A hypoxia injury model based on a bionic intestine-liver organ chip and a preparation method and application thereof

Abstract

The application discloses a hypoxia injury model based on a bionic intestine-liver organ chip and a preparation method and application thereof, and belongs to the technical field of biology. The model comprises a chip base, at least two culture chambers are formed at the top of the chip base, and a bottom liquid flow channel communicating the culture chambers is formed in the inside; an intestine chip module is detachably arranged in the corresponding culture chamber and comprises a first porous membrane for spacing the inner cavity of the intestine chip module and the bottom liquid flow channel; a liver chip module is detachably arranged in the corresponding culture chamber and comprises a second porous membrane for spacing the inner cavity of the liver chip module and the bottom liquid flow channel; and the model is obtained by performing hypoxia treatment on the chip. The application has important scientific value and application prospect for clarifying the mechanism of intestinal-liver axis disorder caused by high-altitude hypoxia, developing drugs for preventing and treating high-altitude diseases and protection strategies.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to a hypoxia injury model based on a biomimetic gut-liver organ chip, its preparation method, and its application. Background Technology

[0002] High-altitude environments are characterized by low air pressure and low oxygen levels. Under hypoxic stress, multiple organ systems can experience functional impairment. Among them, the intestines and liver, as core organs for nutrient metabolism, energy supply, and immune barrier function, are highly sensitive to hypoxia. Currently, there is a lack of in vitro research models that can simulate combined intestinal-liver injury to investigate the multi-organ interactions caused by high-altitude hypoxia, especially the regulatory role of the gut-liver axis in hypoxic injury. Summary of the Invention

[0003] In view of this, in order to at least partially solve at least one of the aforementioned technical problems, this application provides a hypoxia injury model based on a biomimetic gut-liver organ-on-a-chip, its preparation method, and its application.

[0004] According to one embodiment of this application, a hypoxia injury model based on a bionic gut-liver organ chip is provided. The bionic gut-liver organ chip includes:

[0005] The chip base has at least two culture chambers formed on the top, and a bottom liquid flow channel connecting the culture chambers is formed inside;

[0006] The gut microarray module, detachably mounted in the corresponding culture chamber, includes:

[0007] The first porous membrane is used to separate the lumen and bottom fluid flow channel of the intestinal chip module. The inner layer of the first porous membrane is seeded with intestinal epithelial cells, and the outer layer is seeded with vascular endothelial cells.

[0008] The liver chip module, detachably mounted in the corresponding culture chamber, includes:

[0009] The second porous membrane is used to separate the inner lumen and the bottom fluid flow channel of the liver chip module. The inner layer of the second porous membrane is seeded with liver parenchymal cells, and the outer layer is seeded with vascular endothelial cells.

[0010] The hypoxia injury model based on the bionic gut-liver organ chip is obtained by subjecting the bionic gut-liver organ chip to hypoxia in a hypoxic environment.

[0011] According to another embodiment of this application, a method for preparing a hypoxic injury model based on a bionic gut-liver organ chip is provided, comprising: culturing the bionic gut-liver organ chip in a normoxic environment and then transferring it to a hypoxic environment for further culturing, wherein the oxygen concentration of the hypoxic environment is 0.5% to 5%.

[0012] According to another embodiment of this application, an application of a hypoxia injury model based on a biomimetic gut-liver organ-on-a-chip in screening drugs for the prevention and treatment of altitude sickness is provided.

[0013] According to embodiments of this application, the hypoxia injury model provided is suitable for constructing gut-liver interaction models using various cell types. Compared with traditional mouse models, this model is closer to the physiological state of the human gut and liver in terms of cell composition and metabolic pathways, and can more realistically simulate the interaction between the human gut and liver. This model can not only simulate the effects of various extreme environments (such as hypoxia) on human gut and liver tissue, but also be used to establish various gut-liver related disease models, study the damage to gut and liver tissue under pathological conditions, and explore treatment options. Attached Figure Description

[0014] The above and other objects, features and advantages of this application will become clearer from the following description of embodiments with reference to the accompanying drawings, in which:

[0015] Figure 1 A schematic diagram of the biomimetic gut-liver organ-on-a-chip structure constructed in this application.

[0016] Figure 2 This is a schematic diagram of the hypoxia injury model based on the bionic gut-liver organ-on-a-chip proposed in this application.

[0017] Figure 3 This is a flowchart illustrating the construction process of the biomimetic gut-liver organ chip of this application.

[0018] Figure 4 The images show the physiological function characterization of the intestinal chip module after successful construction of the biomimetic intestinal-liver organ chip according to the embodiments of this application; wherein, A is the bright field image of the intestinal chip module and the immunofluorescence image of intestinal epithelial cells after 4 days of co-culture; B is the 3D reconstruction image of the intestinal chip module.

