A dual-chamber hydrogel microsphere-based intestinal organ-on-a-chip model and a method for constructing the same

Abstract

The present application relates to a kind of based on double-chamber hydrogel microsphere intestinal bionic organ model and its construction method, belong to biotechnology field.It is specifically double-chamber hydrogel microsphere with rough pit surface wrapped by intestinal barrier, construction method includes the following steps: the preparation of double-chamber hydrogel microsphere microfluidic device, preparation of rough double-chamber hydrogel microsphere with rough surface and load content, microsphere surface matrix glue modification and surface construction intestinal barrier.It is specifically applied to drug testing and the application research of microorganism and intestinal interaction.The intestinal bionic organ model prepared in the present application has simple manufacturing steps, short manufacturing time, low cost, and the subsequent culture maturation time is greatly shortened compared to other organoid models.The intestinal organ microsphere is expected to become a research platform for intestinal diseases, to promote the research of various intestinal disease treatment methods, and to help cope with the treatment challenges brought by various intestinal complex diseases.

Description

Technical Field

[0001] This invention relates to a biomimetic intestinal organ model based on dual-chamber hydrogel microspheres and its construction method, specifically involving in vitro simulation of human intestinal organs, belonging to the field of biotechnology. Background Technology

[0002] The gut microenvironment primarily refers to the micro-ecosystem within the gut. It includes the gut microbiota, the intestinal mucosal barrier, and also involves various chemical substances. As a crucial component of the complex human ecosystem, the homeostasis of the gut microenvironment's structure and function is of critical scientific and engineering application value for maintaining host health, promoting innovative drug development, and elucidating disease mechanisms. From an engineering perspective, the gut microenvironment can be viewed as a dynamic equilibrium system composed of multiple elements, including microbial communities, metabolites, immune factors, and physicochemical parameters. The efficient operation of this system directly affects the host's physiological functions such as nutrient metabolism, immune regulation, and barrier defense; its imbalance is also closely related to the occurrence and development of various chronic metabolic diseases, immune abnormalities, and malignant tumors. In the field of drug development, the regulation of the gut microenvironment has become a significant breakthrough for novel therapeutic strategies (such as microbiome engineering and targeted delivery systems). In disease mechanism research, system modeling and multi-omics analysis can be used to elucidate the dynamic causal relationship between microenvironmental homeostasis imbalance and pathological processes. Therefore, in-depth research into the composition, interaction networks, and regulatory mechanisms of the gut microenvironment is not only of fundamental scientific value for understanding human physiological and pathological processes, but also provides key theoretical basis and technical pathways for biomedical engineering, drug design, and precision medicine.

[0003] Over the years, various in vivo and in vitro models have been developed to study the pathogenesis of gut-related diseases and test new treatment strategies. The use of animal models faces ethical and low-throughput challenges. Intestinal organoids are expensive, and the apex of the intestinal barrier is enclosed within the organoid, limiting accessibility for manipulation and analysis. Static transwell models cannot fully capture the complex cellular diversity and dynamics of the human gut. Polydimethylsiloxane (PDMS) in intestinal chips absorbs various molecules and suffers from evaporation issues. Therefore, there is an urgent need to develop an intestinal model with advantages such as simple fabrication, lower cost, better biocompatibility, and more accurate simulation of the intestinal barrier, providing a better new platform for research on gut-related diseases and drugs.

[0004] Alginate hydrogel microspheres are safe, multifunctional, and promising biomaterials with excellent biocompatibility and biodegradability, mild gelation conditions, and surface modifiability. They have been widely applied in materials science, pharmacy, and life sciences. Modifying the surface microstructure of alginate microspheres has demonstrated their potential for cell adhesion and cell barrier construction. Furthermore, the development of multi-compartment hydrogel microspheres provides a research foundation for the multifunctionalization of microspheres. Summary of the Invention

[0005] The purpose of this invention is to overcome the above-mentioned shortcomings and provide an intestinal biomimetic organ model based on dual-chamber hydrogel microspheres and its construction method, so as to solve the problem that existing intestinal models have failed to fully reproduce or accurately simulate the complex physiological structure of the intestinal barrier in vivo.

