A microfluidic chip, a multi-organ interaction culture system and a culture method

Abstract

The application discloses a microfluidic chip, a multi-organ interaction culture system and a culture method, relates to the fields of biomedical engineering and cell biology technology, and comprises a first chip layer, a second chip layer and a diaphragm layer, at least two independent cavities for accommodating cultures are arranged in the first chip layer; a main channel, a first sample inlet channel and an outlet channel are arranged in the second chip layer, and the diaphragm layer is arranged between the first chip layer and the second chip layer and is used for allowing soluble signal molecules to diffuse and preventing cells from passing through; wherein the at least two independent cavities of the first chip layer are in fluid communication with the main channel of the second chip layer through the diaphragm layer, the soluble factors can be diffused and dynamically transmitted along the channel gradient by relying on the fluid perfusion of the main channel, and the organ units independently cultured can complete signal interaction and material transmission by relying on the common main channel.

Description

Technical Field

[0001] This application relates to the fields of biomedical engineering and cell biology, and in particular to a microfluidic chip, a multi-organ interaction culture system, and a culture method. Background Technology

[0002] Spinal cord injury (SCI) easily triggers neuro-immune-vascular axis disorders, inducing distal acute kidney injury (AKI), a highly lethal clinical complication. Current research relies on animal models, which have limitations such as large species differences, inability to monitor in real time, and difficulty in precisely controlling single variables. Traditional static co-culture systems lack fluid flow stimulation and cannot simulate the processes of blood flow, fluid shear forces, and the propagation of inflammatory factors in the body fluids. Furthermore, conventional multi-cell co-culture microfluidic chips can only achieve simple cell co-culture. In traditional microfluidic chips, the specific culture media corresponding to different organs are easily mixed, disrupting the exclusive culture microenvironment of each cell and organoid, resulting in reduced cell activity and abnormal physiological function. Signal transduction between multiple organs relies solely on static natural diffusion, which cannot meet the needs of cross-organ inflammatory transmission after spinal cord injury, pathological simulation of kidney injury, and drug efficacy evaluation. Summary of the Invention

[0003] To solve at least one of the above-mentioned technical problems, this application provides a microfluidic chip, a multi-organ interaction culture system, and a culture method, and the technical solutions adopted are as follows.

[0004] The microfluidic chip provided in this application includes: A first chip layer, wherein at least two mutually separated independent cavities are provided within the first chip layer, and the independent cavities are used to contain cultures; The second chip layer has a main channel, a first sample inlet channel and an outlet channel, and the first sample inlet channel and the outlet channel are respectively connected to the main channel; A membrane layer, sandwiched between the first chip layer and the second chip layer, is used to allow soluble signaling molecules to diffuse and prevent cells from passing through; In this configuration, at least two independent cavities of the first chip layer are in fluid communication with the main channel of the second chip layer through the diaphragm layer.

[0005] In some embodiments of this application, a second sample injection channel and a glue injection channel are provided in the second chip layer, and each of the independent cavities is connected to the corresponding second sample injection channel and the glue injection channel.

[0006] In some embodiments of this application, the diaphragm layer is provided with a through hole, and the second sample injection channel and the glue injection channel pass through the through hole and communicate with the independent cavity.

[0007] In some embodiments of this application, the second chip layer is configured with a removable connector for inserting into and blocking the main channel.

[0008] This application also provides a multi-organ interaction culture system, comprising: As described above, microfluidic chips; A spinal cord unit, wherein the spinal cord unit is disposed within at least one of the independent cavities of the first chip layer; A kidney unit, wherein the kidney unit is disposed within at least one other independent cavity of the first chip layer; A vascular unit is disposed on the inner wall of the main channel of the second chip layer and on the surface of the diaphragm layer.

[0009] In some embodiments of this application, the spinal cord unit includes decellularized spinal cord matrix, and the spinal cord unit further includes spinal cord neural stem cells or astrocyte complex; the kidney unit includes decellularized renal medullary matrix, and the kidney unit further includes kidney organoids.

[0010] This application also provides a cultivation method based on the microfluidic chip described above, comprising the following steps: Prepare the microfluidic chip; Temporarily block the main passage; Add the culture to each of the individual cavities; Inoculate each of the individual cavities with the target inoculation material; Release the blockage of the main channel; A vascular unit is constructed in the main channel; Add the target culture medium to each of the independent cavities and place the microfluidic chip in the target environment for incubation.