[0019] Figure 5 The images show the physiological function characterization of the liver chip module of the biomimetic gut-liver organ chip according to the embodiments of this application; where A is a bright field image of the gut chip module after 4 days of co-culture; and B is a confocal microscope image of HepG2 cells after 4 days of co-culture, wherein the staining is albumin (ALB) and cytochrome P450 enzyme (CYP3A4).

[0020] Figure 6 This is a functional characterization diagram of the intestinal chip module of the biomimetic intestinal-liver organ-chip model after 48 hours of hypoxia treatment according to an embodiment of this application; wherein, A is a representative image of intestinal cells under confocal fluorescence microscopy after 48 hours of hypoxia exposure; B is a statistical result of the mRNA expression level of intestinal functional genes after 48 hours of hypoxia exposure.

[0021] Figure 7This is a functional characterization diagram of the liver chip module of the biomimetic gut-liver organ-on-a-chip model after 48 hours of hypoxia treatment according to an embodiment of this application; wherein, A is a representative diagram of hepatocytes after 48 hours of hypoxia exposure; B is a statistical result diagram of the mRNA expression level of liver functional genes after 48 hours of hypoxia exposure.

[0022] Figure 8 This is a comparison of the functional gene expression results of the intestinal chip module, liver chip module, and biomimetic intestinal-liver organ chip cultured separately under normoxic conditions according to embodiments of this application.

[0023] Figure 9 This is a comparison chart showing the detection results of lactate dehydrogenase (LDH) content in the culture medium of the intestinal chip module, liver chip module, and biomimetic intestinal-liver organ chip model cultured separately under hypoxic conditions in this application embodiment.

[0024] Figure 10 This is a comparison of the transmembrane resistance detection results of an individually cultured intestinal chip module and a biomimetic intestinal-liver organ chip under normoxic and hypoxic conditions, as described in the embodiments of this application. Detailed Implementation

[0025] The embodiments of this application will now be described with reference to the accompanying drawings. However, it should be understood that these descriptions are exemplary only and are not intended to limit the scope of this application. In the following detailed description, numerous specific details are set forth to provide a thorough understanding of the embodiments of this application for ease of explanation. However, it will be apparent that one or more embodiments may be implemented without these specific details. Furthermore, descriptions of well-known structures and technologies are omitted in the following description to avoid unnecessarily obscuring the concepts of this application.

[0026] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this application. The term "comprising" as used herein indicates the presence of features, steps, or operations, but does not exclude the presence or addition of one or more other features.

[0027] When using expressions such as "at least one of A, B, and C," the expression should generally be interpreted in accordance with the meaning commonly understood by a person skilled in the art (e.g., "a system having at least one of A, B, and C" should include, but is not limited to, a system having A alone, a system having B alone, a system having C alone, a system having A and B, a system having A and C, a system having B and C, and / or a system having A, B, and C, etc.). When using expressions such as "at least one of A, B, or C," the expression should generally be interpreted in accordance with the meaning commonly understood by a person skilled in the art.

[0028] In realizing the concept of this application, it was found that current research on hypoxia-induced intestinal and liver injury still mainly relies on traditional two-dimensional cell culture and animal models. Two-dimensional cell culture is simple to operate, but it lacks the real three-dimensional microenvironment and intercellular and tissue interactions found in vivo, making it difficult to simulate the complex pathophysiological processes of hypoxia. While animal models can reflect the overall biological effects, they have limitations such as large species differences, high cost, and difficulty in achieving real-time dynamic monitoring and mechanism analysis of hypoxia stress. The rapid development of organ-on-a-chip technology provides new strategies and methods for studying the damage mechanisms of hypoxia to intestinal and liver tissues in vitro. Therefore, constructing a hypoxia injury model based on a biomimetic intestinal-liver organ-on-a-chip has significant scientific value and application prospects for elucidating the mechanism of intestinal-liver axis disorder caused by high-altitude hypoxia and developing drugs and protective strategies for the prevention and treatment of altitude sickness.

[0029] Figure 1 A schematic diagram of the biomimetic gut-liver organ-on-a-chip structure constructed for this application; Figure 2 This is a schematic diagram of the hypoxia injury model based on the bionic gut-liver organ-on-a-chip proposed in this application.

