A microfluidic chip for preparing high-throughput tumor organoids and a preparation method and application thereof

Abstract

The application discloses a microfluidic chip for preparing high-flux tumor organoids and a preparation method and application thereof. The microfluidic chip comprises a top layer and a matched bottom layer connected with the top layer; the top layer contains four hollow areas in the shape of right-angle sectors, and forms liquid storage areas after being connected with the bottom layer; four cell culture areas and four liquid exchange buffer areas are arranged in the areas of the bottom layer corresponding to the hollow areas of the top layer, and one cell culture area and one liquid exchange buffer area form a right-angle sector; the cell culture area is provided with a plurality of small chambers; and the liquid exchange buffer area is connected with the center. The microfluidic chip is provided with the liquid exchange buffer area, which greatly reduces the growth interference and loss of cells during liquid exchange, thereby being beneficial to the formation of uniform tumor organoids and keeping the tumor organoids in situ, facilitating continuous observation; and the top-open structure design is adopted, which is convenient for uniform implantation and dispersion of tumor cells and convenient for recovery of the tumor organoids.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, and specifically relates to a microfluidic chip for preparing high-throughput tumor organoids, its preparation method, and its application. Background Technology

[0002] Targeted therapy is an extremely effective treatment for cancer; however, the lack of effective drug screening models has become a major challenge in the development of targeted cancer drugs. Therefore, the development of targeted cancer drugs urgently requires a tumor model that simulates the in vivo microenvironment in both structure and function.

[0003] Current targeted tumor drug screening models mainly include two-dimensional cultured cell models, animal models, human tissue xenograft (PDX) models, and three-dimensional cultured tumor organoid models. Among these models, two-dimensional cultured cell models cannot accurately reflect the human physiological environment, are oversimplified models, and have stability issues. Animal models and PDX models are complex animal models, with high construction costs, long cycles, difficulty in real-time observation, and difficulty in achieving high-throughput drug screening. Three-dimensional cultured tumor organoid models can assemble different cells into three-dimensional aggregates with specific functions according to a certain structure, offering better simulation and more controllable external conditions, making them more suitable for drug screening and a current research hotspot in drug screening models. Three-dimensional culture of tumor organoids typically involves encapsulating tumor cells in colloids such as Matrigel before culturing. However, this method often results in tumor organoids with inconsistent size and shape. Differences in tumor sphere size lead to differences in cell activity within the tumor sphere and differences in tumor-drug responses due to variations in drug diffusion distance. Studies have reported that when the size of 3D cell structures exceeds 520 μm, varying degrees of cell necrosis occur (Friedrich J, et al. A Reliable Tool to Determine Cell Viability in Complex 3-d Culture: The Acid Phosphatase Assay. J Biomol Screen 2007, 12(7), 925–937.). These issues make it difficult to standardize tumor-drug responses, thus hindering the development of chemotherapy-induced tumor dormancy models.

[0004] The rapid development of microfluidic technology in the field of three-dimensional cell culture has provided a new technological platform for simulating the in vivo environment. This technology promotes the spontaneous aggregation of cells into tumor spheroids through microstructures on microfluidic chips, such as microsieves and micropits, thus avoiding the use of hydrogel scaffolds or rotating devices. The design of microfluidic chips allows for high-throughput, uniformly sized structures, thereby forming high-throughput, uniformly sized tumor spheroids. Although various methods have been developed to construct tumor spheroid arrays using microfluidic chips, they suffer from problems such as low throughput, low space utilization, and complex recovery.For example, Jean-Philippe Frimat's team developed a chip based on cell patterning technology, but it is not suitable for long-term culture of tumor cells (Frimat JP, et al. The Network Formation Assay: A Spatially Standardized Neurite Outgrowth Analytical Display for Neurotoxicity Screening. Lab Chip 2010, 10(6), 701–709.); Eduardo Imanol Agüero's team developed a chip with limited throughput, consisting of 72 wells connected in series (Agüero EI, et al. Microfluidic Devices to Analyze the Response of Sphere-Forming Stem-like Populations to Chemotherapeutic Drugs. J Mol Med (Berl) 2023, 101(11), 1465–1475.); Yu-Chih Chen's team developed a chip that is not convenient for recovering tumor cells (Chen YC, et al. High-Throughput Cancer Cell Sphere Formation for Characterizing the Efficacy of Photo Dynamic Therapy in 3D Cell). Cultures.Sci Rep2015,5,12175.; Yong Hun Jung's team's chip can be adapted to 96-well plates but has low space utilization (Jung YH, et al.Drug Screening by Uniform PatientDerived Colorectal Cancer Hydro-Organoids.Biomaterials 2021,276,121004.); Manjulata Singh's team's chip is already quite ideal but the throughput is still low (Singh M, et al.Production of Uniform 3D Microtumors in Hydrogel Microwell Arrays for Measurement of Viability,Morphology,and Signaling Pathway Activation.Assay Drug Dev Technol2015,13(9),570–583.).To address the aforementioned issues, the development of novel microfluidic chips aims to overcome technical challenges such as low throughput, low space utilization, inconvenient recycling, non-reusability, and the need for frequent culture medium replenishment due to low storage capacity. This new chip will offer higher throughput and space utilization, simplify the recovery process of tumor spheres, allow for chip reuse, and reduce the frequency of culture medium replenishment, providing a more efficient technical platform for three-dimensional cell culture. Summary of the Invention

