A microfluidic chip capable of culturing oral squamous cell carcinoma organoids and a preparation method thereof

Abstract

The application provides a microfluidic chip capable of culturing oral squamous cell carcinoma organoids and a preparation method thereof, relates to the technical field of microfluidic chips, and the chip comprises a four-layer structure: the first layer is a closed layer, the second layer is a culture solution / drug pool layer (containing a concentration gradient filling channel and a slow flow channel), the third layer is a matrix type culture pool layer, and the fourth layer is a matrigel filling layer (containing an S-shaped flow channel); the second layer and the third layer are integrally formed, and the layers are connected through oxygen plasma welding; the problems of easy displacement of matrigel, poor drug gradient regulation, complicated operation, insufficient adaptability and the like in the prior art are solved, the matrigel and the organoids can be stably fixed, the drug concentration gradient can be accurately regulated, the nutrition supply efficiency is enhanced, the structural integration degree and the operation convenience are improved, and the chip is suitable for oral squamous cell carcinoma organoid culture, drug screening and tumor mechanism research.

Description

Technical Field

[0001] This invention relates to the field of microfluidic chip technology, and in particular to a microfluidic chip capable of culturing oral squamous cell carcinoma organoids and its preparation method. Background Technology

[0002] In the basic research and clinical translation of oral squamous cell carcinoma, organoid models have become a core tool for exploring tumorigenesis, drug resistance, and personalized treatment due to their ability to accurately simulate the three-dimensional structure, cellular heterogeneity, and microenvironment characteristics of tumors in vivo. However, current organoid culture technologies still face many bottlenecks, making it difficult to meet the needs of precise research.

[0003] Traditional well plate culture systems have significant drawbacks: First, the culture environment is not uniform, and uneven fluid distribution within the wells leads to large differences in organoid growth conditions and low experimental reproducibility. Second, they cannot simulate in vivo drug concentration gradients, making it difficult to achieve accurate drug efficacy assessment. Third, the fixation effect between the matrix gel and organoids is poor, and displacement is easily caused by liquid replacement. Furthermore, the reagent consumption is huge, resulting in increased research costs.

[0004] Existing microfluidic chip technology also has significant shortcomings: First, it has poor compatibility with matrix gel. Most chips adopt an open or suspended design, making the matrix gel prone to breakage, displacement, or blockage of channels, leading to organoid culture failure. Second, it has weak concentration gradient control capabilities. Drug delivery is mostly in a single-concentration mode, which cannot simulate the real microenvironment of drug diffusion from high concentration to low concentration in in vivo tumor tissue, reducing the clinical relevance of drug efficacy assessment. Third, it has low structural integration. The functions of culture, drug delivery, and observation are scattered, making operation cumbersome and prone to contamination. Fourth, it lacks a dedicated design for oral squamous cell carcinoma organoids. Existing chips are mostly applicable to general cells or other tumor types, making it difficult to match the growth characteristics of oral squamous cell carcinoma organoids (such as high invasiveness and strong matrix dependence), resulting in insufficient model stability and physiological relevance.

[0005] Therefore, developing a highly integrated microfluidic chip specifically for oral squamous cell carcinoma organoids that combines stable fixation with precise concentration gradient control has become a key issue that urgently needs to be addressed in the field. Summary of the Invention

[0006] To achieve the above objectives, this invention provides a microfluidic chip for culturing oral squamous cell carcinoma organoids. It employs a four-layer vertically stacked integrated structure, with each layer forming a fluid-connected functional system through microfluidic channels. This chip enables organoid culture while ensuring stable fixation with matrix gel and precise concentration gradient control. Specifically, it includes:

[0007] The fourth layer has an inlet and an outlet, with an S-shaped flow channel between them. The third layer has multiple culture tanks arranged in a matrix. These culture tanks are located above the flow channel and are connected to it. Above the third layer is a second layer. The second layer has storage tanks corresponding to the culture tanks, and these storage tanks are connected to the culture tanks. The multiple horizontal storage tanks are interconnected. These horizontal storage tanks are grouped together, with multiple groups converging on one side and connected to a second opening via a channel. On the other side, the multiple groups of horizontal storage tanks are respectively connected to a concentration gradient infusion channel. The concentration gradient infusion channel has a first opening and a second opening at the end furthest from the storage tank. The first layer covers the upper part of the second layer.