[0030] Specifically, according to an embodiment of one aspect of this application, such as Figure 1 and Figure 2 As shown, a hypoxic injury model based on a bionic gut-liver organ-on-a-chip is provided. This model is obtained by subjecting the bionic gut-liver organ-on-a-chip to hypoxia in a hypoxic environment. The bionic gut-liver organ-on-a-chip includes:

[0031] The chip substrate has at least two culture chambers formed on its top, and a bottom fluid flow channel connecting the culture chambers is formed inside. The intestinal chip module is detachably disposed in the corresponding culture chamber and includes: a first porous membrane for separating the inner lumen and the bottom fluid flow channel of the intestinal chip module, wherein intestinal epithelial cells are seeded on the inner layer of the first porous membrane and vascular endothelial cells are seeded on the outer layer; the liver chip module is detachably disposed in the corresponding culture chamber and includes: a second porous membrane for separating the inner lumen and the bottom fluid flow channel of the liver chip module, wherein liver parenchymal cells are seeded on the inner layer of the second porous membrane and vascular endothelial cells are seeded on the outer layer.

[0032] According to embodiments of this application, the hypoxia injury model provided is suitable for constructing gut-liver interaction models using various cell types. Compared with traditional mouse models, this model is closer to the physiological state of the human gut and liver in terms of cell composition and metabolic pathways, and can more realistically simulate the interaction between the human gut and liver. This model can not only simulate the effects of various extreme environments (such as hypoxia) on human gut and liver tissue, but also be used to establish various gut-liver related disease models, study the damage to gut and liver tissue under pathological conditions, and explore treatment options.

[0033] According to embodiments of this application, intestinal epithelial cells include intestinal absorptive cells and goblet cells.

[0034] According to the embodiments of this application, by adding intestinal absorptive cells and goblet cells, the physiological structure and function of the intestinal mucosa in vivo can be simulated more accurately. This enables the model to not only have the basic absorption barrier function, but also to simulate the innate defense mechanism of mucus secretion. As a result, the constructed hypoxia injury model can more realistically reflect key pathological processes such as the destruction of the integrity of the intestinal barrier, the loss of the mucus layer, and the interaction with immune cells.

[0035] According to an embodiment of this application, the pore size of the first porous membrane is 0.4~3μm; the pore size of the second porous membrane is 0.4~3μm.

[0036] According to embodiments of this application, the pore size of the first porous membrane can be 0.4 μm, 1 μm, 1.5 μm, 2 μm, 2.5 μm, or 3 μm, but is not limited to the listed values; other unlisted values ​​within this range are also applicable. Alternatively, it can be within a range consisting of any two values, such as 1 μm to 2 μm, 2 μm to 3 μm, etc.

[0037] According to embodiments of this application, the pore size of the second porous membrane can be 0.4 μm, 1 μm, 1.5 μm, 2 μm, 2.5 μm, or 3 μm, but is not limited to the listed values; other unlisted values ​​within this range are also applicable. Alternatively, it can be within a range consisting of any two values, such as 1 μm to 2 μm, 2 μm to 3 μm, etc.

[0038] According to the embodiments of this application, the pore size range of the porous membrane can effectively separate the upper layer cells and the lower layer endothelial cells to prevent cell migration and mixing, while ensuring that biomolecules such as cytokines and inflammatory mediators can diffuse freely between the two layers, thereby more accurately simulating the physiological exchange of intercellular signaling pathways in the intestinal barrier in vivo.

[0039] In one or more specific embodiments of this application, the pore sizes of the first porous membrane and the second porous membrane may be the same, or different pore sizes may be set according to specific circumstances. This application does not impose any restrictions on this.

[0040] According to embodiments of this application, the materials of the first porous membrane and the second porous membrane are polycarbonate.

[0041] According to embodiments of this application, the porous membrane material is polycarbonate, which has superior chemical stability, mechanical strength and precise processability, and can ensure the integrity of the porous membrane structure during long-term culture and low-oxygen treatment; its good surface properties are beneficial to cell adhesion, spreading and differentiation.

[0042] According to embodiments of this application, the inner cavity of the intestinal chip module is filled with intestinal cell culture medium; the inner cavity of the liver chip module is filled with hepatocyte culture medium; and the bottom fluid channel of the chip base is filled with endothelial cell culture medium.

[0043] According to the embodiments of this application, the selection of different culture media provides the most suitable growth and differentiation conditions for the three types of cells, namely intestinal cells, hepatocytes and vascular endothelial cells, thereby maintaining the function of each tissue without interfering with each other; at the same time, the endothelial cell culture medium with bottom co-flow realizes the simulation of the circulatory system while physically isolating the cell layer, creating a biomimetic fluid environment for communication between organs.

[0044] According to the embodiments of this application, the volume ratio of intestinal cell culture medium to endothelial cell culture medium is (1~3):(10~20); the volume ratio of hepatocyte culture medium to endothelial cell culture medium is (1~3):(10~20).