[0011] In some embodiments of this application, after adding the culture to each of the independent cavities, the mixture is kept at a constant temperature until it is completely solidified, and then the target inoculum is inoculated into each of the independent cavities.

[0012] In some embodiments of this application, the construction of the vascular unit in the main channel includes: seeding and culturing human umbilical vein endothelial cells into the main channel and the diaphragm layer from the first injection channel.

[0013] In some embodiments of this application, the target environment is a 37°C atmosphere with 5% CO2.

[0014] This application has at least the following beneficial effects: The first chip layer of this application is provided with at least two completely separated independent cavities, each cavity is independent and does not interfere with each other, and can be individually adapted to the specific culture media and culture conditions of different organs; the second chip layer integrates the main channel, the first sample inlet channel and the outlet channel to form a complete fluid perfusion path. At the same time, the diaphragm allows soluble cytokines and inflammatory factors to diffuse freely, while blocking cell migration across regions, realizing physical isolation and biochemical signal communication. It can realize the diffusion and dynamic transmission of soluble factors along the channel gradient by relying on the fluid perfusion of the main channel, so that independently cultured organ units can complete signal interaction and material transmission by relying on the common main channel.

[0015] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0016] The present application will be further illustrated below with reference to the accompanying drawings and embodiments. It should be noted that the embodiments illustrated in the following drawings are exemplary and are only used to explain the present application, and should not be construed as limiting the present application.

[0017] Figure 1 This is a schematic diagram of a microfluidic chip in one embodiment of this application; Figure 2 This is a perspective view of a microfluidic chip in one embodiment of this application; Figure 3 This is a schematic diagram of the first chip layer and the separator layer in one embodiment of this application; Figure 4 This is a schematic diagram of the first chip layer in one embodiment of this application.

[0018] Reference numerals: First chip layer 100; Independent cavity 110; Chamber 111; Second chip layer 200; main channel 210; first injection channel 211; outlet channel 212; second injection channel 220; dispensing channel 230; connecting channel 240; 300mm membrane layer. Detailed Implementation

[0019] The following is combined Figures 1 to 4 The embodiments of this application are described in detail below, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0020] In the description of this application, it should be understood that the terms "center", "middle", "longitudinal", "transverse", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "axial", "radial", "circumferential", etc., indicating the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this application.

[0021] In the description of this application, "several" means one or more, "multiple" means two or more, "greater than," "less than," and "exceeding" are understood to exclude the stated number, while "above," "below," and "within" are understood to include the stated number. The use of "first" and "second" in the description is merely for distinguishing technical features and should not be construed as indicating or implying relative importance, or implicitly indicating the number of indicated technical features, or implicitly indicating the order of the indicated technical features.

[0022] In the description of this application, unless otherwise expressly specified and limited, the terms "set up," "install," "connect," and "link" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this application based on the specific circumstances.

[0023] In the description of this application, the use of terms such as "one embodiment," "some embodiments," "an example," "some instances," "some embodiments," "illustrative embodiment," "example," "specific example," and "some examples" indicates that the specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0024] Combination Figure 1 , Figure 2 , Figure 3 , Figure 4 As shown, the microfluidic chip provided in this application includes: The first chip layer 100 has at least two mutually separated independent cavities 110, which are used to contain cultures. The second chip layer 200 has a main channel 210 and a first sample inlet channel 211 and an outlet channel 212, which are respectively connected to the main channel 210. A membrane layer 300 is sandwiched between the first chip layer 100 and the second chip layer 200. The membrane layer 300 is capable of being diffused by soluble signal molecules and also serves to prevent cells from passing through. In this process, at least two independent cavities 110 of the first chip layer 100 are fluidly connected to the main channel 210 of the second chip layer 200 through the diaphragm layer 300.

[0025] The microfluidic chip of this application adopts a three-layer stacked closed-layer chip structure, which consists of a second chip layer 200, a membrane layer 300, and a first chip layer 100 from top to bottom.

[0026] The first chip layer 100 has a flat plate structure, and the independent cavity 110 has a groove structure that is recessed inward from the surface of the first chip layer 100 facing the membrane layer 300. The first chip layer 100 is provided with at least two mutually separated independent cavities 110 for accommodating various cultures such as organoids, composite cells, decellularized matrix, and special culture media, providing independent and dedicated culture space for different organ cultures.