[0008] Preferably, the diameter of the storage tank is larger than the diameter of the culture tank.

[0009] Preferably, the concentration gradient infusion channel consists of multiple sets of parallel channels with S-shaped bends, and the number of parallel channels increases as they approach the storage tank.

[0010] Preferably, there is a slow-flow channel between the second opening and multiple sets of transverse liquid storage tanks, and the number of the slow-flow channels increases exponentially with distance from the second opening until each outlet of the slow-flow channel is connected to a liquid storage tank.

[0011] Preferably, one of the first or third opening is a drug inlet and the other is a culture medium outlet, and the second opening is a culture medium inlet.

[0012] Preferably, the first layer completely covers the upper end of the second layer, and the first layer has a standard injection hole; the standard injection hole of the first layer corresponds to the first opening, the second opening and the third opening.

[0013] Preferably, the second layer and the third layer are integrally formed; the second layer is connected to the first layer, and the third layer is connected to the fourth layer by oxygen plasma welding.

[0014] Based on the aforementioned microfluidic chip, this application also discloses a microfluidic chip kit for culturing oral squamous cell carcinoma organoids, including the aforementioned microfluidic chip, as well as a warming device, an injection pump, an inverted microscope, a vacuum degassing device, a chip fixation platform, and a pipette; the warming device includes a constant temperature incubator and a temperature-controlled water bath.

[0015] Based on the aforementioned microfluidic chip, this application also discloses a method for using a microfluidic chip capable of culturing oral squamous cell carcinoma organoids, as detailed below:

[0016] The first step is to inject the organoid-containing matrix gel into the inlet of the fourth layer until the flow channel is filled, and then backflow into the culture tank of the third layer, where the matrix gel solidifies.

[0017] The second step involves injecting culture medium through the second opening of the second layer. The culture medium enters the reservoir to provide nutrients for the organoids.

[0018] The third step involves delivering drugs or nutrient solutions through the first or third opening of the second layer, and adjusting the flow rate through the concentration gradient infusion channel to deliver drugs or nutrient solutions of different concentrations.

[0019] Based on the aforementioned microfluidic chip, this application also discloses a method for preparing a microfluidic chip capable of culturing oral squamous cell carcinoma organoids, as detailed below:

[0020] S1, Construct a four-layer chip model, including the first, second, third, and fourth layers arranged sequentially in the top and bottom directions;

[0021] S2, the second, third and fourth layers are molded to form a mold for the microfluidic chip channel;

[0022] S3, mix PDMS and curing agent, pour the mixed solution into each mold, and remove the mold after curing;

[0023] S4, using oxygen plasma to treat the surface of each finished product;

[0024] S5 uses oxygen plasma welding technology to weld the first and fourth layers to the top of the second layer and the bottom of the third layer, respectively, to form a complete microfluidic chip.

[0025] Beneficial effects

[0026] 1. In this invention, the S-shaped flow channel in the fourth layer and the matrix-type culture tank in the third layer form a "reverse suction-limiting" synergistic structure, which can precisely control the filling morphology of the matrix gel in the culture tank. Combined with the diameter difference design between the culture tank and the reservoir, the matrix gel solidifies into a stable columnar structure, effectively avoiding displacement or leakage caused by fluid scouring. At the same time, this structure significantly reduces the shear force between the matrix gel and the culture medium / drug, protecting the three-dimensional structural integrity of the organoid and solving the problems of easy blockage of matrix gel and easy damage to organoids in existing chips.