[0045] According to embodiments of this application, the volume ratio of intestinal cell culture medium to endothelial cell culture medium can be 1:10, 1:15, 1:20, 2:10, 2:15, 2:20, 3:10, 3:15, or 3:20, but is not limited to the listed values; other unlisted values ​​within this range are also applicable. Alternatively, it can be within a range consisting of any two values, such as (1~2):(10~15), (2~3):(15~20), etc.

[0046] According to embodiments of this application, the volume ratio of hepatocyte culture medium to endothelial cell culture medium can be 1:10, 1:15, 1:20, 2:10, 2:15, 2:20, 3:10, 3:15, or 3:20, but is not limited to the listed values; other unlisted values ​​within this range are also applicable. Alternatively, it can be within a range consisting of any two values, such as (1~2):(10~15), (2~3):(15~20), etc.

[0047] According to embodiments of this application, a gradient for material exchange and signal transmission similar to the tissue-circulation relationship in vivo is established in the chip system. By controlling the culture medium volume on the tissue side (intestine, liver) to a smaller value, while setting the culture medium volume on the circulation side (endothelium) to a larger value, the difference in quantity between interstitial fluid and blood can be simulated. Thus, under low flow rate culture conditions, a concentration difference and clearance efficiency close to physiological can be formed, ensuring that metabolites, drugs or damage signals can be transmitted between tissues and blood vessels at a reasonable speed and proportion, thereby improving the physiological relevance and data reliability of the model in studies such as hypoxia injury response.

[0048] According to an embodiment of this application, the chip base further includes at least two culture chambers, which are respectively disposed on both sides of the culture chambers where the intestinal chip module and the liver chip module are located; the culture chambers are filled with endothelial cell culture medium.

[0049] In one or more specific embodiments of this application, multiple liquid culture chambers can be provided, and the number of liquid culture chambers on both sides of the intestinal chip module and the liver chip module can be the same, for example, two liquid culture chambers can be provided on each side, or they can be different, for example, one liquid culture chamber can be provided on one side and two liquid culture chambers can be provided on the other side. This application does not limit this.

[0050] According to embodiments of this application, the culture chamber can be used to independently control the supply and drainage of endothelial cell culture medium in the substrate liquid flow channel, facilitating the replacement of the endothelial cell culture medium.

[0051] According to embodiments of this application, a dual-end liquid supply / drainage structure with independently controllable liquid is constructed inside the chip, providing a structural basis for simulating bidirectional material exchange and circulation in organs within the body. This design allows the intestinal and liver tissue sides to form controllable liquid flow paths with the two end reservoirs, facilitating directional perfusion, continuous renewal, and dynamic sampling of the culture medium during experiments.

[0052] According to an embodiment of this application, the method for preparing a hypoxic injury model based on a bionic gut-liver organ chip is as follows: after the bionic gut-liver organ chip is cultured in a normoxic environment, it is transferred to a hypoxic environment for further culture, and the oxygen concentration of the hypoxic environment is 0.5%~5%.

[0053] According to embodiments of this application, the oxygen concentration in a low-oxygen environment can be 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5%, but is not limited to the listed values; other unlisted values ​​within this range are also applicable. Alternatively, it can be within a range consisting of any two values, such as 1%~3%, 2%~4%, etc.

[0054] Figure 3 This is a flowchart illustrating the construction process of the biomimetic gut-liver organ chip of this application.

[0055] In some specific embodiments of this application, such as Figure 3 As shown, an intestinal-on-a-chip module is first constructed, followed by a liver-on-a-chip module, which is then cultured for a period of time to obtain a bionic intestinal-liver organ chip. After exposing the bionic intestinal-liver organ chip to a hypoxic environment, a hypoxic injury model based on the bionic intestinal-liver organ chip can be obtained.

[0056] According to the embodiments of this application, the normoxic pre-culture stage provides the necessary time for each cell layer to form a stable barrier and establish intercellular and transmembrane signaling networks. The subsequent hypoxic treatment can effectively trigger a series of key pathological events such as hypoxic stress, oxidative damage, barrier disruption and metabolic function changes in the functionalized model, thereby improving the realism and reproducibility of the model in the study of the mechanism of hypoxic injury in the intestine and liver.

[0057] According to the embodiments of this application, the culture time under normal oxygen conditions is 24 to 72 hours; the culture time under low oxygen conditions is 24 to 72 hours.

