Micro-fluidic chip capable of culturing oral squamous cell carcinoma organoid and preparation method of micro-fluidic chip

Abstract

The invention provides a micro-fluidic chip capable of culturing oral squamous cell carcinoma organoid and a preparation method thereof, and relates to the technical field of micro-fluidic chips, 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 injection channel and a slow flow channel), the third layer is a matrix culture pool layer, and the fourth layer is a matrix culture pool layer. The fourth layer is a matrigel inflow layer (containing an S-shaped flow channel); the second layer and the third layer are integrally formed, and all the layers are connected through oxygen plasma welding; the problems that in the prior art, matrigel is prone to shifting, drug gradient regulation and control is poor, operation is tedious, and adaptability is insufficient are solved, the matrigel and organoids can be stably fixed, the drug concentration gradient can be precisely regulated and controlled, the nutrition supply efficiency is enhanced, the structural integration level and operation convenience are improved, and the application prospect is wide. The method is suitable for oral squamous cell carcinoma organoid culture, drug screening and tumor mechanism research.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microfluidic chips, in particular to a microfluidic chip capable of culturing oral squamous carcinoma organoids and a preparation method thereof. BACKGROUND

[0002] In the basic research and clinical transformation of oral squamous carcinoma, organoid models have become a core tool for exploring tumor occurrence mechanisms, drug resistance and individualized treatment because they can accurately simulate the three-dimensional structure of in vivo tumors, cell heterogeneity and microenvironment characteristics. However, the existing organoid culture technology still has many bottlenecks and cannot meet the needs of accurate research:

[0003] The traditional well plate culture system has significant defects: first, the uniformity of the culture environment is poor, and the uneven distribution of fluid in the wells leads to large differences in organoid growth conditions and low experimental repeatability; second, it cannot simulate the in vivo drug concentration gradient, making it difficult to achieve accurate drug efficacy evaluation; third, the fixing effect of the matrix glue and the organoids is poor, and displacement is easily caused by liquid replacement, and the consumption of reagents is huge, causing research costs to rise.

[0004] The existing microfluidic chip technology also has obvious deficiencies: first, the matrix glue has poor adaptability, and most chips use open or suspended design, which easily causes the matrix glue to rupture, displace or block the channel, leading to failure of organoid culture; second, the concentration gradient regulation ability is weak, and drug delivery is mostly in 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 evaluation; third, the structure integration is low, and the functions of culture, drug administration and observation are scattered, which is cumbersome to operate and easy to introduce pollution; fourth, there is no special design for oral squamous carcinoma organoids, and the existing chips are mostly suitable for general cells or other tumor types, which are difficult to match the growth characteristics of oral squamous carcinoma organoids (such as high invasiveness and strong matrix dependence), resulting in insufficient model stability and physiological relevance.

[0005] Therefore, it is a key problem to be solved in the field to develop a high-integration oral squamous carcinoma organoid-specific microfluidic chip that has stable matrix glue fixation and precise concentration gradient regulation. SUMMARY

[0006] To achieve the above-mentioned purpose, the present application provides a microfluidic chip capable of culturing oral squamous carcinoma organoids, which adopts a four-layer vertical integrated structure, and each layer forms a functional system in fluid communication through a microfluidic channel, for realizing the culture of organoids under the premise of ensuring the stable fixation of matrix glue and the precise regulation of concentration gradient, specifically comprising:

[0007] The fourth layer is provided with a liquid inlet and a liquid outlet, and a flow channel with an S-shaped bend is arranged between the liquid inlet and the liquid outlet; the third layer is provided with a plurality of culture pools arranged in a matrix; the plurality of culture pools are all located at the upper end of the flow channel and are in communication with the flow channel; the second layer is arranged above the third layer; the second layer is provided with liquid storage pools corresponding to the culture pools, and the liquid storage pools are in communication with the culture pools; the plurality of liquid storage pools in the lateral direction are in communication with each other; the plurality of liquid storage pools in the lateral direction are arranged as a group, the plurality of groups of liquid storage pools in the lateral direction converge at one side and are in communication with the second opening through a channel, and the plurality of groups of liquid storage pools in the lateral direction are in communication with the concentration gradient filling channels at the other side; the concentration gradient filling channels are provided with a first opening and a second opening at the end away from the liquid storage pools. The first layer is arranged at the upper end of the second layer.