[0027] 2. In this invention, the concentration gradient infusion channel of the second layer adopts an "S-shaped bend + parallel channel increment" design. By adjusting the flow rate of different channels, a continuous and stable drug concentration gradient can be formed, which can accurately simulate the drug diffusion microenvironment of tumor tissue in vivo.

[0028] 3. In this invention, the four-layer integrated structure (first layer for sealing, second layer for drug delivery / storage, third layer for culture, and fourth layer for matrix gel injection) achieves a high degree of integration of the "matrix gel fixation-culture-drug delivery-observation" function. Combined with the standard injection port design of the first layer, it avoids the risk of contamination caused by frequent cap opening. The integrated molding of the second and third layers and the oxygen plasma welding technology ensure the chip's high airtightness, further improving system stability.

[0029] 4. Designed to address the high matrix dependence and growth characteristics of oral squamous cell carcinoma organoids, this chip can stably simulate the three-dimensional microenvironment of tumors, providing an ideal model for research on the pathogenesis, metastasis pathways, and targeted therapy of oral squamous cell carcinoma. Simultaneously, the matrix-style culture pool supports multi-omics experiments (such as gene sequencing and proteomics analysis), and combined with supporting temperature control and monitoring equipment, it can be extended to the field of automated high-throughput screening, showing broad application prospects. Attached Figure Description

[0030] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings of the embodiments will be briefly described below.

[0031] The accompanying drawings described below are only related to some embodiments of the invention and are not intended to limit the invention.

[0032] In the attached diagram:

[0033] Figure 1 A perspective view of the three-dimensional structure of a microfluidic chip according to an embodiment of the present invention is shown.

[0034] Figure 2 An exploded view of a microfluidic chip according to an embodiment of the present invention is shown.

[0035] Figure 3 A three-dimensional structural diagram of the fourth layer of a microfluidic chip according to an embodiment of the present invention is shown.

[0036] Figure 4 Cross-sectional views of the second and third layers of a microfluidic chip according to an embodiment of the present invention are shown.

[0037] Figure 5 Cross-sectional views of the second and third layers of a microfluidic chip according to an embodiment of the present invention are shown.

[0038] Figure 6 A three-dimensional structural diagram of the first layer of a microfluidic chip according to an embodiment of the present invention is shown.

[0039] Figure 7 A schematic diagram illustrating a method of using a microfluidic chip according to an embodiment of the present invention is shown.

[0040] Figure 8A schematic diagram illustrating the shear force of the culture medium on a matrix gel containing organoids during the use of a microfluidic chip according to an embodiment of the present invention is shown.

[0041] Figure 9 Another schematic diagram illustrates the shear force of the culture medium on a matrix gel containing organoids during the use of a microfluidic chip according to an embodiment of the present invention.

[0042] Figure 10 A flowchart illustrating a method for fabricating a microfluidic chip according to an embodiment of the present invention is shown.

[0043] List of main reference numerals

[0044] 1. First layer;

[0045] 2. Second layer; 201. First opening; 202. Concentration gradient infusion channel; 203. Storage tank; 204. Second opening; 205. Third opening;

[0046] 3. Third layer; 301. Culture pool;

[0047] 4. Fourth layer; 401. Liquid inlet; 402. Flow channel; 403. Liquid outlet. Detailed Implementation

[0048] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions of this application will be clearly and completely described below in conjunction with specific embodiments and corresponding drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of them. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0049] In this document, “illustrative” means “serving as an example, illustration or description”, and any illustration or implementation described herein as “illustrative” should not be construed as a more preferred or advantageous technical solution.

[0050] To keep the drawings concise, only the parts relevant to this application are shown schematically in each drawing, and they do not represent the actual structure of the product. In addition, to make the drawings concise and easy to understand, in some drawings, only one of the components with the same structure or function is shown schematically, or only one of them is labeled.

[0051] In this document, unless otherwise expressly specified and limited, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance; unless otherwise specified or explained, the term "multiple" refers to two or more; the terms "connected," "fixed," etc., should be interpreted broadly. For example, "connected" can be a fixed connection, a detachable connection, an integral connection, or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium. Those skilled in the art can understand the specific meaning of the above terms in this application according to the specific circumstances.