[0058] According to embodiments of this application, the incubation period under normal aerobic conditions can be 24 hours, 28 hours, 32 hours, 36 hours, 40 hours, 44 hours, 48 ​​hours, 60 hours, 66 hours, or 72 hours, but is not limited to the listed values; other unlisted values ​​within this range are also applicable. Alternatively, it can be within a range consisting of any two values, such as 24~32 hours, 28~40 hours, etc.

[0059] According to embodiments of this application, the incubation period under hypoxic conditions can be 24 hours, 30 hours, 36 hours, 42 hours, 48 ​​hours, 54 hours, 60 hours, 66 hours, or 72 hours, but is not limited to the listed values; other unlisted values ​​within this range are also applicable. Alternatively, it can be within a range consisting of any two values, such as 24~48 hours, 36~72 hours, etc.

[0060] According to the embodiments of this application, culture in the normoxic phase allows intestinal, liver, and vascular endothelial cells to complete adhesion, morphological stability, and functional maturation, providing a reliable physiological baseline for subsequent injury response; while the gradient design of the hypoxic phase of 24 to 72 hours can cover different injury stages from early stress and initial barrier function impairment to obvious metabolic disorders and inflammatory cascade amplification, thereby improving the controllability and applicability of the model in simulating the dynamic process and mechanism analysis of hypoxic intestinal-liver injury.

[0061] According to embodiments of this application, the application of a hypoxia injury model based on a biomimetic gut-liver organ-on-a-chip in screening drugs for the prevention and treatment of altitude sickness is described.

[0062] According to embodiments of this application, a biomimetic gut-liver organ-on-a-chip-based hypoxia injury model provides a biomimetic, quantifiable, and high-throughput in vitro evaluation platform for altitude sickness (especially types related to gut-liver hypoxia injury). This model can reproduce key pathological processes such as intestinal barrier disruption and abnormal liver metabolism and detoxification function under high-altitude hypoxic conditions. This allows drug developers to rapidly and accurately evaluate the effects and mechanisms of candidate compounds in protecting the intestinal barrier, reducing liver damage, and improving hypoxia tolerance in a controlled in vitro environment, thereby improving the efficiency, reproducibility, and clinical translational value of drug screening.

[0063] The following will further explain the solution of this application with reference to specific embodiments. Unless otherwise stated, the reagents used in the following embodiments are all commercially available reagents.

[0064] Example 1: Construction of Intestinal-Liver Organ-on-a-Chip

[0065] like Figure 3 As shown, the main steps are as follows:

[0066] (1) Construction of intestinal microarray module: Intestinal epithelial Caco2 and HT29 cells were seeded on the upper layer of the intestinal microarray module. The cells were mixed at a ratio of 9:1 and then seeded onto the upper layer of the plug-in porous membrane. The total number of cells was 8×10. 4 After the cells adhered to the wells, intestinal cell culture medium was added, and the chip was dynamically cultured on a precision shaker for 7 days at a shaking angle of 5 degrees and a shaking speed of 2 rpm to allow it to differentiate into intestinal villi structures. Subsequently, vascular endothelial cells (HUVECs) were seeded into the porous submembrane layer of the intestinal chip module at a cell number of 5 × 10⁶ cells / well. 4 / well; add endothelial cell culture medium to the lower layer, and incubate overnight to form a single layer of dense endothelial tissue barrier.

[0067] (2) Liver chip module construction: HepG2 cells were seeded on the upper layer of the liver chip module, with a cell number of 8×10⁶ cells. 4 / well; After the upper layer of hepatocytes adheres, HUVECs are seeded in the lower layer of the liver microarray module at a cell count of 5 × 10⁶ cells. 4 / well. After the chip adheres to the wall, hepatocyte culture medium is added to the upper layer and endothelial cell culture medium is added to the lower layer. The chip is then placed on a precision shaker for dynamic culture for 2 days. The shaking angle of the shaker is 5 degrees and the shaking speed is 2 rpm, so that it forms a liver chip module containing a single layer of dense endothelial barrier and hepatocyte layer.

[0068] (3) Construction of biomimetic intestinal-liver microarray: Differentiated intestinal and liver microarray modules were placed in the wells of the culture chamber of the same culture unit for co-culture. The microarray was placed on a shaker and rocked up and down, providing a fluid culture environment for the intestinal and liver microarray modules and promoting nutrient exchange. After 4 days of co-culture, samples were collected for subsequent analysis.