[0005] The primary objective of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a microfluidic chip for fabricating high-throughput tumor organoids.

[0006] Another object of the present invention is to provide a method for preparing the above-mentioned microfluidic chip for preparing high-throughput tumor organoids.

[0007] Another object of the present invention is to provide the application of the above-mentioned microfluidic chip for the fabrication of high-throughput tumor organoids.

[0008] The objective of this invention is achieved through the following technical solution: a microfluidic chip for preparing high-throughput tumor organoids, comprising a top layer and a matching bottom layer connected to the top layer; the top layer contains four right-angled fan-shaped hollow areas, which form a liquid storage area after being connected to the bottom layer; four cell culture areas and four medium exchange buffers are provided in the bottom layer area corresponding to the hollow areas of the top layer, with one cell culture area and one medium exchange buffer forming a right-angled fan; the cell culture area is provided with several small chambers; the medium exchange buffer is connected to the center of the circle, and the setting of the medium exchange buffer avoids the cells cultured in the small chambers being aspirated during medium exchange, thereby avoiding the loss of cultured cells and interference with the formation of tumor organoids.

[0009] The microfluidic chip is preferably made of polydimethylsiloxane.

[0010] The microfluidic chip used for preparing high-throughput tumor organoids is preferably circular in shape.

[0011] The thickness of the top layer is preferably 4 mm.

[0012] The four right-angled sectors are formed by two vertically connected partitions.

[0013] The cell culture zone is preferably fan-shaped.

[0014] The fluid exchange buffer is preferably a right-angled sector shape, with its arc matching the inner arc of the cell culture zone.

[0015] The chamber is preferably a light-transmitting chamber, which is beneficial for observation.

[0016] The inner surface of the chamber is coated with a hydrophilic polymer coating.

[0017] The hydrophilic polymer is obtained by polymerizing N,N-dimethylacrylamide and glycidyl methacrylate; preferably, it is prepared by the following steps: N,N-dimethylacrylamide, glycidyl methacrylate and degassed pure water are mixed, and after degassed, tetramethylethylenediamine and an initiator solution are added to obtain a reaction solution. The reaction is carried out under degassed conditions, and the reaction product is dialyzed to obtain the hydrophilic polymer; the contents of each substance in the reaction solution are as follows: N,N-dimethylacrylamide 4.8-5.0% v / v, glycidyl methacrylate 0.09-0.1% v / v, tetramethylethylenediamine 0.09-0.1% v / v, and initiator 0.05% (w / v, g / mL).

[0018] The preferred volume ratio is 50:1:1:1:900 to 1000.

[0019] The initiator is preferably potassium persulfate.

[0020] The preferred reaction time is 120 min.

[0021] The solution used in the dialysis is preferably pure water.

[0022] The preferred specification for the dialysis tubing used in the dialysis is a 3500 Da cut-off dialysis tubing.

[0023] The chamber is hexagonal prism-shaped with a regular hexagonal base, which facilitates computer image recognition and processing, and distinguishes it from round tumor organoids.

[0024] The chamber has a side length of 86.66 μm and a depth of 250 μm.

[0025] The distance between the chambers is 80 μm.

[0026] Each of the cell culture zones is equipped with 10,000 chambers.

