacy detection after the drug acts. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0044] Figure 3 This is an exploded view of the structure of the multi-layer chip in Example 1, and the structural layers from top to bottom of the chip correspond from left to right.

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

[0046] Figure 5 Schematic diagram of the structure of the cell culture layer in Example 1.

[0047] Figure 6 A perspective view of a high-throughput organ chip for multi-drug combination screening in an embodiment.

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

[0049] Figure 8This is an exploded view of the structure of the multi-layer chip in Example 3, which corresponds to the structural layers of the chip from top to bottom from left to right.

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

[0051] Fig.10 is a schematic diagram of the structure of the cell culture layer in Example 3;

[0052] Fig.11 It is a schematic diagram of the partial structure of the cell culture chamber in the embodiment.

[0053] Description of reference numerals:

[0054] 1 single drug concentration gradient generating plate, 11 drug adding hole, 12 concentration gradient drug generating hole, 13 mixing channel; 2 drug mixing plate, 21 concentration gradient drug transition hole, 22 drug mixing hole, 23 distribution channel, 231 primary channel, 232 secondary channel, 233 damper; 3 cell culture plate, 31 cell culture chamber, 32 cell channel, 321 cell adding port, 33 drug channel, 34 interval, 35 sampling hole. DETAILED DESCRIPTION

[0055] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0056] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0057] Example 1

[0058] like Figure 1 As shown, this embodiment provides a high-throughput organ chip for multi-drug combination screening, which functionally includes a single drug concentration gradient generating part, a drug combination concentration generating part, a drug mixing part, a cell culture part and a detection part; structurally, the chip is a multi-layer structure, which includes, from top to bottom, a single drug concentration gradient generating plate 1, a cell culture plate 3 and a plurality of drug mixing plates 2.

[0059] The single concentration gradient generating part is located on the single drug concentration gradient generating plate 1 (cover layer) of the chip, and adopts a "Christmas tree" structure to generate m concentration gradients of n drugs; in this example, n=3, m=3. Figure 2As shown, three groups of "Christmas tree" structures are arranged side by side in the circumference to generate the concentration gradients of the three drugs. Among them, the first-stage mixing channel is provided with three mixing channels. The mixer in the mixing channel adopts a mixer with a spiral channel structure. If the number of concentration gradients needs to be increased, one more mixing channel can be added to each subsequent stage compared with the previous stage. The center position of any two mixing channels is the input port of the mixing channel of this stage. Here we refer to the spiral mixer. The spiral mixer is similar to the damper structure designed by me, but the application scenarios and principles are different. The spiral mixer uses repeated opposite curved channels to mix liquids through the action of Dean flow. The principle of the damper is to increase the local resistance through relatively small channel size and repeatedly curved channels, so as to ignore the resistance of other parts of the channel and balance the flow resistance of each drug entering the cell culture chamber.

[0060] The drug combination concentration generating part adopts a multi-layer asymmetric flow channel structure design, which requires a total of n*m layers, and each layer is used to distribute n*m ​​drugs of different concentrations and types generated in the single concentration gradient generating part to a specific cell culture chamber. In this example, n=3, m=3, so the drug combination concentration generating part requires a total of 9 layers of flow channel structure, such as Figure 3 To ensure that the mixing ratio of the drugs is controllable, Figure 4 As shown, in each layer of the flow channel structure, the distribution flow channel is divided into three flow channels from the inlet, and then the outlets of the three flow channels are used as inlets to connect the three flow channels respectively. A damper is designed at the terminal of the flow channel to balance the flow resistance to control the flow rate and control the mixing ratio.

[0061] The drug mixing part is located in the cell culture layer of the chip, and a serpentine mixer is used to achieve uniform mixing of three drugs of different proportions flowing from the drug combination concentration generating part, such as Figure 5 As shown, the serpentine mixer directly connects the drug combination generated in the drug combination concentration generating part to the cell culture chamber after mixing.

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

[0063] The cell culture part is located in the cell culture layer of the chip and consists of n^m cell culture chambers. In this example, n=3, m=3, so the cell culture part requires 27 cell culture chambers. Each cell culture chamber is composed of a three-channel structure, the middle channel is a cell channel, and the two sides are drug channels. The three channels are composed of trapezoidal microcolumns in the middle, the distance between the microcolumns 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 of the trapezoid and the hypotenuse is 70°-80°, preferably 79.4°.

[0064] The detection part is located in the cell culture layer of the chip, at the end of the cell culture chamber, and is a circular structure. In the circular detection part, the carboxyl groups on the surface are first activated by PLASMA treatment, and then the amino groups are chemically grafted by a silane coupling agent, and then the quantum dots are grafted on the chip by EDC solution, and then the nucleic acid aptamer of the target detection protein is connected to the chip by EDC solution and NHs solution. At the same time, the antibody of the target protein is grafted on the gold nanoparticles by PEG. In this example, liver cancer is selected as the target disease for the evaluation of the combined drug regimen, so the specific nucleic acid aptamer and specific antibody for liver cancer are selected.