[0069] (4) After the chip was constructed, the supernatant was collected, and the cells in the upper and lower layers of the insert were washed three times with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde at room temperature for 30 minutes. Then, the cells were permeabilized with 0.25% Triton X-100 in PBS (PBST buffer) for 10 minutes, and washed three times with PBST for 5 minutes each time. The cells were blocked with PBST buffer containing 5% normal goat serum for 1 hour at room temperature. The primary antibody was diluted 1:200 with antibody dilution buffer, and the cells were stained with the corresponding primary antibody overnight at 4°C. The cells were then washed three times with PBST for 5 minutes each time. The cells were stained with secondary antibody (1:500 diluted in 1% bovine serum albumin BSA) at room temperature for 1 hour. After staining with secondary antibody, the cell nuclei were counterstained with 4′,6-diamidinyl-2-phenylindole (DAPI), incubated at room temperature in the dark for 10 minutes, and then washed three times with PBS buffer for 5 minutes each time. Remove the chip insert, place it flat on a glass slide, seal it with an anti-fluorescence quencher, and observe and photograph it using a confocal fluorescence microscope. The results are as follows: Figure 4 and Figure 5 As shown.

[0070] Figure 4 The images show the physiological function characterization of the intestinal chip module after successful construction of the biomimetic intestinal-liver organ chip according to the embodiments of this application; wherein, A is the bright field image of the intestinal chip module and the immunofluorescence image of intestinal epithelial cells after 4 days of co-culture; B is the 3D reconstruction image of the intestinal chip module; Figure 5 The images show the physiological function characterization of the liver chip module of the biomimetic gut-liver organ chip according to the embodiments of this application; where A is a bright field image of the gut chip module after 4 days of co-culture; and B is a confocal microscope image of HepG2 cells (stained with ALB and CYP3A4) after 4 days of co-culture.

[0071] according to Figure 4 and Figure 5 It can be seen that the prepared intestinal-liver organ-on-a-chip reproduces the key physiological characteristics of the human intestine and liver to a high degree in multiple dimensions such as tissue morphology, barrier function and metabolic activity.

[0072] Example 2: Study on hypoxia injury based on a biomimetic gut-liver organ-on-a-chip model

[0073] The specific steps are as follows:

[0074] (1) A hypoxic injury model of the intestinal-liver organ-on-a-chip was established by exposing the biomimetic intestinal-liver organ-on-a-chip to a hypoxic environment for 48 hours. The control group was an intestinal-liver organ-on-a-chip placed in a normoxic environment. The oxygen concentration in the hypoxic environment was 1%.

[0075] (2) The bionic gut-liver organ chip of the control group and the hypoxia simulation group were placed on a shaker for dynamic culture. After 48 hours, the samples were collected for subsequent detection.

[0076] (3) After the chip was constructed, the supernatant was collected, and the cells in the upper and lower layers of the insert were washed three times with PBS and fixed with 4% paraformaldehyde at room temperature for 30 minutes. Then, the cells were permeabilized with 0.25% Triton X-100 in PBS (PBST buffer) for 10 minutes, and washed three times with PBST for 5 minutes each time. The cells were blocked with PBST buffer containing 5% normal goat serum for 1 hour at room temperature. The primary antibody was diluted 1:200 with antibody dilution buffer, and the cells were stained with the corresponding primary antibody overnight at 4°C. The cells were then washed three times with PBST for 5 minutes each time. The cells were stained with secondary antibody (1:500 diluted in 1% BSA) at room temperature for 1 hour. After staining with secondary antibody, the cell nuclei were counterstained with 4′,6-diamidinyl-2-phenylindole (DAPI), incubated at room temperature in the dark for 10 minutes, and then washed three times with PBS buffer for 5 minutes each time. The chip insert was removed, placed flat on a glass slide, mounted with an anti-fluorescence quencher, and observed and photographed using a confocal fluorescence microscope. The results are as follows: Figure 6 and Figure 7 As shown.

[0077] Figure 6 This is a functional characterization diagram of the intestinal chip module of the biomimetic intestinal-liver organochip model after 48 hours of hypoxia treatment according to an embodiment of this application; wherein, A is a representative image of intestinal cells under confocal fluorescence microscopy after 48 hours of hypoxia exposure; B is a statistical result of the mRNA expression level of intestinal functional genes after 48 hours of hypoxia exposure; Figure 7 This is a functional characterization diagram of the liver chip module of the biomimetic gut-liver organ-on-a-chip model after 48 hours of hypoxia treatment according to an embodiment of this application; wherein, A is a representative diagram of hepatocytes after 48 hours of hypoxia exposure; B is a statistical result diagram of the mRNA expression level of liver functional genes after 48 hours of hypoxia exposure.

[0078] according to Figure 6 A and Figure 7 As can be seen from A, the intestinal microarray module showed significant structural damage after 2 days of hypoxia treatment: the expression levels of tight junction protein ZO-1 and brush border marker Villin decreased, and their continuous distribution pattern on intestinal villi was disrupted.