[0052] Example: Please refer to Figures 1 to 10 :

[0053] This invention proposes a microfluidic chip for culturing oral squamous cell carcinoma organoids, comprising:

[0054] The fourth layer 4 has an inlet 401 and an outlet 403, and an S-shaped flow channel 402 exists between the inlet 401 and the outlet 403; see reference. Figure 3 and Figure 7 In the presence of organoids, hereinafter referred to as matrix colloids (in Figure 7 The substance (labeled as Matrigel+PDO) is injected into the flow channel 402 through the inlet 401, fills the flow channel 402, and finally flows out from the outlet 403. It should be noted that in this application, the first layer 1 is called the sealing layer, the second layer 2 is called the culture medium / drug pool layer, the third layer 3 is called the Matrigel containing organoid culture pool layer, and the fourth layer 4 is the Matrigel inflow layer. For the sake of simplicity, they are all described as the first layer 1, the second layer 2, the third layer 3, and the fourth layer 4.

[0055] The third layer (3) contains multiple culture tanks (301) arranged in a matrix. Each culture tank (301) is located at the upper end of and connected to the flow channel (402). Each culture tank (301) is a main incubation chamber. During the injection of the matrix gel, the matrix gel is drawn back into the culture tank (301) as it passes through it. (See reference...) Figure 4 , Figure 5 and Figure 7The culture tank 301 has a columnar structure. After the matrix gel enters the culture tank 301, the matrix gel forms a liquid column under the restriction of the culture tank 301, so that the organoids are fully encapsulated in the matrix gel. Due to the influence of the matrix gel's own gravity, the matrix gel flowing in the flow channel 402 has little impact on the matrix gel in the culture tank 301, preventing displacement or leakage of the matrix gel during the flow process, and reducing the risk of blockage of the flow channel 402. After the matrix gel solidifies, it forms a columnar structure containing organoids. At the same time, due to the tension of the matrix gel itself, an arc-shaped protrusion will form at the top of the column as a contact surface to contact the culture medium or drug solution in the storage tank 203.

[0056] Above the third layer 3 is a second layer 2; the second layer 2 has a storage tank 203 corresponding to the culture tank 301, and the storage tank 203 is connected to the culture tank 301; multiple horizontal storage tanks 203 are interconnected; multiple horizontal storage tanks 203 are set as a group, and multiple groups of horizontal storage tanks 203 converge on one side and are connected to the second opening 204 through a channel, and multiple groups of horizontal storage tanks 203 are respectively connected to the concentration gradient inlet channel 202 on the other side; the concentration gradient inlet channel 202 has a first opening 201 and a second opening 204 at the end away from the storage tank 203; the second opening 204 is the culture medium inlet, and the culture medium is injected through the second opening 204, and the culture medium will flow into the storage tank 203, which is the culture tank 301. The matrix gel containing organoids provides nutrients to meet the long-term culture requirements of organoids. The first opening 201 and the third opening 205 can be used as the inlet of the drug solution or as the outlet of the culture medium, and the specific choice is determined by the specific experimental procedure. It should be noted that the flow direction of different fluids in the second layer 2 is as follows: Case 1: Culture medium is dripped into one end of the second opening 204, enters multiple reservoirs 203 through the slow flow channel, and flows out from the first opening 201 or the third opening 205 after passing through the concentration gradient infusion channel 202; Case 2: Drug solution is dripped into one end of the first opening 201 or the third opening 205, enters the reservoir 203 after passing through the concentration gradient infusion channel 202 to achieve different drug concentration gradients.

[0057] The first layer, 1, covers the top of the second layer, 2; see reference. Figure 1 , Figure 2 and Figure 6 The first layer 1 can be a glass substrate or another layer of PDMS covering the upper end of the second layer 2, sealing the channels of the microfluidic chip proposed in this application, and ensuring the operation of fluid in the channels. It should be noted that the first layer 1 can only cover all the channels and the liquid reservoir 203 of the second layer 2, or it can completely cover the upper end of the second layer 2, and standard liquid injection holes are opened at the first opening 201, the second opening 204 and the third opening 205 of the second layer 2.