[0027] The second chip layer 200 is disposed corresponding to the first chip layer 100. The main channel 210 is a groove channel structure with an inwardly recessed surface of the second chip layer 200 facing the separator layer 300, and its opening side faces the separator layer 300. The main channel 210 serves as a common fluid transport and signal conduction path for the entire chip.

[0028] A membrane layer 300 is disposed between the first chip layer 100 and the second chip layer 200. The membrane layer 300 allows soluble signaling molecules to diffuse through and also prevents cells from passing through. It can be understood that the membrane layer 300 is a semi-permeable membrane structure with a fixed pore size. The pore size allows for the free diffusion of signaling substances such as soluble proteins, inflammatory factors, cytokines, and small nutrient molecules, while the pore size is smaller than the minimum particle size of conventional somatic cells, stem cells, and organoid units, thus blocking the migration of various cells across membranes and cavities. The pore size of the membrane layer and the activation treatment method of the pores can be adjusted to control the diffusion rate of cytokines.

[0029] The projection of the main channel 210 on the diaphragm layer 300 overlaps with the projection of each independent cavity 110 on the diaphragm layer 300, so that at least two independent cavities 110 of the first chip layer 100 are in fluid communication with the main channel 210 of the second chip layer 200 through the diaphragm layer 300.

[0030] Understandably, at least two completely separate independent cavities 110 can respectively correspond to the culture of different organ units such as spinal cord and kidney, while the main channel 210 supports the adhesion and growth of vascular endothelial cells on its inner wall and the surface of the diaphragm layer, forming a continuous endothelial barrier. At the same time, the diaphragm layer 300 is a semi-permeable membrane structure, which can allow soluble signaling molecules such as cytokines and chemokines to diffuse freely, while preventing endothelial cells or other cells in the cavity from passing through. This can simulate the process by which metabolic products, hormones or cytokines from multiple organs in the human body enter the blood circulation system and are transported to distant organs through the blood flow.

[0031] The first chip layer 100 of this application is provided with at least two completely separated independent cavities 110. Each cavity is independent and does not interfere with the others, and can be individually adapted to the specific culture media and culture conditions of different organs. The second chip layer 200 integrates a main channel, a first sample inlet channel and an outlet channel to form a complete fluid perfusion path. At the same time, the diaphragm allows soluble cytokines and inflammatory factors to diffuse freely, while blocking cell migration across regions, realizing physical isolation and biochemical signal exchange. It can realize the diffusion and dynamic transmission of soluble factors along the channel gradient by relying on the fluid perfusion of the main channel, so that independently cultured organ units can complete signal interaction and material transport by relying on the common main channel.

[0032] In some embodiments, the second chip layer 200 is provided with a second sample inlet channel 220 and a glue injection channel 230, and each independent cavity 110 is connected to its corresponding second sample inlet channel 220 and glue injection channel 230. For each independent cavity 110 of the first chip layer 100, the second chip layer 200 is equipped with an independently provided set of second sample inlet channels 220 and glue injection channels 230. It can be understood that the output ports of the second sample inlet channels 220 and glue injection channels 230 can be located on the side of the second chip layer 200 facing away from the diaphragm layer 300. The second sample inlet channel 220 is directly connected to the independent cavity 110, and the glue injection channel 230 is connected to the independent cavity 110 through a connecting channel 240. The gel injection channel 230 is mainly used to inject decellularized matrix, hydrogel and other solid matrix materials into the corresponding independent chamber 110. The second sample injection channel 220 is specifically used to seed cells and supplement the dedicated culture medium into the corresponding independent chamber 110. The second sample injection channel 220 and gel injection channel 230 of each chamber operate independently and do not interfere with each other.

[0033] It is understood that an independent cavity 110 has two chambers 111 within the first chip layer 100. The input end of the connecting channel 240 is connected to the second sample inlet channel 220, and the output end of the connecting channel 240 is connected to the two chambers 111 respectively. Specifically, the two chambers 111 are connected in the middle, and the projection of the main channel 210 on the first chip layer 100 intersects with the part connecting the two chambers 111. Furthermore, the projection of the main channel 210 on the first chip layer 100 is located at the midpoint of the part connecting the two chambers 111, enabling a single second sample inlet channel 220 to simultaneously perform synchronous feeding, inoculation, and liquid exchange operations on the two chambers 111 within the same independent cavity 110 through the connecting channel 240.