[0006] The technical solution of the present invention is an intestinal biomimetic organ model based on dual-chamber hydrogel microspheres, specifically a dual-chamber hydrogel microsphere with a rough pitted surface wrapped by the intestinal barrier; the dual-chamber hydrogel microsphere is a micron-sized microsphere with two equal compartments, one compartment containing M0 macrophages and the other containing Fe3O4 nanoparticles.

[0007] The apparatus for fabricating a biomimetic intestinal organ model based on dual-chamber hydrogel microspheres includes a microfluidic liquid supply system, a dispersion and cutting system, an aerosol spraying system, and a collection system. The four systems are arranged sequentially from top to bottom. Liquid is introduced through the microfluidic liquid supply system, followed by microsphere cutting through the dispersion and cutting system. During the falling process, the microspheres are modified through the aerosol spraying system, and finally, the dual-chamber hydrogel microspheres are collected through the collection system.

[0008] Furthermore, the microfluidic liquid supply system includes a liquid supply pump, a fixing mechanism, a microfluidic chip, a first liquid inlet tube, and a second liquid inlet tube; the microfluidic chip is a dual-channel microfluidic chip, with the first and second liquid inlets located on the two side channels for sample introduction, and the middle channel located in the dual-chamber outlet for sample exit; the two sample inlets of the microfluidic chip are respectively connected to the first and second liquid inlet tubes, and both the first and second liquid inlet tubes are supplied with liquid by the liquid supply pump; the microfluidic chip is fixed by the fixing mechanism; The dispersion cutting system includes a conductive needle, a high-voltage DC power supply, and a conductive ring; one end of the conductive needle extends into the sample outlet of the microfluidic chip for drainage, and the conductive needle is perpendicular to the microfluidic chip; a conductive ring is set below the conductive needle, and both the conductive needle and the conductive ring are powered by the high-voltage DC power supply; The aerosol spraying system includes aerosol spraying mechanisms on both sides, both of which are located below the conductive ring. The intersection of the aerosol spraying mechanisms on both sides is located on the same vertical plane as the conductive needle. The collection system includes a collector located below the intersection of the two aerosol spraying mechanisms; Furthermore, the spray centers of the aerosol spraying mechanisms on both sides are located on the same horizontal plane, and the center of the conductive ring is on the same plane as the spray contact surface of both; the plane where the spray nozzle of the aerosol spraying mechanism is located is between the conductive ring and the collector.

[0009] The connection between the conductive needle and the dual-channel microfluidic chip involves connecting the conductive needle to the fluid convergence outlet of the chip, vertically holding the entire dispersion cutting system in the holder, and connecting a high-voltage power supply to control droplet manufacturing.

[0010] Another technical solution of the present invention, a method for constructing an intestinal biomimetic organ model based on dual-chamber hydrogel microspheres, comprises the following steps: S1. Preparation of the dual-chamber hydrogel microsphere microfluidic device: The dual-chamber hydrogel microsphere microfluidic device is used; a hole is drilled at the intersection of the two channels, and a conductive needle is inserted into the fluid convergence outlet to form a 3D microfluidic system; finally, the positive terminal of the DC high voltage power supply is connected to the conductive needle, and the negative terminal is connected to the conductive ring to obtain the dual-chamber hydrogel microsphere microfluidic device. S2. Preparation of roughened double-chamber hydrogel microspheres with rough surfaces and loaded with contents: Alginate solutions containing M0 macrophages and alginate solutions containing Fe3O4 nanoparticles were injected into the left and right channel inlets of a microfluidic chip, respectively. Dual-chamber hydrogel microspheres loaded with contents on both sides were prepared using a dual-chamber hydrogel microsphere microfluidic device prepared in S1. During the falling of the microspheres, calcium ion aerosols were sprayed on both sides through an aerosol spraying component, causing the alginate and calcium ions to cross-link, changing the surface microstructure of the dual-chamber hydrogel microspheres, forming a pitted structure on the surface, and obtaining rough dual-chamber hydrogel microspheres. S3, Microsphere surface matrix modification: The rough double-chamber hydrogel microspheres prepared by S2 were placed in a 4-6 mM CaCl2 culture medium solution for swelling treatment, and then transferred to a culture medium solution containing matrix gel for surface modification to obtain matrix gel-rough double-chamber hydrogel microspheres. S4. Surface construction of intestinal barrier: The prepared matrix gel-rough dual-chamber hydrogel microspheres were dropped onto the bottom of a culture dish to form droplets containing individual microspheres. A uniform Caco-2 cell suspension was added to the droplets containing individual microspheres. After incubating upside down in a cell culture incubator for a period of time, the culture dish was placed upright, fresh culture medium was added, and culture was continued for 7 days to obtain intestinal microspheres, which is an intestinal biomimetic organ model based on dual-chamber hydrogel microspheres.