[0008] Preferably, the diameter of the liquid storage pool is greater than the diameter of the culture pool.

[0009] Preferably, the concentration gradient filling channel is composed of a plurality of groups of S-shaped parallel channels, and the number of parallel channels increases as it approaches the liquid storage pool.

[0010] Preferably, a slow flow channel exists between the second opening and the plurality of groups of liquid storage pools in the lateral direction, the number of slow flow channels increases exponentially away from the second opening, and each outlet of the slow flow channel is in communication with the liquid storage pool.

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

[0012] Preferably, the first layer completely covers the upper end of the second layer, and the first layer has a standard liquid injection hole; the standard liquid 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 with the first layer and the third layer by oxygen plasma welding.

[0014] Based on the above-mentioned microfluidic chip, the application further discloses a microfluidic chip kit capable of culturing oral squamous carcinoma organoids, which comprises the above-mentioned microfluidic chip, and further comprises a heat preservation device, a syringe pump, an inverted microscope, a vacuum defoaming device, a chip fixing platform and a pipettor; the heat preservation device comprises a constant temperature incubator and a temperature control water bath.

[0015] Based on the above-mentioned microfluidic chip, the application further discloses a use method of the microfluidic chip capable of culturing oral squamous carcinoma organoids, which is as follows:

[0016] First step, inject the Matrigel containing organoids into the liquid inlet through the fourth layer until the flow channel is filled, and backflow into the culture pool in the third layer, and make the Matrigel solidify;

[0017] Second step, inject the culture solution through the second opening of the second layer, and the culture solution enters the storage pool to provide nutrition for the organoids;

[0018] Third step, realize the delivery of drugs or nutrient solution through the first opening or the third opening of the second layer, and realize the delivery of drugs or nutrient solution with different concentrations by adjusting the flow rate and filling the channel by concentration gradient.

[0019] Based on the above-mentioned microfluidic chip, the application further discloses a preparation method of a microfluidic chip capable of culturing oral squamous cell carcinoma organoids, and specifically as follows:

[0020] S1, a chip model with a four-layer structure is constructed, including a first layer, a second layer, a third layer and a fourth layer arranged in the up-down direction in turn;

[0021] S2, the second layer and the third layer, and the fourth layer are flipped and made into a mold of a microfluidic chip channel;

[0022] S3, mix PDMS with a curing agent, pour the mixed solution into each mold, and take out after solidification;

[0023] S4, the surface of each finished product is treated by oxygen plasma;

[0024] S5, using oxygen plasma welding technology, the first layer and the fourth layer are welded at the upper end of the second layer and the lower end of the third layer respectively to form a complete microfluidic chip.

[0025] Advantages

[0026] 1. In the present application, the S-shaped flow channel of the fourth layer and the matrix type culture pool of the third layer form a "backflow-limiting" cooperative structure, which can accurately control the filling form of the Matrigel in the culture pool. Combined with the diameter difference design of the culture pool and the storage pool, the Matrigel forms a stable columnar structure after solidification, effectively avoiding displacement or leakage caused by fluid flushing. At the same time, this structure significantly reduces the shear force between the Matrigel and the culture solution / drug, protects the three-dimensional structural integrity of the organoids, and solves the problems of easy blockage of Matrigel and easy damage of organoids in the existing chip;

[0027] 2. In the present application, the concentration gradient filling channel of the second layer adopts "S-shaped bending + parallel channel increment" design, which can form a continuous and stable drug concentration gradient by adjusting the flow rate of different channels, and accurately simulate the drug diffusion microenvironment of tumor tissue in vivo;

[0028] 3. In the present application, the four-layer integrated structure (first layer closed, second layer drug delivery / reservoir, third layer culture, fourth layer Matrigel injection) realizes the high integration of the "Matrigel fixation-culture-drug delivery-observation" function, and cooperates with the standard liquid injection hole design of the first layer to avoid the pollution risk caused by frequent opening of the cover. The integrated molding of the second layer and the third layer and the oxygen plasma welding technology ensure the high airtightness of the chip, further improving the system stability;

[0029] 4. According to the high matrix dependence and growth characteristics of oral squamous carcinoma organoids, the chip can stably simulate the three-dimensional microenvironment of tumors, and provide an ideal model for the occurrence mechanism, metastasis path and targeted therapy research of oral squamous carcinoma. At the same time, the matrix culture pool supports multi-omics experiments (such as gene sequencing, proteomics analysis), combined with the supporting temperature control and observation equipment, can be expanded to the field of automated high-throughput screening, and has wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0030] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings of the embodiments will be briefly introduced below.