[0034] In some embodiments, the diaphragm layer 300 is provided with through holes, through which the second sample injection channel 220 and the dispensing channel 230 pass and communicate with the independent cavity 110. The diaphragm layer 300 has through holes corresponding to the projection positions of the second sample injection channel 220 and the dispensing channel 230 on the diaphragm layer 300, penetrating the second chip layer 200. The second sample injection channel 220 passes through the through holes and directly communicates with the independent cavity 110. The dispensing channel 230 passes through the corresponding through holes and communicates with the connecting channel 240, and also leads to the independent cavity 110. The through holes correspond only to dedicated operating channels and do not affect the overall isolation and permeability functions of the diaphragm layer 300. While ensuring independent operation of the cavity, the overall sealing structure and signal communication performance of the chip are maintained.

[0035] In some embodiments, the second chip layer 200 is equipped with a detachable connector (not shown in the figure), which is used to insert into and seal the main channel 210. A detachable connector is also configured at the outlet channel 212 of the second chip layer 200. The connector adopts a columnar sealing structure that matches the inner diameter of the main channel 210 and the outlet channel 212. The connector can be inserted from the outside of the outlet channel 212 to completely seal the end passage of the main channel 210, achieving temporary sealing of the main channel 210. Simultaneously, the connector can be manually removed at any time to release the seal on the main channel 210 and restore its open state. The disassembly and assembly operation is simple, and the sealing effect is stable. The detachable connector allows for flexible switching between temporary sealing and open operation of the main channel. During matrix injection and curing, it can block the flow of matrix fluid into the main channel 210, effectively preventing the hydrogel or decellularized matrix from clogging the main channel 210 after curing.

[0036] This application also provides a multi-organ interaction culture system, comprising: Such as the microfluidic chip mentioned above; A spinal cord unit, wherein the spinal cord unit is disposed within at least one independent cavity 110 of the first chip layer 100; The kidney unit is disposed within at least one other independent cavity 110 of the first chip layer 100; The vascular unit is disposed on the inner wall of the main channel 210 of the second chip layer 200 and on the surface of the diaphragm layer 300.

[0037] A spinal cord unit is placed in one of the independent cavities 110 of the first chip layer 100, forming a dedicated spinal cord culture microenvironment based on the independent cavity 110; a kidney unit is placed in any other independent cavity 110 of the first chip layer 100, physically separated from the spinal cord unit and cultured independently; vascular units are placed on the entire inner wall surface of the main channel 210 of the second chip layer 200 and the lower surface of the diaphragm layer 300 facing the main channel 210, so that the vascular units fit into the fluid area of ​​the main channel 210, forming a biomimetic vascular barrier structure.

[0038] The multi-organ interaction culture system of this application integrates three major functional units—spinal cord, blood vessels, and kidneys—into an integrated spinal cord-blood vessel-kidney three-unit structured co-culture system in a microfluidic chip. Relying on the chip's layered and partitioned structure, the three major units are arranged in a structured and modular manner. This system can replicate the anatomical distribution relationship of the human spinal cord-blood circulation-kidney system and can realistically simulate the cross-organ pathological process of inflammatory signals being transmitted through vascular pathways after spinal cord injury, inducing kidney damage. It provides a standardized in vitro pathological modeling and drug screening platform.

[0039] In some embodiments, the spinal cord unit includes decellularized spinal cord matrix (DSCM), and further includes spinal cord neural stem cells or an astrocyte complex; the kidney unit includes decellularized renal medullary matrix (dECM), and further includes kidney organoids. The spinal cord unit is composed of decellularized spinal cord matrix, spinal cord neural stem cells, and an astrocyte complex, with the cell complex embedded within the decellularized spinal cord matrix and implanted in a corresponding independent cavity 110, mimicking the native microenvironment of spinal cord tissue using a specific matrix. The kidney unit is composed of decellularized renal medullary matrix and kidney organoids, with kidney organoids possessing renal tubule and podocyte structures embedded within the decellularized renal medullary matrix and implanted in a corresponding independent cavity 110, restoring the physiological structure and functional characteristics of kidney tissue, suitable for long-term pathological modeling and drug evaluation experiments.

[0040] This application also provides a cultivation method based on the microfluidic chip described above, comprising the following steps: Prepare microfluidic chips; Temporarily block main passage 210; Add culture to each independent chamber 110; Inoculate each independent cavity 110 with the target inoculation material; Release the blockage of main passage 210; A vascular unit is constructed in the main channel 210; Add the target culture medium to each independent cavity 110 and place the microfluidic chip in the target environment for culture.