[0079] (4) Gene expression detection: After the chip was constructed, the upper layer cells were collected and subjected to real-time quantitative PCR detection. The results are as follows: Figure 6 and Figure 7 As shown.

[0080] according to Figure 6 B and Figure 7 As shown in Figure B, after 2 days of hypoxia treatment, the expression of Villin in intestinal villi decreased, while the expression of mucin MUC2 increased abnormally after hypoxia culture, suggesting that the intestinal epithelium may experience mucus secretion dysfunction under stress. The decreased expression levels of hepatocyte albumin ALB and the drug-metabolizing enzyme CYP3A4 indicate that hypoxia inhibits the core functions of hepatocytes.

[0081] Example 3 compares the functional differences between intestinal-on-a-chip modules, liver-on-a-chip modules, and biomimetic intestinal-liver organ-on-a-chip models cultured alone under normoxic and hypoxic conditions.

[0082] The specific steps are as follows:

[0083] (1) Construction of intestinal microarray module: Caco2 and HT29 cells were seeded on the upper layer of the intestinal microarray module. After being mixed evenly at a ratio of 9:1, the mixture was seeded onto the upper layer of the plug-in porous membrane, with a total cell count of 8 × 10⁶ cells. 4 After the cells adhered to the wells, intestinal cell culture medium was added, and the intestinal microarray module was placed on a precision shaker for dynamic culture for 7 days. The shaker was set at a 5-degree angle and a 2 rpm speed to allow the cells to differentiate into intestinal villi. Subsequently, HUVECs were seeded into the porous submembrane layer of the intestinal microarray module at a cell number of 5 × 10⁶ cells / well. 4 / well; add endothelial cell culture medium to the lower layer, and incubate overnight to form a single layer of dense endothelial tissue barrier.

[0084] (2) Liver microarray module construction: HepG2 cells were seeded on the upper layer of the liver microarray module, with a cell count of 8 × 10⁶ cells / year. 4 / well; After the upper layer of hepatocytes adheres, HUVECs are seeded in the lower layer of the liver microarray module at a cell count of 5 × 10⁶ cells. 4 / well. After the chip adheres to the wall, hepatocyte culture medium is added to the upper layer and endothelial cell culture medium is added to the lower layer. The chip is then placed on a precision shaker for dynamic culture for 2 days. The shaking angle of the shaker is 5 degrees and the shaking speed is 2 rpm, so that a liver chip module containing a single layer of dense endothelial barrier and hepatocyte layer is formed.

[0085] (3) Construction of biomimetic intestinal-liver organ-on-a-chip: Differentiated intestinal-on-a-chip modules and liver tissue modules were placed in the wells of the culture chamber of the same culture unit for co-culture. The biomimetic intestinal-liver organ-on-a-chip was placed on a shaker, which provided a fluid culture environment for the intestinal and liver modules and promoted nutrient exchange. After 4 days of co-culture, the samples were collected for subsequent detection.

[0086] (4) Separate culture of intestinal and liver microarray modules: The differentiated intestinal microarray module and liver tissue module were placed in the wells of the culture chambers of different culture units and cultured separately. The microarray was placed on a shaker and shaken up and down to promote differentiation. After dynamic culture for 4 days, the samples were collected for subsequent detection.

[0087] (5) Construction of relevant hypoxia model: The intestinal chip module, liver chip module and bionic intestinal-liver organ chip were cultured separately under normoxic conditions for 2 days, then transferred to a hypoxia incubator to simulate a hypoxia environment. After dynamic culture for 48 hours, the samples were collected for subsequent detection.

[0088] (6) Gene expression detection: After the chip was constructed, the upper layer cells were collected and subjected to real-time quantitative PCR detection. The results are as follows: Figure 8 As shown.

[0089] Figure 8 This is a comparison of the functional gene expression results of the intestinal chip module, liver chip module, and biomimetic intestinal-liver organ chip model cultured separately under normoxic conditions in the embodiments of this application.

[0090] according to Figure 8 It can be seen that, compared with the biomimetic gut-liver organ-on-a-chip model, the expression of intestinal mucoprotein MUC2 and liver drug-metabolizing enzyme CYP1A2 in the co-culture group of the gut-on-a-chip module and liver-on-a-chip module cultured separately under normoxic conditions was significantly increased, indicating that after co-culture of the intestine and liver, there is signal exchange between the two, which promotes their respective functions to be more complete.

[0091] (7) Cell viability detection: After the chip was constructed, the upper and lower culture media were collected, and their LDH content was detected separately. The results are as follows: Figure 9 As shown.