[0031] The drawings in the following description only relate to some embodiments of the present application, and are not a limitation of the present application.

[0032] In the drawings:

[0033] Figure 1 A perspective structure perspective schematic diagram of a microfluidic chip according to an embodiment of the present application is shown.

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

[0035] Figure 3 A perspective structure schematic diagram of the fourth layer of a microfluidic chip according to an embodiment of the present application is shown.

[0036] Figure 4 A cross-sectional view of the second layer and the third layer of a microfluidic chip according to an embodiment of the present application is shown.

[0037] Figure 5 A sectional view of the second layer and the third layer of a microfluidic chip according to an embodiment of the present application is shown.

[0038] Figure 6 A perspective structure schematic diagram of the first layer of a microfluidic chip according to an embodiment of the present application is shown.

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

[0040] Figure 8A schematic diagram showing the shear force of culture solution on Matrigel containing organoids during the use of a microfluidic chip according to an embodiment of the present application.

[0041] Figure 9 Another schematic diagram showing the shear force of culture solution on Matrigel containing organoids during the use of a microfluidic chip according to an embodiment of the present application.

[0042] Figure 10 A flow chart showing a method of manufacturing a microfluidic chip according to an embodiment of the present application.

[0043] List of main reference signs

[0044] 1, first layer;

[0045] 2, second layer; 201, first opening; 202, concentration gradient filling channel; 203, liquid storage pool; 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 DESCRIPTION

[0048] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions of the present application will be described below in connection with specific embodiments of the present application and corresponding drawings. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of protection of the present application.

[0049] In this document, "schematic" means "serves as an example, instance or illustration", and any diagram, embodiment described as "schematic" in this document should not be interpreted as a more preferred or more advantageous technical solution.

[0050] In order to make the drawings simple and clear, only the parts related to the present application are schematically shown in the drawings, which do not represent the actual structure of the product. In addition, in order to make the drawings simple and easy to understand, in some drawings, only one of the parts with the same structure or function is schematically shown, or only one of them is marked.

[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 7, the culture pool 301 is in a columnar structure, and after the matrix glue enters the culture pool 301, the matrix glue forms a liquid column under the restriction of the culture pool 301, so that the organoids are fully wrapped in the matrix glue, and due to the influence of the self-gravity of the matrix glue, the flowing matrix glue in the flow channel 402 has little influence on the matrix glue in the culture pool 301, preventing displacement or leakage of the matrix glue during flow, while reducing the risk of flow channel 402 blockage; after the matrix glue solidifies, a columnar structure containing organoids is formed, and due to the self-tension of the matrix glue, an arc-shaped protrusion is formed at the top of the columnar structure as a contact surface, which contacts the culture solution or drug solution in the liquid storage pool 203;

[0056] The third layer 3 is provided above the second layer 2; the second layer 2 is provided with a liquid storage pool 203 corresponding to the culture pool 301, and the liquid storage pool 203 is in communication with the culture pool 301; the plurality of transverse liquid storage pools 203 are in communication with each other; the plurality of transverse liquid storage pools 203 are arranged as a group, and the plurality of groups of transverse liquid storage pools 203 converge on one side and are connected to the second opening 204 through a channel, and the plurality of groups of transverse liquid storage pools 203 are connected to the concentration gradient filling channel 202 on the other side respectively; the concentration gradient filling channel 202 is provided with a first opening 201 and a second opening 204 at an end away from the liquid storage pool 203; the second opening 204 is an inlet for the culture solution, and the culture solution is injected through the second opening 204 and flows into the liquid storage pool 203 to provide nutrient components for the matrix glue containing organoids in the culture pool 301, meeting the long-term culture requirements of the organoids, and the first opening 201 and the third opening 205 can be selected as the inlet for the drug solution or as the outlet for the culture solution, and the specific selection is determined by the specific process of the experiment; it should be noted that the flow directions of different fluids in the second layer 2 are as follows: case one, the culture solution is dropped into one end of the second opening 204, enters the plurality of liquid storage pools 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 filling channel 202; case two, the drug solution is dropped into one end of the first opening 201 or the third opening 205, and after passing through the concentration gradient filling channel 202 to achieve different drug concentration gradients, enters the liquid storage pool 203.