[0041] First, the microfluidic chip is pre-processed and prepared. Then, the main channel 210 of the second chip layer 200 is temporarily blocked using a connector to interrupt the fluid flow. Through the dedicated channels of each independent cavity 110, corresponding cultures, including organ-specific decellularized matrix and basic culture systems, are added to each independent cavity 100. After culture addition, the corresponding target inoculation material, including nerve cells and kidney organoids, is precisely inoculated into each independent cavity 110. After the inoculation of the independent cavities 110 is completed, the connector is removed, releasing the temporary blockage of the main channel 210 and allowing it to be fully open. The vascular unit is then constructed within the open main channel 210. Finally, target-specific culture media matching organ characteristics are added to each independent cavity 110, and the entire microfluidic chip is placed in a constant temperature and humidity cell culture environment for long-term culture. The culture method provided in this application can be adapted to the specific culture media and culture conditions of different organs, and the independently cultured organ units can complete signal interaction and material transmission by relying on the common main channel 210.

[0042] In some embodiments, after adding culture to each independent cavity 110, the mixture is kept at a constant temperature until it is completely solidified, and then the target inoculation material is inoculated into each independent cavity 110. After adding culture, such as decellularized matrix, hydrogel, or other solid matrix culture, to each independent cavity 110, the microfluidic chip is placed in a room temperature or constant temperature environment for a preset time until the liquid matrix in each independent cavity 110 is completely solidified. Then, the corresponding target cells, organoids, or other inoculation materials are inoculated into each independent cavity 110 to ensure that the matrix forms a stable structure and provides a stable attachment and growth carrier for cells and organoids.

[0043] It can adjust the concentration of decellularized matrix and the thickness of the gel layer, thereby regulating matrix permeability.

[0044] In some embodiments, constructing a vascular unit in the main channel 210 includes: seeding and culturing human umbilical vein endothelial cells into the main channel 210 and the diaphragm layer 300 through the first injection channel 211. After the main channel 210 is unblocked and fully connected, the prepared human umbilical vein endothelial cell suspension is injected into the main channel 210 through the first injection channel 211 of the second chip layer 200, while simultaneously wetting the exposed surface of the diaphragm layer 300, ensuring that the endothelial cells uniformly cover the inner wall of the main channel 210 and the surface of the diaphragm layer 300; subsequently, the cells are statically cultured under conventional cell culture conditions to allow the endothelial cells to adhere, proliferate, and fuse, ultimately forming a dense and complete vascular endothelial barrier structure, completing the construction of the vascular unit, conforming to the physiological structure of human vascular endothelium, forming a stable vascular barrier and fluid pathway, simulating the material permeability and signal transduction functions of human blood vessels, and improving the biomimetic realism of the multi-organ interaction model.

[0045] In some embodiments, the target environment is 37°C and a 5% CO2 atmosphere. The target environment for chip culture is strictly set to a constant temperature of 37°C and a 5% CO2 cell culture atmosphere. The microfluidic chip with culture medium added is placed in this culture environment. 37°C and 5% CO2 is the optimal standard culture environment for the growth of human cells and organoids, which can simulate the physiological temperature and gas environment in the human body, effectively maintain the proliferation, differentiation and normal physiological functions of various organ unit cells, and ensure the accuracy and stability of experimental data.

[0046] Understandably, in the spinal cord-vascular-kidney three-unit structured co-culture system, the spinal cord unit is formed by co-culturing human neural stem cells (HNSCs) and human spinal cord astrocytes (HSP-ASCs) in a specific ratio to create a neural network, which is then embedded in decellularized spinal cord matrix (DSCM) and implanted into the corresponding independent cavity 110 of the first chip layer 100. The vascular unit is constructed from human umbilical vein endothelial cells (HUVECs) to create a double-layer endothelial barrier structure, implanted on both sides of the diaphragm layer 300 and in the main channel region of the second chip layer 200, simulating the endothelial barrier and blood flow channels in vivo. The kidney unit uses kidney organoids obtained from stem cell differentiation, embedded in decellularized renal medullary matrix (dECM), and implanted into another independent cavity 110 of the first chip layer 100; the kidney organoids include proximal tubules, distal tubules, podocytes, and endothelial-like structures, possessing the basic physiological tissue characteristics of the kidney.