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

[0066] In this example, the micro-column structure in the cell culture part can prevent the gel structure from invading the culture medium flow channel when the cells are added through the surface tension, and can also be easily processed by soft lithography or DLP photocuring printing.

[0067] The method for using the high-throughput organ chip for multi-drug combination screening in this embodiment is as follows:

[0068] 1) Add hydrogels with cell clusters into 27 cell culture chambers through the cell injection channel.

[0069] 2) Cross-linked hydrogel.

[0070] 3) A medium without drugs and a medium with different drugs are respectively inputted from the medium inlet and the drug inlet at the same time, and the gold nanoparticles modified with antibodies are mixed in the medium.

[0071] 4) Continue to 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 solution every day, and irradiate it with a laser emitter. Take pictures with a camera and analyze the fluorescence intensity using image processing software.

[0073] Example 2

[0074] In this embodiment, based on the structure of the first embodiment, the three-channel structure of the cell culture part is separated by a half-height wall structure in the middle to replace the trapezoidal micro-columns, and the height of the half-height wall is 50 μm-100 μm, preferably 100 μm.

[0075] The half-height wall design in this embodiment can prevent the gel structure from invading the culture medium flow channel when cells are added through the surface tension effect, and is convenient for CNC milling machine processing.

[0076] Example 3

[0077] This embodiment provides a high-throughput organ chip for detecting two drugs and four gradients for multi-drug combination screening. The cell culture layer of the chip has 8 (2x4) cell culture chambers; the chip includes:

[0078] The drug combination concentration generating part adopts a multi-layer asymmetric flow channel structure design, which requires a total of n*m layers, and each layer is used to distribute n*m ​​drugs of different concentrations and types generated in the single concentration gradient generating part to a specific cell culture chamber. In this example, n=2, m=4, so the drug combination concentration generating part requires a total of 8 layers of flow channel structure, such as Figure 3 To ensure that the mixing ratio of the drugs is controllable, Figure 4 As shown, in each layer of the flow channel structure, the distribution flow channel is divided into three flow channels from the inlet, and then the outlets of the three flow channels are used as inlets to connect the three flow channels respectively. A damper is designed at the terminal of the flow channel to balance the flow resistance to control the flow rate and control the mixing ratio.

[0079] The drug mixing part is located in the cell culture layer of the chip, and a serpentine mixer is used to achieve uniform mixing of three drugs of different proportions flowing from the drug combination concentration generating part, such as Figure 5 As shown, the serpentine mixer directly connects the drug combination generated in the drug combination concentration generating part to the cell culture chamber after mixing.

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

[0081] The cell culture part is located in the cell culture layer of the chip and consists of m^n cell culture chambers. In this example, n=3, m=3, so the cell culture part requires 27 cell culture chambers. Each cell culture chamber is composed of a three-channel structure, the middle channel is a cell channel, and the two sides are drug channels. The three channels are composed of trapezoidal microcolumns in the middle, the distance between the microcolumns 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 of the trapezoid and the hypotenuse is 70°-80°, preferably 79.4°.

[0082] The detection part is located in the cell culture layer of the chip, at the end of the cell culture chamber, and is a circular structure. In the circular detection part, the carboxyl groups on the surface are first activated by PLASMA treatment, and then the amino groups are chemically grafted by a silane coupling agent, and then the quantum dots are grafted on the chip by EDC solution, and then the nucleic acid aptamer of the target detection protein is connected to the chip by EDC solution and NHs solution. At the same time, the antibody of the target protein is grafted on the gold nanoparticles by PEG. In this example, liver cancer is selected as the target disease for the evaluation of the combined drug regimen, so the specific nucleic acid aptamer and specific antibody for liver cancer are selected.

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

[0084] In this example, the micro-column structure in the cell culture part can prevent the gel structure from invading the culture medium flow channel when the cells are added through the surface tension, and can also be easily processed by soft lithography or DLP photocuring printing.

[0085] In the above embodiment, the chip design method is:

[0086] 1) Setting the flow rate of the target solution input and the length, width, spacing of each mixing channel of the organ chip, the type of drug, the number of concentration gradients of each drug, etc.;

[0087] 2) Modeling using solidworks software;

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

[0089] 4) comparing the concentration of each drug in each cell culture chamber in step 3) with a preset concentration, wherein the concentration of each drug in each cell culture chamber refers to the drug concentration in each cell culture chamber simulated by comsol fluid mechanics in step 3); the preset concentration refers to the concentration obtained by theoretical calculation;