[0027] The above-mentioned method for fabricating microfluidic chips for high-throughput tumor organoids includes the following steps:

[0028] (1) Mask fabrication: The top and bottom structural images of the microfluidic chip used to prepare high-throughput tumor organoids were drawn using software and printed as a film mask with local pattern light transmission.

[0029] (2) Mold making:

[0030] A. Pour the photoresist onto a single-sided polished silicon wafer and use a spin coater to obtain a uniform photoresist layer;

[0031] B. Heat the photoresist layer to obtain a hardened photoresist layer;

[0032] C. Apply the mask obtained in step (1) onto the hardened photoresist layer and perform ultraviolet lithography using an ultraviolet lithography machine;

[0033] D. After photolithography, heat the photoresist until it hardens to room temperature, then use a developer to dissolve and wash away the photoresist in the non-patterned areas, and let it air dry.

[0034] E. Heat the template silicon wafer dried in step D to fix the pattern, clean and dry it, fumigate it with dichlorodimethylsilane, and rinse it with isopropanol to obtain the mold.

[0035] (3) Chip fabrication:

[0036] A. After sealing the mold edges with aluminum foil, pour the solution obtained by mixing polydimethylsiloxane and initiator into the mold. After it is evenly spread and free of bubbles, heat it to obtain top-layer cured PDMS and bottom-layer cured PDMS.

[0037] B. After removing excess material and cleaning, the treated top-layer cured PDMS and bottom-layer cured PDMS are bonded together to obtain the chip;

[0038] C. Drop the hydrophilic polymer into the chip obtained in step B, ensuring that the hydrophilic polymer enters the chamber. After standing, remove the hydrophilic polymer, dry it, and then soak it in pure water for sterilization to obtain a microfluidic chip for preparing high-throughput tumor organoids.

[0039] The preferred heating conditions in step (2)B are heating at 90-100°C for 20-40 minutes.

[0040] In step (2)C, the ultraviolet energy intensity of the ultraviolet lithography is set to 15mw / cm2 and the duration is 30s.

[0041] The preferred heating conditions in step (2)D are heating at 90-100°C for 20-40 minutes.

[0042] The developer used in step (2)D is preferably PGMEA developer.

[0043] The heating conditions described in step (2)E are preferably 130-150°C for 40-50 minutes.

[0044] The polydimethylsiloxane and initiator in the solution described in step (3) can be prepared according to the instructions; preferably, the polydimethylsiloxane and initiator are mixed in a mass ratio of 10:1.

[0045] The heating conditions described in step (3) are preferably 130-150°C for 5-15 minutes.

[0046] The above-mentioned microfluidic chip for preparing high-throughput tumor organoids is applied in tumor organoid culture.

[0047] The application of the microfluidic chip for preparing high-throughput tumor organoids in drug screening preferably includes the following steps: tumor cells are non-adherently multi-cell selectively amplified in the microfluidic chip for preparing high-throughput tumor organoids. When the cells are amplified to a sufficient size, the culture medium is replaced with a culture medium containing the relevant test drug. After the drug treatment is completed, the cells are detected.

[0048] The aforementioned detection methods include measuring cell viability or gently aspirating tumor organoids for immunohistochemical analysis.

[0049] The present invention has the following advantages and effects compared with the prior art:

[0050] 1. This invention provides a microfluidic chip for preparing high-throughput tumor organoids. The microfluidic chip is equipped with high-throughput cell culture chambers of the same size, which is conducive to the culture of high-throughput and uniform tumor organoids. In addition, a medium exchange buffer is set up, which greatly reduces the interference and loss of cell growth during medium exchange, thus facilitating the formation of uniform tumor organoids and keeping the tumor organoids in situ for continuous observation.

[0051] 2. The microfluidic chip for preparing high-throughput tumor organoids provided by the present invention adopts a top-open structural design, which facilitates the uniform seeding and dispersion of tumor cells and the recovery of tumor organoids. Attached Figure Description

[0052] Figure 1 This is a schematic diagram of the structure of the microfluidic chip for preparing high-throughput tumor organoids according to the present invention; wherein, 1 is the cell culture area with a side length of 86.66 μm and a depth of 250 μm; 2 is the medium exchange buffer with a depth of 4 mm; 3 is the partition; 4 is the top layer; and 5 is a 100 mm culture dish.