[0057] The first layer 1 covers the upper end of the second layer 2; for 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 to seal the channels of the microfluidic chip proposed in the present application and ensure the operation of the fluid in the channels; it should be noted that the first layer 1 can cover only all the channels and liquid storage pools 203 of the second layer 2, or can completely cover the upper end of the second layer 2 and be provided with standard liquid injection holes corresponding to the first opening 201, the second opening 204 and the third opening 205 of the second layer 2.

[0058] Wherein, the diameter of the liquid pool 203 is greater than the diameter of the culture pool 301; please refer to Figure 5 and Figure 7 As described above, when the diameter of the liquid pool 203 is greater than the diameter of the culture pool 301, the level of the matrix glue sucked into the culture pool 301 will rise to exceed the height of the culture pool 301, and then an arc-shaped protrusion will be formed under the action of the self-tension of the matrix glue. This arc-shaped protrusion will increase the contact area between the matrix glue and the culture solution or the drug solution, effectively increasing the nutrient supply to the organoids in the matrix glue; at the same time, please refer to Figure 8 and Figure 9 The flow direction of the culture solution or the drug solution is marked as Figure 8 Stream line in the figure, and the shear force direction between the culture solution or the drug solution and the matrix glue is marked as Figure 8 Shear Force in the figure. After using the culture pool 301 and the liquid pool 203 structure in the present application, the shear force between the culture solution or the drug solution and the matrix glue can be effectively reduced, ensuring the stability of the matrix glue and the internal organoids; it should be noted that the pipe diameter of the flow channel 402 is much smaller than the pipe diameter of the culture pool 301, so that when the culture pool 301 sucks the matrix glue, the level of the matrix glue rises more slowly, thereby ensuring that the amount of the matrix glue entering the culture pool 301 is more controllable.

[0059] Wherein, the concentration gradient filling channel 202 is composed of multiple groups of S-shaped bending parallel channels, and the number of parallel channels increases as it approaches the liquid pool 203; please refer to Figure 2 and Figure 4 Each transverse liquid pool 203 is in communication with a separate parallel channel, as shown in Figure 4 The number of longitudinal parallel channels gradually decreases as the parallel channels move away from the liquid pool 203, as in the present embodiment, four parallel channels are directly connected to the four liquid pools 203, and after the four parallel channels converge, they are connected to one end of three parallel channels, and the other end of the three parallel channels is 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, thereby flexibly adjusting the flow rate of the liquid and the composition of the drug in the parallel channels, and realizing the establishment of a stable drug concentration gradient in the organoid region in the transverse direction, to meet the high-throughput screening experiments under multiple components and multiple conditions.

[0060] Wherein, there is a slow-flow channel between the second opening 204 and multiple transverse liquid pools 203, and the number of slow-flow channels increases exponentially away from the second opening 204 until each outlet of the slow-flow channel is in communication with the liquid pool 203; please refer to Figure 2 and Figure 5The sustained supply and dynamic replacement of the culture solution are supported through the slow-flow channel and the plurality of liquid storage pools 203, so as to meet the long-term culture requirement of the organoids.

[0061] The first opening 201 or the third opening 205 is the medicine solution inlet, and the other is the culture solution outlet, and the second opening 204 is the culture solution inlet.

[0062] The first layer 1 is completely overlaid on the upper end of the second layer 2, and the first layer 1 has a standard liquid injection hole; the standard liquid 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 is integrally formed with the third layer 3; the second layer 2 and the first layer 1, and the third layer 3 and the fourth layer 4 are connected through oxygen plasma welding; for reference Figure 1 Based on the difficulty of product preparation, the second layer 2 and the third layer 3 can be integrally formed together for preparation, and 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 through oxygen plasma welding, so as to ensure the sealing of the channel.