[0011] Furthermore, in S2: the M0 macrophages and the alginate solution containing Fe3O4 nanoparticles are specifically a mixture of M0 macrophages / Fe3O4 nanoparticles and a 1%-2% alginate solution at a volume ratio of 1:9; during mixing, the solution can be gently stirred clockwise to achieve the purpose of mixing without damaging the cells.

[0012] Preferably, the volume-to-mass ratio of the alginate solution is 1.25%.

[0013] Furthermore, in S2: the flow rate of the alginate aqueous solution in the dual-channel system is 2500 μL / h, and the voltage of the high-voltage DC power supply is 5500 V, so as to facilitate the fabrication of dual-chamber microspheres with a diameter of 400 μm.

[0014] Furthermore, the calcium ion aerosol sprayed in S2 is specifically a 1M calcium chloride aqueous solution, and the spraying speed should be greater than 1.7 m / s.

[0015] Furthermore, the specific method for surface modification in S3 is as follows: the microspheres obtained in S2 are placed in a 0.5 mg / mL matrix gel solution, and then incubated at 37℃ and 5% CO2 for 2 hours. During the matrix gel incubation process, the microspheres are inverted or shaken slightly 1-3 times. Preferably, the swelling time of the microspheres in S3 is greater than 1 hour.

[0016] Furthermore, the specific method for preparing the intestinal microspheres in S4 is as follows: a uniform Caco-2 cell suspension is added to a droplet containing individual microspheres, and then the droplet is inverted and incubated in a cell culture incubator for 2-4 hours. After inverting the culture dish, fresh culture medium is added, and the culture is continued for 6-8 days to prepare biomimetic intestinal organ microspheres with basic intestinal functions. During the culture of the intestinal microspheres, the culture medium used is MEM medium containing 4-6 mM CaCl2 and 20% FBS.

[0017] In the above scheme, two pre-prepared alginate solutions are used to fabricate dual-chamber hydrogel microspheres. Within the channels of the microfluidic chip, the liquid flow rate is slow, exhibiting a laminar flow effect. Therefore, at the fluid convergence outlet of the dual channels of the microfluidic chip, and in the conductive needles following entry, the two alginate aqueous solutions maintain a laminar flow state throughout the entire process; that is, the different contents encapsulated in the two alginate aqueous solutions do not mix. Subsequent cross-linking with CaCl2 causes the alginate to form a stable network structure, fixing the contents within their respective chambers and further ensuring the stability of the dual-chamber structure.

[0018] The third technical solution of the present invention is to conduct applied research on drug testing based on the application of the intestinal biomimetic organ model of dual-chamber hydrogel microspheres. Specifically, it involves culturing drugs with the intestinal biomimetic organ model and observing the metabolic effect of the intestinal biomimetic organ model on the drugs in order to elucidate the mechanism of action of intestinal metabolic drugs.