[0053] Figure 2 This is a schematic diagram of the fabrication process of the microfluidic chip for preparing high-throughput tumor organoids according to the present invention.

[0054] Figure 3 This is a schematic diagram of the process for obtaining tumor organoids by inoculating tumor cell suspension according to the present invention.

[0055] Figure 4 These are bright-field photographs of continuous cultures of tumor cells from different cell sources in this invention.

[0056] Figure 5 This is a graph showing the statistical results of the diameter of multicellular tumor organoids from different regions in this invention.

[0057] Figure 6 This image shows the retention of tumor organoids in a micro-pit array using an open high-throughput microporous array microfluidic chip under conditions of no or no liquid flow buffer.

[0058] Figure 7 This is a graph showing the statistical results of tumor cell survival rate after drug treatment of tumor organoids obtained in this invention.

[0059] Figure 8 These are photographs showing the recovery status of tumor organoids from the microfluidic chip used in this invention. Detailed Implementation

[0060] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the cell culture conditions in the following embodiments are 37°C and 5% CO2. For conditions not specifically specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. For reagents or instruments whose manufacturers are not specified, they are all conventional products that can be purchased commercially.

[0061] The main sources of materials involved in the embodiments of this invention are:

[0062] DMEM medium and penicillin-streptomycin were purchased from Gibco, USA.

[0063] Fetal bovine serum and pancreatin / EDTA digestion solution were purchased from Thermo Fisher Scientific, USA.

[0064] Single-displacement silicon wafers, purchased from Suzhou Ruicai Semiconductor Co., Ltd., China;

[0065] Plasma cleaner, purchased from Chengdu Mingheng Technology Development Co., Ltd., China;

[0066] SU-8 adhesive, purchased from Nantong Zhongxin Qiheng Energy Technology Co., Ltd., China;

[0067] The spin coater and baking coater were purchased from Jiangsu Leibo Scientific Instruments Co., Ltd.

[0068] The ultraviolet lithography machine was purchased from Chengdu Feicheng Optoelectronic Technology Co., Ltd.

[0069] PGMEA developer solution, purchased from ELECTRONIC MATERIALS;

[0070] Dichlorodimethylsilane, purchased from Macklin;

[0071] PDMS kit, purchased from Dow Corning, USA.

[0072] Example 1

[0073] A microfluidic chip for fabricating high-throughput tumor organoids, such as Figure 1 As shown, it includes a top layer 4 and a matching bottom layer connected to the top layer; the top layer 4 contains raised partitions 3, forming four right-angled fan-shaped hollow areas, which connect to the bottom layer 5 to form a liquid storage area, and the raised partitions 3 divide the bottom layer into four right-angled fan-shaped areas; in the bottom layer area corresponding to the hollow area of ​​the top layer, four cell culture zones 1 and four medium exchange buffer zones 2 are set, with one cell culture zone and one medium exchange buffer zone forming a right-angled fan shape; the cell culture zone is set with several small chambers; the medium exchange buffer zone is connected to the center; in cell culture, the microfluidic chip is placed in a culture dish 5 to avoid cell contamination. Its preparation process is as follows: Figure 2 As shown, the specific steps are as follows:

[0074] I. Microfluidic Chip Fabrication

[0075] 1. Mask Fabrication: The combined upper and lower layer structural pattern of the chip was drawn using AutoCAD 2022 software and printed as a film mask for partial pattern transparency. The lower layer structural pattern of this mask is a transparent fan-shaped array with four missing corners. Each array contains 10,000 transparent regular hexagons (the shape of the lower layer chambers) circumscribed by a 150μm circle. There are 4 such arrays on one chip, resulting in a total of 40,000 regular hexagonal chambers. The side length of each hexagon is 86.66μm, and the row and column spacing is 80μm. At the missing corners of the fan-shaped array, there is a blank area of ​​isosceles right triangles with a side length of 10mm. This area does not contain hexagonal chambers and serves as a liquid buffer area during media replacement to reduce disturbance to the tumor organoids cultured in the chambers during media replacement. The upper structure consists of four 30mm side-length translucent right-angled fan-shaped structures to correspond to the lower chamber structure for holding culture medium, and a translucent ring with an inner diameter of 85mm and an outer diameter of 88mm is set as the chip boundary for chip post-processing.