[0092] Figure 9 This is a comparison of the detection results of lactate dehydrogenase (LDH) content in the culture medium of the intestinal chip module, liver chip module, and biomimetic intestinal-liver organ-on-a-chip model cultured separately under hypoxic conditions in this application embodiment.

[0093] according to Figure 9 It can be seen that, compared with the biomimetic gut-liver organ-on-a-chip model, the LDH content of the gut-on-a-chip module and liver-on-a-chip module cultured separately under hypoxic conditions was significantly increased in the co-culture group. This indicates that both the intestine and liver were damaged to some extent after co-culture under hypoxic conditions, and there was also cytokine exchange between them, which may have caused "secondary damage".

[0094] (8) Transmembrane resistance detection: The transmembrane resistance (TEER) of the intestinal barrier in the isolated and co-culture models was detected using a transmembrane resistance meter. The result was calculated using the following formula: TEER = TEER 检测 × Surface area; where the surface area is 0.33 cm²2 The result is as follows Figure 10 As shown.

[0095] Figure 10 This is a comparison of the transmembrane resistance detection results of an individually cultured intestinal chip module and a biomimetic intestinal-liver organ chip under normoxic and hypoxic conditions, as described in the embodiments of this application.

[0096] according to Figure 10 It can be seen that there was no significant difference in TEER between the single culture group and the co-culture group under normoxic conditions. However, after 2 days of hypoxia treatment, the transepithelial resistance of the intestinal barrier in the co-culture group decreased significantly. This is because the release of cytokines after liver injury further damages the intestinal barrier.

[0097] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of this application. It should be understood that the above descriptions are merely specific embodiments of this application and are not intended to limit this application. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A hypoxia injury model based on a biomimetic gut-liver organ-on-a-chip, characterized in that, The biomimetic gut-liver organ chip includes: The chip base has at least two culture chambers formed on the top, and a bottom liquid flow channel connecting the culture chambers is formed inside. An intestinal microarray module, detachably disposed within the corresponding culture chamber, includes: A first porous membrane is used to separate the inner lumen of the intestinal chip module from the bottom fluid flow channel. The inner layer of the first porous membrane is seeded with intestinal epithelial cells, and the outer layer is seeded with vascular endothelial cells. A liver chip module, detachably disposed within the corresponding culture chamber, includes: The second porous membrane is used to separate the inner cavity of the liver chip module and the bottom fluid flow channel. The inner layer of the second porous membrane is seeded with liver parenchymal cells, and the outer layer is seeded with vascular endothelial cells. The hypoxia injury model based on the bionic gut-liver organ chip was obtained by subjecting the bionic gut-liver organ chip to hypoxia treatment in a hypoxic environment.

2. The hypoxia injury model according to claim 1, characterized in that, The intestinal epithelial cells include intestinal absorptive cells and goblet cells.

3. The hypoxia injury model according to claim 1, characterized in that, The pore size of the first porous membrane is 0.4~3μm; The pore size of the second porous membrane is 0.4~3μm.

4. The hypoxia injury model according to claim 1 or 3, characterized in that, The first porous membrane and the second porous membrane are made of polycarbonate.

5. The hypoxia injury model according to claim 1, characterized in that, The lumen of the intestinal chip module is filled with intestinal cell culture medium; The inner cavity of the liver chip module is filled with hepatocyte culture medium; The bottom fluid channel of the chip base is filled with endothelial cell culture medium.

6. The hypoxia injury model according to claim 5, characterized in that, The volume ratio of the intestinal cell culture medium to the endothelial cell culture medium is (1~3):(10~20). The volume ratio of the hepatocyte culture medium to the endothelial cell culture medium is (1~3):(10~20).

7. The hypoxia injury model according to claim 5, characterized in that, The chip base also includes at least two liquid storage culture chambers, which are respectively disposed on both sides of the culture chamber where the intestinal chip module and the liver chip module are located; The culture chamber is filled with the endothelial cell culture medium.

8. A method for preparing a hypoxia injury model based on a biomimetic gut-liver organ-on-a-chip as described in any one of claims 1 to 7, characterized in that, include: The biomimetic gut-liver organ chip was cultured in a normoxic environment and then transferred to a hypoxic environment for further culture, wherein the oxygen concentration of the hypoxic environment was 0.5% to 5%.

9. The preparation method according to claim 8, characterized in that, The co-culture time under the normoxic environment is 24-72 hours; The co-culture time under the hypoxic environment is 24-72 hours.

10. The application of the hypoxia injury model based on a biomimetic gut-liver organ-on-a-chip as described in any one of claims 1 to 7 in screening drugs for the prevention and treatment of altitude sickness.

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