[0058] The diameter of the storage tank 203 is larger than the diameter of the culture tank 301; see reference. Figure 5 and Figure 7 As mentioned above, when the diameter of the storage tank 203 is larger than the diameter of the culture tank 301, after the level of the matrix gel drawn back into the culture tank 301 rises above the height of the culture tank 301, an arc-shaped bulge will form under the action of the matrix gel's own tension. This arc-shaped bulge increases the contact area between the matrix gel and the culture medium or drug solution, effectively increasing the nutrient supply to the organoids within the matrix gel; simultaneously, refer to... Figure 8 and Figure 9 (The flow direction of the culture medium or drug solution is marked as follows) Figure 8 The direction of shear force between the culture medium or drug solution and the matrix gel is marked as Stream line. Figure 8 The shear force in the culture medium or drug solution and the matrix gel can be effectively reduced by using the structure of culture tank 301 and reservoir 203 in this application, thus ensuring the stability of matrix gel and internal organoids. It should be noted that the diameter of the flow channel 402 is much smaller than the diameter of the culture tank 301. This makes the rise of the matrix gel level more slow when the culture tank 301 backflows matrix gel, thereby ensuring that the amount of matrix gel entering the culture tank 301 is more controllable.

[0059] The concentration gradient infusion channel 202 consists of multiple sets of parallel channels with S-shaped bends, and the number of parallel channels increases as they approach the storage tank 203; see reference. Figure 2 and Figure 4 Each group of horizontal storage tanks 203 is connected to a separate parallel channel, such as Figure 4 As shown, the number of longitudinal parallel channels gradually decreases as the parallel channels move further away from the reservoir 203. In this embodiment, four parallel channels are directly connected to the four reservoirs 203. After these four parallel channels converge, they are connected to one end of three parallel channels. The other ends of these three parallel channels are connected to the first opening 201 and the third opening 205. It should be noted that in the actual preparation process, the number of parallel channels can be increased or decreased according to the specific concentration gradient requirements. Thus, by using multiple parallel channels, the flow rate of the liquid and the composition of the drug in the parallel channels can be flexibly adjusted to establish a stable drug concentration gradient laterally in the organoid region, which can meet the requirements of high-throughput screening experiments under multiple components and conditions.

[0060] In this design, a slow-flow channel exists between the second opening 204 and multiple sets of transverse liquid storage tanks 203. The number of slow-flow channels increases exponentially with distance from the second opening 204, until each outlet of the slow-flow channel is connected to a liquid storage tank 203. (See reference...) Figure 2 and Figure 5Through the slow-flow channel and multiple liquid storage tanks 203, it supports the continuous supply and dynamic replacement of culture medium, meeting the needs of long-term culture of organoids.

[0061] Among them, the first opening 201 or the third opening 205 is either the drug inlet or the culture medium outlet, and the second opening 204 is the culture medium inlet.

[0062] The first layer 1 completely covers the upper end of the second layer 2, and the first layer 1 has a standard injection hole; the standard injection hole of the first layer 1 corresponds to the first opening 201, the second opening 204 and the third opening 205.

[0063] The second layer 2 and the third layer 3 are integrally formed; the second layer 2 is connected to the first layer 1, and the third layer 3 is connected to the fourth layer 4 by oxygen plasma welding; see reference. Figure 1 Considering the difficulty of product preparation, in this application, the second layer 2 and the third layer 3 of the four-layer composite structure can be prepared together by integral molding, while the first layer 1 and the fourth layer 4 need to be prepared separately. At the same time, the first layer 1 and the fourth layer 4 are connected to the second layer 2 and the third layer 3 by oxygen plasma welding to ensure the sealing of the channel.