[0047] Understandably, inflammation modeling and dynamic perfusion can be performed in a three-unit structured co-culture system of spinal cord-vascular-kidney. Inflammatory factors are applied to the spinal cord unit, while a constant low-speed fluid perfusion is performed through the first inlet into the main channel, simulating the in vivo blood flow microenvironment and promoting the transmission of inflammatory signals from the vascular unit to the kidney unit. The concentration and duration of inflammatory factor stimulation can be altered to regulate the intensity of inflammatory signal release; the perfusion rate of the microfluidic channel can be optimized to control convective transport efficiency. Furthermore, kidney organoids at different differentiation stages can be selected to match the sensitivity of the inflammatory injury response.

[0048] Those skilled in the art can achieve controllable inflammatory stress, apoptosis, and damage marker expression responses in the renal unit by optimizing the above-mentioned conventional parameters, without the need for additional creative experiments.

[0049] The culture system provided in this application is a multi-organ interaction culture system, which can be used to study the cross-organ interaction mechanism of nerve-vascular-kidney after spinal cord injury, analyze the cross-tissue propagation pathway of inflammatory signals, screen anti-inflammatory candidate drugs and evaluate their efficacy and safety, evaluate the effects of cell therapy regimens such as mesenchymal stem cells, replace some animal models, and carry out in vitro pathophysiological simulation experiments.

[0050] The embodiments of this application have been described in detail above with reference to the accompanying drawings. However, this application is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of this application. Furthermore, unless otherwise specified, the embodiments and features described in the embodiments of this application can be combined with each other.

Claims

1. A microfluidic chip, characterized in that, include: A first chip layer, wherein at least two mutually separated independent cavities are provided within the first chip layer, and the independent cavities are used to contain cultures; The second chip layer has a main channel, a first sample inlet channel and an outlet channel, and the first sample inlet channel and the outlet channel are respectively connected to the main channel; A membrane layer, sandwiched between the first chip layer and the second chip layer, is used to allow soluble signaling molecules to diffuse and prevent cells from passing through; In this configuration, at least two independent cavities of the first chip layer are in fluid communication with the main channel of the second chip layer through the diaphragm layer.

2. The microfluidic chip according to claim 1, characterized in that: The second chip layer is provided with a second sample injection channel and a glue injection channel, and each independent cavity is connected to its corresponding second sample injection channel and glue injection channel.

3. The microfluidic chip according to claim 2, characterized in that: The diaphragm layer is provided with through holes, and the second sample injection channel and the glue injection channel pass through the through holes and are connected to the independent cavity.

4. The microfluidic chip according to claim 1, characterized in that: The second chip layer is configured with a removable connector for inserting into and blocking the main channel.

5. A multi-organ interaction culture system, characterized in that, include: The microfluidic chip as described in any one of claims 1 to 4; A spinal cord unit, wherein the spinal cord unit is disposed within at least one of the independent cavities of the first chip layer; A kidney unit, wherein the kidney unit is disposed within at least one other independent cavity of the first chip layer; A vascular unit is disposed on the inner wall of the main channel of the second chip layer and on the surface of the diaphragm layer.

6. The multi-organ interaction culture system according to claim 5, characterized in that: The spinal cord unit includes acellular spinal cord matrix, and the spinal cord unit also includes spinal cord neural stem cells or astrocyte complex; the kidney unit includes acellular renal medullary matrix, and the kidney unit also includes kidney organoids.

7. A cultivation method based on a microfluidic chip as described in any one of claims 1 to 4, characterized in that, Includes the following steps: Prepare the microfluidic chip; Temporarily block the main passage; Add the culture to each of the individual cavities; Inoculate each of the individual cavities with the target inoculation material; Release the blockage of the main channel; A vascular unit is constructed in the main channel; Add the target culture medium to each of the independent cavities and place the microfluidic chip in the target environment for incubation.

8. The cultivation method according to claim 7, characterized in that: After adding the culture to each of the independent cavities, the mixture is kept at a constant temperature until it is completely solidified, and then the target inoculum is inoculated into each of the independent cavities.

9. The cultivation method according to claim 7, characterized in that: The construction of the vascular unit in the main channel includes: seeding and culturing human umbilical vein endothelial cells into the main channel and the diaphragm layer from the first injection channel.

10. The cultivation method according to claim 7, characterized in that: The target environment is 37°C and 5% CO2 atmosphere.

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