[0019] The application of the intestinal biomimetic organ model based on dual-chamber hydrogel microspheres is used to conduct applied research on the interaction between microorganisms and the intestine; specifically, bacteria and the intestinal biomimetic organ model can be co-cultured, and the interaction between microorganisms and the intestinal biomimetic organ model can be studied by using the detection methods of genes, proteins and metabolites.

[0020] The beneficial effects of this invention are as follows: By modifying and altering the internal multi-chamber design and surface microstructure of alginate microspheres, this invention develops an intestinal organ microsphere with a dual-chamber hydrogel microsphere as its core scaffold. The fabrication process for these intestinal microspheres is simple, time-efficient, and low-cost, and the subsequent culturing and maturation time is significantly shortened compared to organoid models. Intestinal organ microspheres hold promise as a research platform for intestinal diseases, promoting research into treatment methods for various intestinal diseases and helping to address the treatment challenges posed by complex intestinal diseases. Intestinal microspheres can also be used to detect the efficacy of drugs used to treat intestinal diseases, providing a novel and reliable platform for research into treatment strategies. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a three-dimensional structural diagram of the device described in this invention.

[0023] Figure 2 This is a three-dimensional structural schematic diagram of the device described in this invention from another perspective.

[0024] Figure 3 This is a schematic diagram of the microfluidic chip described in this invention.

[0025] Figure 4 This is a schematic diagram of the intestinal microsphere construction method described in this invention.

[0026] Figure 5 This is a microscopic characterization result of the surface-roughened double-chamber hydrogel microspheres of Example 2.

[0027] Figure 6 This is a schematic diagram of the microsphere size influencing factors in Example 3; a. Curves showing the effect of voltage and flow rate of alginate aqueous solution on the diameter of microspheres; b. Schematic diagram of microsphere particle size distribution.

[0028] Figure 7 These are bright-field microscopic images of the intestinal microspheres in Example 4, showing both successes and failures.

[0029] Figure 8 This is a graph showing the intestinal microsphere cell viability characterization data from Example 4; a) Results of cell viability staining experiment; b) Statistical data on cell viability.

[0030] Explanation of reference numerals in the attached drawings: 1. Microfluidic liquid supply system; 11. Liquid supply pump; 12. Fixing mechanism; 13. Microfluidic chip; 131. First liquid inlet; 132. Second liquid inlet; 133. Dual-chamber liquid outlet; 14. First liquid inlet tube; 15. Second liquid inlet tube; 2. Dispersion and cutting system; 21. Conductive needle; 22. High-voltage DC power supply; 23. Conductive ring; 3. Aerosol spraying system; 31. Aerosol spraying mechanism; 4. Collection system; 41. Collector.

[0031] In the accompanying drawings, the same parts use the same reference numerals. The drawings are not to scale. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] This application provides an intestinal biomimetic organ model based on dual-chamber hydrogel microspheres and its construction method, which solves the problem that existing intestinal models have failed to fully reproduce or accurately simulate the complex physiological structure of the intestinal barrier in vivo.

[0034] To better understand the above technical solutions, the following will provide a detailed explanation of the technical solutions in conjunction with the accompanying drawings and specific implementation methods.

[0035] Example 1: Fabrication of a dual-chamber microfluidic chip like Figure 2 As shown, the microfluidic chip 13 is a dual-channel microfluidic chip. Samples are introduced into the two side channels, where the first inlet 131 and the second inlet 132 are located, and samples are discharged from the dual-chamber outlet 133 located in the middle channel. The microfluidic chip is fabricated using soft photolithography, and the specific fabrication steps are as follows: (1) SU-8 2050 photoresist was uniformly applied to a 75 mm silicon wafer at 1250 rpm for 90 s using a spin coater to obtain a 140 μm thick photoresist layer. After soft baking at 70°C for 5 minutes, the silicon wafer was patterned by UV exposure through a transparent photomask.