[0076] 2. Mold Fabrication: After surface treatment of a 4-inch single-sided polished silicon wafer for 2 minutes using a plasma cleaner, SU-8 photoresist is poured onto the wafer at room temperature and a spin coater is used at 500 rpm for 1 minute to obtain a SU-8 photoresist layer with a thickness of approximately 250 μm. Then, the wafer is heated to 95°C for approximately 30 minutes using a baking oven. After returning to room temperature and hardening, ultraviolet lithography is performed using a UV lithography machine and a pre-drawn mask, with the UV energy intensity set to 15 mW / cm². 2The curing time is 30 seconds. Then, heat at 95°C for approximately 30 minutes using a glue oven. After returning to room temperature and hardening, use PGMEA developer (propylene glycol monomethyl ether acetic acid, 1-methoxy-2-acetoxypropane) in a fume hood, shaking at approximately 30 rpm for about 15 minutes on a horizontal shaker to dissolve and remove the SU-8 glue from the non-patterned areas, then air dry. Heat the developed template silicon wafer at 140°C for approximately 45 minutes to harden the film and fix the pattern. Finally, the silicon wafer undergoes a plasma cleaner surface treatment for 2 minutes, then is placed in a glass desiccator in a fume hood. Dimethyldichlorosilane is added to the bottom of the desiccator at a rate of 1.5 mL per wafer, and the wafer is fumigated for approximately 1 hour. Afterward, rinse the surface with isopropanol and air dry to obtain the desired silicon wafer mold.

[0077] 3. PDMS Chip Fabrication: After sealing the mold with aluminum foil, pour the degassed liquid (PMDS and initiator in a 10:1 weight ratio) of polydimethylsiloxane (PDMS) and its matching initiator into the mold. The bottom chip should contain approximately 10 mL, and the top chip approximately 20 mL. After evenly spreading and removing air bubbles, heat at 140℃ for about 10 minutes to obtain two cured PDMS sheets. Post-process using an 80mm circular scalpel and surgical blade to remove excess material. Then, surface treat with a plasma cleaner for 2 minutes before bonding the two chips together to obtain the desired PDMS chip.

[0078] II. Hydrophilic polymer coating for microfluidic chips

[0079] 1. Degas approximately 9.5 mL of pure water, then add 0.5 mL of N,N-dimethylacrylamide and 10 μL of glycidyl methacrylate, mix gently by inverting, and degas for another 10 min. Next, add 10 μL of tetramethylethylenediamine (TEMED) and 100 μL of pure water containing 0.005 g of potassium persulfate to the mixture to obtain the reaction system. React under degassing conditions for 120 min; the reaction system will become slightly viscous. Finally, to remove unpolymerized monomers and small compound molecules to prevent cell damage, dialyze the polymer solution using a 3500 Da cutoff dialysis tube, changing the pure water every 12 hours. After dialysis for 3 days, collect the solution from the dialysis tube; this is a hydrophilic polymer solution, stored at 4°C for later use.

[0080] 2. After chip sealing, immediately add an appropriate amount of hydrophilic polymer solution diluted 20 times with pure water to the chip and let it stand for 10 minutes. If air blocks the liquid from entering the micro-pits, gently scrape the air-containing chambers with a blunt object such as a scraper to expel the air disturbance in the chamber array, ensuring that the hydrophilic copolymer liquid enters the chambers. Afterward, remove the hydrophilic polymer from the chip and dry it at 80°C for 30 minutes.

[0081] 3. After coating, the chip is placed in a petri dish and immersed in pure water. It is then sterilized at 134°C for 30 minutes to completely remove the gas from the micro-pits and sterilize it before use.

[0082] Example 2

[0083] The specific steps for preparing tumor organoids derived from multiple cells of colon cancer are as follows:

[0084] (1) Cell preparation:

[0085] 1) HCT116 colon cancer cells and HT29 colon cancer cells (purchased from Wuhan Procell Biotechnology Co., Ltd.) were cultured separately in culture flasks and recovered when the coverage reached 85%.

[0086] 2) Trypsin digestion was used to prepare a single-cell suspension. After centrifugation, the cells were resuspended in DMEM medium containing 10% v / v fetal bovine serum to obtain a concentration of approximately 1×10⁻⁶ cells / mL. 6 Cell suspension of cells / mL.