[0064] A microfluidic chip kit capable of culturing oral squamous cell carcinoma organoids comprises the above-mentioned microfluidic chip, and further comprises a heat preservation device, a syringe pump, an inverted microscope, a vacuum degassing device, a chip fixing platform and a pipette; the heat preservation device comprises a constant temperature incubator and a temperature-controlled water bath; wherein the constant temperature incubator is used to maintain the temperature and humidity environment required for cell growth, the syringe pump controls the injection rate of the matrigel and the drug solution, and the inverted microscope is used to observe the growth state of the organoids in real time. The vacuum degassing device is used for bubble removal during the preparation of PDMS or matrigel, and the temperature-controlled water bath can be used to keep the matrigel in a liquid state to ensure smooth injection. The chip clamp or fixing platform helps to maintain the stability of the chip during microscopic operation and fluid processing.

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

[0066] In the first step, the matrigel containing the organoids is injected into the liquid inlet 401 through the fourth layer 4, until the flow channel 402 is filled, and the matrigel is sucked into the culture pool 301 in the third layer 3, and the matrigel is solidified;

[0067] In the second step, the culture solution is injected through the second opening 204 of the second layer 2, and the culture solution enters the liquid storage pool 203 to provide nutrition for the organoids;

[0068] Thirdly, the drug or nutrient solution is delivered through the first opening 201 or the third opening 205 of the second layer 2, and is filled into the channel 202 through the concentration gradient, and the flow rate is adjusted to deliver the drug or nutrient solution with different concentrations.

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

[0070] For reference Figure 10 The preparation method of the microfluidic chip is as follows:

[0071] S1, a chip model with a four-layer structure is constructed, including a first layer 1, a second layer 2, a third layer 3 and a fourth layer 4 arranged in the upper-lower direction; in this step, the construction of the chip model relies on the support of a three-dimensional modeling software, and the second layer 2 and the third layer 3 are modeled as a whole, and the first layer 1 and the fourth layer 4 are modeled separately, so that the model as a whole can be exploded and unfolded from top to bottom in the order of the first layer 1, the second layer 2 / third layer 3 and the fourth layer 4;

[0072] S2, the second layer 2 and the third layer 3 and the fourth layer 4 are flipped and made into a mold of the microfluidic chip channel; in this step, according to the data of the model in the three-dimensional modeling software, a transparent acrylic material is processed into a mold of the chip by laser cutting or numerical control processing; it should be noted that some three-dimensional modeling software supports software-in-mold flipping and supports 3D printing technology to directly prepare the mold;

[0073] S3, mix PDMS with a curing agent, pour the mixed solution into each mold, and take out after curing; in this step, the mixed PDMS and curing agent must be treated by vacuum to remove bubbles to ensure the smoothness of the channel; it should be noted that PDMS refers to polydimethylsiloxane;

[0074] S4, the surface of each finished product is treated by oxygen plasma; before this step, the surface of the finished product needs to be trimmed to remove irregular structures such as burrs; at the same time, the surface of the finished product treated by oxygen plasma has hydrophilicity, which can enhance the adhesion between the surface of the finished product and other materials such as matrix glue and culture solution;

[0075] S5, using oxygen plasma welding technology, the first layer 1 and the fourth layer 4 are welded at 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 use a glass substrate or another layer of PDMS to ensure the sealing and stability of the microfluidic system.

[0076] The above merely provides a specific implementation of the present application, and on the basis of the above teaching, those skilled in the art can make other improvements or modifications on the basis of the above embodiments. Those skilled in the art should understand that the above specific description is only for better explaining the purpose of the present application, and the protection scope of the present application should be subject to the protection 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).

2. The microfluidic chip for culturing oral squamous cell carcinoma organoids according to claim 1, characterized in that, The diameter of the storage tank (203) is larger than the diameter of the culture tank (301).

3. The microfluidic chip for culturing oral squamous cell carcinoma organoids according to claim 1, characterized in that, 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).

4. The microfluidic chip for culturing oral squamous cell carcinoma organoids according to claim 1, characterized in that, 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).

5. A 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.

6. A 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).

7. A 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.

8. A microfluidic chip kit for culturing oral squamous cell carcinoma organoids, comprising the microfluidic chip according to any one of claims 1-7, 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.

9. The method of using the microfluidic chip according to any one of claims 1-7 is 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.

10. The method for fabricating a microfluidic chip according to any one of claims 1-7 is 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.

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