[0036] (2) The obtained pattern was baked at 75°C for another 20 minutes and then developed with SU-8 developer. After the microstructure was formed, the mold was treated with a silylating agent (1H,1H,2H,2H-perfluorooctyltrichlorosilane) under vacuum for 6 hours. Subsequently, a 10:1 mixture of degassed PDMS and initiator was poured onto the mold and cured at 65°C for 3 hours.

[0037] (3) After curing, the PDMS imprint is peeled off from the mold pattern, and a precision stamping press with an XY platform is used to generate channel inlets and fluid convergence outlets. In order to create a fluid network consisting of a single-layer chip, the PDMS chip is tightly sealed onto a glass substrate after oxygen plasma activation on both surfaces and cured at 65°C for at least one hour.

[0038] The dual-channel microfluidic chip 13 has a channel width of 100μm.

[0039] Example 2: Construction of a device for fabricating a biomimetic intestinal organ model based on dual-chamber hydrogel microspheres like Figure 1-2 As shown, the microfluidic device includes a microfluidic liquid supply system 1, a dispersion and cutting system 2, an aerosol spraying system 3, and a collection system 4. The four systems are arranged sequentially from top to bottom. Liquid is introduced through the microfluidic liquid supply system 1, followed by microsphere cutting through the dispersion and cutting system 2. The microspheres are modified through the aerosol spraying system 3 during the falling process, and finally the dual-chamber hydrogel microspheres are collected through the collection system 4.

[0040] The microfluidic liquid supply system 1 includes a liquid supply pump 11, a fixing mechanism 12, a microfluidic chip 13, a first liquid inlet tube 14, and a second liquid inlet tube 15. The microfluidic chip is a dual-channel microfluidic chip, with the first liquid inlet 131 and the second liquid inlet 132 located on both sides for sample introduction, and the middle channel located at the dual-chamber outlet 133 for sample exit. The two sample inlets of the microfluidic chip 13 are respectively connected to the first liquid inlet tube 14 and the second liquid inlet tube 15, and both the first liquid inlet tube 14 and the second liquid inlet tube 15 are supplied with liquid by the liquid supply pump 11. The microfluidic chip 13 is fixed by the fixing mechanism 14. The dispersion cutting system 2 includes a conductive needle 21, a high-voltage DC power supply 22, and a conductive ring 23. One end of the conductive needle 21 extends into the dual-chamber outlet 133 of the microfluidic chip 13 for drainage. The conductive needle 21 is perpendicular to the microfluidic chip 13 and is fixed by a holding mechanism. The conductive ring 23 is arranged below the conductive needle 21. Both the conductive needle 21 and the conductive ring 23 are powered by the high-voltage DC power supply 22. The aerosol spraying system 3 includes aerosol spraying mechanisms 31 disposed on both sides. Both aerosol spraying mechanisms 31 are located below the conductive ring 23, and the intersection surface of the aerosol spraying mechanisms 31 on both sides is located on the same vertical plane as the conductive needle 21. The collection system 4 includes a collector 41, which is located below the intersection of the two aerosol spraying mechanisms 31.

[0041] The spray centers of the aerosol spraying mechanisms 31 on both sides are located on the same horizontal plane, and the center of the conductive ring 23 is on the same plane as the spray contact surface of the two. The plane where the spray nozzle of the aerosol spraying mechanism 31 is located is between the conductive ring 23 and the collector 41.

[0042] The conductive needle 21 is connected to the dual-channel microfluidic chip 13 by connecting the conductive needle 21 to the dual-chamber outlet 133 of the chip, holding the entire dispersion cutting system 2 vertically in the holder, and connecting the high-voltage DC power supply 22 to control the droplet manufacturing.

[0043] In the structure described in this embodiment, the microfluidic liquid supply system 1 is used to input the dispersed phase solution of the dual-chamber microspheres containing the load; the conductive needle 21 is used to combine with the dual-channel microfluidic chip at the fluid convergence outlet to output two dispersed phase solutions in a laminar flow state; the high-voltage DC power supply 22 and the conductive ring 23 are used to cut the dispersed phase solution to form hydrogel droplets with a dual-chamber structure; the CaCl2 aerosol spray is used to create a rough pit structure on the surface of the microspheres; the collection bath in the collector 41 is used to ultimately cross-link the microspheres to form a relatively stable spherical structure.