[0064] A microfluidic chip kit for culturing oral squamous cell carcinoma organoids includes the aforementioned microfluidic chip, as well as a warming device, an injection pump, an inverted microscope, a vacuum degassing device, a chip fixation platform, and a pipette. The warming device includes a constant-temperature incubator and a temperature-controlled water bath. The constant-temperature incubator maintains the temperature and humidity environment required for cell growth, the injection pump controls the injection rate of matrix gel and drug solution, and the inverted microscope allows for real-time observation of the organoid's growth status. The vacuum degassing device removes air bubbles during PDMS or matrix gel preparation, and the temperature-controlled water bath maintains the matrix gel in a liquid state, ensuring smooth injection. The chip fixture or fixation platform helps maintain chip stability during micromanipulation and fluid handling.

[0065] For reference Figure 7 The specific usage method of the above-mentioned microfluidic chip is as follows:

[0066] The first step is to inject the organoid-containing matrix gel into the inlet 401 of the fourth layer 4 until the flow channel 402 is filled, and then backflow into the culture tank 301 of the third layer 3, and allow the matrix gel to solidify.

[0067] The second step involves injecting culture medium through the second opening 204 of the second layer 2, which then enters the storage tank 203 to provide nutrients for the organoids.

[0068] The third step is to deliver drugs or nutrient solutions through the first opening 201 or the third opening 205 of the second layer 2, and to adjust the flow rate through the concentration gradient infusion channel 202 to deliver drugs or nutrient solutions of different concentrations.

[0069] Through the above steps, it is ensured that organoids can grow in a controlled environment and can be used for drug screening and cancer treatment research.

[0070] For reference Figure 10 The specific fabrication method of the above-mentioned microfluidic chip is as follows:

[0071] S1, Construct a four-layer chip model, including layer 1, layer 2, layer 3, and layer 4 arranged sequentially from top to bottom; In this step, the chip model is constructed with the support of 3D modeling software. Layer 2 and layer 3 are modeled as a whole, while layer 1 and layer 4 are modeled separately, so that the model can be exploded from top to bottom in the order of layer 1, layer 2 / layer 3, and layer 4;

[0072] S2, the second layer 2, the third layer 3, and the fourth layer 4 are molded to form a mold for the microfluidic chip channel; in this step, based on the data of the model in the 3D modeling software, the transparent acrylic material is processed into a chip mold using laser cutting or CNC machining. It should be noted that some 3D modeling software supports in-software molding and supports 3D printing technology to directly prepare the mold.

[0073] S3, mix PDMS and curing agent, pour the mixed solution into each mold, and remove it after curing; in this step, the mixed PDMS and curing agent must be vacuum treated to remove air bubbles and ensure the surface of the channel is smooth; it should be noted that PDMS refers to polydimethylsiloxane;

[0074] S4. Use oxygen plasma to treat the surface of each finished product. Before this step, the surface of the finished product needs to be trimmed to remove burrs and other irregular structures. At the same time, the surface of the finished product after oxygen plasma treatment is hydrophilic, which can enhance the adhesion between the surface of the finished product and other materials, such as matrix adhesives and culture solutions.

[0075] S5. Using oxygen plasma welding technology, the first layer 1 and the fourth layer 4 are welded to the upper end of the second layer 2 and the lower end of the third layer 3, respectively, to form a complete microfluidic chip. In this step, the first layer 1 can be a glass substrate or another layer of PDMS to ensure the sealing and stability of the microfluidic system.