[0087] (2) Multi-cell culture in a microfluidic chip, the process is as follows: Figure 3 As shown:

[0088] 1) Take 4000 μL of cell suspension and evenly seed it into the four cell culture zones of the microfluidic chip prepared in Example 1, 1000 μL in each cell culture zone, and let it stand for 1 hour.

[0089] 2) Tumor cells that did not fall into the microwells were aspirated by changing the culture medium. The culture medium was changed through the medium exchange buffer.

[0090] 3) Continuous culture allows for the production of 40,000 multi-cell-derived tumor organoids of varying sizes, depending on the time elapsed. Figure 4 If tumor organoids need to be extracted, all tumor organoids on the chip can be easily aspirated using a pipette. The diameter of the tumor organoids was counted by sampling from each of the four cell culture zones. Five samples were taken from each cell culture zone, with each sample containing 25 tumor organoids. The results are as follows: Figure 5 As shown, the tumor organoids exhibit relatively uniform growth and have similar diameters.

[0091] Example 3

[0092] A microfluidic chip without a media exchange buffer is provided. Its structure is basically the same as that of the microfluidic chip provided in Example 1, except that its cell culture area is a right-angled sector and does not have a media exchange buffer. Figure 6 As shown in (B) in the diagram.

[0093] Cell culture was performed according to the steps in Example 2, and the tumor organoid occupancy rate was as follows: Figure 6 As shown, based on the tumor organoids obtained from the microfluidic chip provided in Example 1, the tumor organoid yield using the microfluidic chip without a media exchange buffer is only about 35% in the same area. It is evident that although the microfluidic chip without a media exchange buffer has more cell culture chambers, media exchange may significantly interfere with the retention and formation of tumor organoids. Therefore, the presence of a media exchange buffer is more conducive to obtaining a large number of tumor organoids.

[0094] Example 4

[0095] (1) Drug treatment: Prepare relevant test drugs. FOLFIRINOX (1×) represents a combination chemotherapy regimen of fluorouracil (4μM), oxaliplatin (0.5μM) and SN-38 (12.5nM). This regimen can be combined with Conteltinib (MCE, HY-109084), PF-562271 (MCE, HY-10459), Ifebemtinib (MCE, HY-122844), Defactinib (MCE, HY-12289), and Verteporfin (MCE, HY-B0146) at a concentration of 10μM according to the instructions. Test the drug response of tumor organoids to different drug combinations. Tumor cell viability was assessed using Calcein-AM / PI staining of both live and dead cells. The principle is that Calcein-AM in live cells can cross the cell membrane via esterase action to remove the AM group, after which Calcein emits strong green fluorescence, making it detectable under a fluorescence microscope. PI (propidium iodide) can penetrate damaged cell membranes into dead cells, embedding itself in the cell's DNA double helix structure and emitting red fluorescence, thus making dead cells detectable as red fluorescence. Statistical analysis was performed based on the co-staining results, as shown below. Figure 7 As shown, the FOLFIRINOX (1×) chemotherapy regimen combined with Verteporfin (10μM) can cause greater damage to tumor cells compared to other regimens.

[0096] (4) Tumor organoid recovery:

[0097] Prepare a 1% bovine serum albumin (BSA) solution for rinsing pipette tips and centrifuge tubes.

[0098] Use a pipette to draw up the cell culture medium, gently wash three times with PBS, gently aspirate the tumor organoids, collect them into a centrifuge tube, centrifuge at 300g for 2 minutes, and discard the supernatant for later use.

[0099] Following the steps in Example 2, a single culture can recover approximately 40,000 tumor organoids, with a recovery rate as follows: Figure 8 As shown, the microfluidic chip provided by this invention is beneficial for the recycling of tumor organoids.

[0100] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A microfluidic chip for fabricating high-throughput tumor organoids, characterized in that: It includes a top layer and a matching bottom layer connected to the top layer; the top layer contains 4 right-angled fan-shaped hollow areas, which form a liquid storage area after connecting with the bottom layer; in the bottom area corresponding to the hollow area of ​​the top layer, there are 4 cell culture areas and 4 medium exchange buffers, one cell culture area and one medium exchange buffer form a right-angled fan; the cell culture area is set with several small chambers; the medium exchange buffer is connected to the center.