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

[0091] In the above embodiment, the distribution mechanism of the culture chambers is: the culture chambers are radially distributed from the center to the outside, and m circles are provided, and each circle is provided with m^(n-1) culture chambers. For the first drug, the culture chambers of each circle of culture chambers flow into the drug of the drug in increasing order from the inside to the outside, that is, the first circle of culture medium flows into the lowest concentration of the first drug, the second circle of culture chambers flows into the second concentration of the first drug, the third circle of culture chambers flows into the third concentration of the first drug, and so on. For the remaining drugs, each column of culture chambers radially distributed from the inner circle to the outer circle flows into the same concentration of the same drug. The distribution rule of each drug is that for the yth concentration of the xth drug, starting from the (y-1)*m^(nx)+1th column of culture chambers, the continuous m^(nx) columns of culture chambers are allocated with the concentration of the drug, and then every (m-1)*m^(nx)th column of culture chambers, the continuous m^(nx) columns of culture chambers are allocated with the concentration of the drug. For example, for two drugs, four concentration gradient combinations are used for high-throughput testing of the culture chamber design. A represents the first drug, B represents the second drug, A1 is the lowest concentration of drug A, and B4 is the highest concentration of drug B. The design rules are as follows:

[0092] Column 1 Column 2 Column 3 Column 4 Lap 1 A1B1 A1B2 A1B3 A1B4 Lap 2 A2B1 A2B2 A2B3 A2B4 Lap 3 A3B1 A3B2 A3B3 A3B4 Lap 4 A4B1 A4B2 A4B3 A4B4

[0093] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A high-throughput organ chip for multi-drug combination screening, characterized in that: The chip is a multi-layer structure, which includes, 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 comprises a drug adding hole and a concentration gradient drug generating hole, and the drug adding hole and the concentration gradient drug generating hole are connected through a mixing channel; The cell culture plate is provided with a plurality of cell culture chambers, each of which is connected to a drug mixing hole; 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 holes correspond to and are connected with the concentration gradient drug generation holes one by one; the concentration gradient drug transition holes and the drug mixing holes are connected through a distribution flow channel; the drug mixing holes are connected with the cell culture chamber of the cell culture plate.

2. The high-throughput organ chip for multi-drug combination screening according to claim 1, characterized in that: When testing m concentration gradients of n drugs; the number of drug addition holes on the single drug concentration gradient generating plate is 2n, and the number of concentration gradient drug generating holes is m*n, wherein every two drug addition holes are connected to the m concentration gradient drug generating holes through a mixing flow channel to obtain m different concentrations of a drug.

3. The high-throughput organ chip for multi-drug combination screening according to claim 2, characterized in that: The mixing channel is an asymmetric channel.

4. The high-throughput organ chip for multi-drug combination screening according to claim 2, characterized in that: The drug mixing plate has n*m layers, and each layer of the drug mixing plate is used to distribute n*m ​​drugs of different concentrations and types generated in a single concentration gradient generating part into a specific cell culture chamber.

5. The high-throughput organ chip for multi-drug combination screening according to claim 4, characterized in that: The inlet of the distribution channel is connected to the concentration gradient drug generation hole, and the distribution channel is connected to m^(n-1) cell culture chambers through the channel from the inlet.

6. The high-throughput organ chip for multi-drug combination screening according to any one of claims 1 to 5, characterized in that: A mixer is provided between the mixing channel and the concentration gradient drug generating hole; a damper is provided between the distribution channel and the drug mixing hole; and a mixer is provided between the drug mixing hole and the cell culture chamber.

7. The high-throughput organ chip for multi-drug combination screening according to claim 6, characterized in that: The cell culture chamber comprises three flow channel structures, wherein the middle flow channel is a cell flow channel, and the two sides are drug flow channels, and the intervals between the three flow channels are composed of trapezoidal microcolumns or half-height walls; a cell adding hole is arranged at a position corresponding to the two ends of the cell flow channel on a single drug concentration gradient generating plate, and a sampling hole is arranged at one end at a position corresponding to the two ends of the drug flow channel on the single drug concentration gradient generating plate, and the other end is connected to the drug mixing hole.

8. A method for designing a high-throughput organ chip for multi-drug combination screening according to any one of claims 1 to 7, characterized in that: The steps include: 1) Setting the flow rate of the target solution input and the length, width, spacing of each mixing channel of the organ chip, the type of drug, the number of concentration gradients of each drug, etc.; 2) Modeling using solidworks software; 3) Import into the COMSO simulation software and input parameters such as initial solution concentration and viscosity; 4) comparing the concentration of each drug in each cell culture chamber obtained by simulation in step 3) with the preset concentration to obtain a solution concentration difference, 5) If the solution concentration differences are all 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 size parameters of the flow channel are modified and steps 2-4 are repeated until the solution concentrations are all less than or equal to the error threshold.

9. A high-throughput screening method for multi-drug combination, characterized in that: Using the high-throughput organ chip for multi-drug combination screening according to any one of claims 1 to 7 comprises the following steps: 1) adding the hydrogel with cell clusters into the cell culture chamber through the cell injection hole; 2) Cross-linked hydrogel; 3) simultaneously inputting a medium without drugs and a medium with different drugs into two drug inlets; 4) continuously inputting the culture medium at a constant drug addition flow rate until the flow rate of the culture medium flowing out of the sampling hole stabilizes; 5) Place the chip in a cell culture incubator, change the culture medium every day, repeat steps 3-4 and perform culture testing.

10. Application of the chip according to any one of claims 1 to 7 in any of the following: A1) Application in drug combination screening; A2) Application in drug sensitivity testing.

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