[0076] The above description is merely a specific embodiment of this application. Under the guidance of the above teachings, those skilled in the art can make other improvements or modifications based on the above embodiments. Those skilled in the art should understand that the above specific description is only to better explain the purpose of this application, and the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A microfluidic chip for culturing oral squamous cell carcinoma organoids, characterized in that, include: The fourth layer (4) is provided with an inlet (401) and an outlet (403), and there is an S-shaped flow channel (402) between the inlet (401) and the outlet (403). The third layer (3) is provided with multiple culture pools (301) arranged in a matrix; all of the culture pools (301) are located at the upper end of the flow channel (402) and are connected to the flow channel (402); Above the third layer (3) is a second layer (2); the second layer (2) is provided with a storage tank (203) corresponding to the culture tank (301), and the storage tank (203) is connected to the culture tank (301); the multiple storage tanks (203) in the horizontal direction are interconnected; the multiple storage tanks (203) in the horizontal direction are set as a group, and the multiple groups of storage tanks (203) in the horizontal direction converge on one side and are connected to the second opening (204) through a channel, and the multiple groups of storage tanks (203) in the horizontal direction are respectively connected to the concentration gradient infusion channel (202) on the other side; the concentration gradient infusion channel (202) has a first opening (201) and a second opening (204) at the end away from the storage tank (203); The first layer (1) covers the top of the second layer (2); The diameter of the storage tank (203) is larger than the diameter of the culture tank (301); The concentration gradient infusion channel (202) is composed of multiple sets of parallel channels with S-shaped bends, and the number of parallel channels increases as they approach the storage tank (203); There is a slow-flow channel between the second opening (204) and multiple sets of transverse liquid storage tanks (203). The number of the slow-flow channels increases exponentially with distance from the second opening (204) until each outlet of the slow-flow channel is connected to the liquid storage tank (203).

2. The microfluidic chip for culturing oral squamous cell carcinoma organoids according to claim 1, characterized in that, The first opening (201) or the third opening (205) is either a drug inlet or a culture medium outlet, and the second opening (204) is a culture medium inlet.

3. The microfluidic chip for culturing oral squamous cell carcinoma organoids according to claim 1, characterized in that, The first layer (1) completely covers the upper end of the second layer (2), and the first layer (1) has a standard injection hole; the standard injection hole of the first layer (1) corresponds to the first opening (201), the second opening (204) and the third opening (205).

4. The microfluidic chip for culturing oral squamous cell carcinoma organoids according to claim 1, characterized in that, The second layer (2) and the third layer (3) are integrally formed; the second layer (2) and the first layer (1), and the third layer (3) and the fourth layer (4) are connected by oxygen plasma welding.

5. A microfluidic chip kit for culturing oral squamous cell carcinoma organoids, comprising the microfluidic chip according to any one of claims 1-4, characterized in that, It also includes a heat preservation device, an injection pump, an inverted microscope, a vacuum degassing device, a chip fixation platform, and a pipette; the heat preservation device includes a constant temperature incubator and a temperature-controlled water bath.

6. The method of using the microfluidic chip according to any one of claims 1-4, characterized in that, The specific steps are as follows: The first step is to inject the organoid-containing matrix gel into the inlet (401) of the fourth layer (4) until the flow channel (402) is filled, and then backflow into the culture tank (301) of the third layer (3) to allow the matrix gel to solidify. The second step is to inject culture medium through the second opening (204) of the second layer (2), and the culture medium enters the reservoir (203) to provide nutrition for the organoids; The third step is to deliver drugs or nutrient solutions through the first opening (201) or the third opening (205) of the second layer (2), and to adjust the flow rate through the concentration gradient infusion channel (202) to deliver drugs or nutrient solutions of different concentrations.

7. The method for fabricating a microfluidic chip according to any one of claims 1-4, characterized in that, Specifically as follows: S1, construct a four-layer chip model, including the first layer (1), the second layer (2), the third layer (3) and the fourth layer (4) arranged in the top and bottom directions. S2, the second layer (2), the third layer (3), and the fourth layer (4) are molded and made into a mold for the microfluidic chip channel; S3, mix PDMS and curing agent, pour the mixed solution into each mold, and remove the mold after curing; S4, using oxygen plasma to treat the surface of each finished product; S5, using oxygen plasma welding technology, the first layer (1) and the fourth layer (4) are welded to the upper end of the second layer (2) and the lower end of the third layer (3) respectively to form a complete microfluidic chip.

Images