2. The microfluidic chip for fabricating high-throughput tumor organoids according to claim 1, characterized in that: The microfluidic chip is made of polydimethylsiloxane; The aforementioned small chamber is a light-transmitting chamber; The inner surface of the chamber is coated with a hydrophilic polymer coating.

3. The microfluidic chip for fabricating high-throughput tumor organoids according to claim 2, characterized in that: The hydrophilic polymer is obtained by polymerizing N,N-dimethylacrylamide and glycidyl methacrylate.

4. The microfluidic chip for fabricating high-throughput tumor organoids according to claim 3, characterized in that: The hydrophilic polymer was prepared by the following steps: N,N-dimethylacrylamide, glycidyl methacrylate, and degassed pure water were mixed, and after degassing, tetramethylethylenediamine and an initiator solution were added to obtain a reaction solution. The reaction was carried out under degassed conditions, and the reaction product was dialyzed to obtain the hydrophilic polymer. The contents of each substance in the reaction solution were as follows: N,N-dimethylacrylamide 4.8-5.0% v / v, glycidyl methacrylate 0.09-0.1% v / v, tetramethylethylenediamine 0.09-0.1% v / v, and initiator 0.05% (w / v, g / mL).

5. The microfluidic chip for fabricating high-throughput tumor organoids according to claim 1, characterized in that: The microfluidic chip used for preparing high-throughput tumor organoids is circular in shape. The cell culture area is fan-shaped; The fluid exchange buffer zone is a right-angled sector, and its arc matches the inner arc of the cell culture zone; The chamber is hexagonal prism-shaped.

6. The microfluidic chip for fabricating high-throughput tumor organoids according to claim 1, characterized in that: The chamber has a side length of 86.66 μm and a depth of 250 μm; The distance between the cells is 80 μm; Each of the cell culture zones is equipped with 10,000 chambers.

7. The method for fabricating a microfluidic chip for high-throughput tumor organoids according to any one of claims 1 to 6, characterized in that... Includes the following steps: (1) Mask fabrication: The top and bottom structural images of the microfluidic chip used to prepare high-throughput tumor organoids were drawn using software and printed as a film mask with local pattern light transmission. (2) Mold making: A. Pour the photoresist onto a single-sided polished silicon wafer and use a spin coater to obtain a uniform photoresist layer; B. Heat the photoresist layer to obtain a hardened photoresist layer; C. Apply the mask obtained in step (1) onto the hardened photoresist layer and perform ultraviolet lithography using an ultraviolet lithography machine; D. After photolithography, heat the photoresist until it hardens to room temperature, then use a developer to dissolve and wash away the photoresist in the non-patterned areas, and let it air dry. E. Heat the template silicon wafer dried in step D to fix the pattern, clean and dry it, fumigate it with dichlorodimethylsilane, and rinse it with isopropanol to obtain the mold. (3) Chip fabrication: A. After sealing the mold edges with aluminum foil, pour the solution obtained by mixing polydimethylsiloxane and initiator into the mold. After it is evenly spread and free of bubbles, heat it to obtain top-layer cured PDMS and bottom-layer cured PDMS. B. After removing excess material and cleaning, the treated top-layer cured PDMS and bottom-layer cured PDMS are bonded together to obtain the chip; C. Drop the hydrophilic polymer into the chip obtained in step B, ensuring that the hydrophilic polymer enters the chamber. After standing, remove the hydrophilic polymer, dry it, and then soak it in pure water for sterilization to obtain a microfluidic chip for preparing high-throughput tumor organoids.

8. The method for fabricating a microfluidic chip for high-throughput tumor organoids according to claim 7, characterized in that: The heating conditions described in step (2)B are heating at 90-100℃ for 20-40 minutes; The ultraviolet energy intensity of the ultraviolet lithography described in step (2)C is set to 15mw / cm2 and the duration is 30s; The heating conditions described in step (2)D are 90-100℃ for 20-40 minutes; The developer mentioned in step (2)D is PGMEA developer; The heating conditions described in step (2)E are 130-150℃ for 40-50 minutes; The heating conditions described in step (3) are 130-150℃ for 5-15 minutes.

9. The application of the microfluidic chip for preparing high-throughput tumor organoids as described in any one of claims 1 to 6 in tumor organoid culture.

10. The application of the microfluidic chip for preparing high-throughput tumor organoids as described in any one of claims 1 to 6 in drug screening.

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