[0044] Example 3: Preparation and characterization of two-chamber hydrogel microspheres with rough surfaces and loaded contents.

[0045] The preparation process of the dual-chamber hydrogel microspheres is as follows: S1. Preparation of the dual-chamber hydrogel microsphere microfluidic device: The dual-chamber hydrogel microsphere microfluidic device described in Example 2 is used; a hole is drilled at the intersection of the two channels, and a conductive needle is inserted into the fluid convergence outlet to form a 3D microfluidic system; finally, the positive terminal of the DC high voltage power supply 22 is connected to the conductive needle 21, and the negative terminal is connected to the conductive ring 23 to obtain the dual-chamber hydrogel microsphere microfluidic device. S2. Preparation of roughened double-chamber hydrogel microspheres with roughened surfaces and loaded with contents: Microspheres containing M0 macrophages (5 × 10⁶ cells / mL) were prepared. 6 A solution containing 1.25% Fe3O4NPs (cells / ml) and an alginate solution containing 1.25% Fe3O4NPs by mass volume were injected into the first inlet tube 14 and the second inlet tube 15, respectively, to prepare two-chamber microspheres loaded with M0 macrophages and Fe3O4 on the left and right sides, respectively.

[0046] In this process, droplet fabrication is controlled by a high-voltage power supply, and the flow rate of the alginate solution is controlled by a pump. During the fabrication of the dual-chamber hydrogel microspheres, the flow rate of the alginate aqueous solution in the dual channels is 2500 μL / h, and the voltage of the high-voltage DC power supply is 5500 V, in order to fabricate dual-chamber microspheres with a diameter of 400 μm.

[0047] Meanwhile, on both sides of the path where the droplets fall, the CaCl2 aerosol injector begins to generate CaCl2 (1 M) mist and controls the injection speed to be greater than 1.7 m / s; the generated alginate droplets form pit structures on the surface under the impact of the CaCl2 mist, and then fall into the collection bath (100 mM CaCl2) in the collector 41 to form rough microspheres.

[0048] Characterization of dual-chamber hydrogel microspheres: The overall morphology of unloaded meteorite crater microspheres and double-chambered rough microspheres loaded with M0 macrophages and Fe3O4 nanoparticles was photographed using bright-field microscopy and phase-contrast microscopy. The results are as follows: Figure 5 As shown, this embodiment demonstrates the successful preparation of meteorite crater-double-chamber hydrogel microspheres with good surface roughness.

[0049] Example 4: Controllable Adjustment of Microsphere Particle Size like Figure 6 As shown, the microsphere size can be extensively adjusted by controlling the voltage and the flow rate of the alginate aqueous solution, indicating a customizable model size. Figure 6 a). For example Figure 6 As shown in Figure a, a voltage range of 4000-8000 V was selected as the experimental conditions to fabricate dual-chamber microspheres, aiming to observe the relationship between the voltage magnitude and the diameter of the fabricated microspheres. Simultaneously, as... Figure 6 As shown in Figure a, three flow rates of 25 μL / min, 35 μL / min, and 50 μL / min were selected as experimental conditions to fabricate dual-chamber microspheres, aiming to observe the relationship between the alginate flow rate and the diameter of the fabricated microspheres. During this experiment, the CaCl2 (1M) mist injection velocity was kept constant at 1.7 m / s. Figure 6As can be seen, the microsphere size is inversely proportional to the voltage and directly proportional to the flow rate of the alginate aqueous solution. The microsphere diameter can be controllably adjusted between 100 and 2000 μm using different experimental conditions.

[0050] The experimental conditions were fixed at a voltage of 5500 V and a flow rate of 35 μL / min for the alginate aqueous solution. A large number of microspheres were prepared, and 100 microspheres were randomly selected for statistical analysis of particle size distribution. Figure 6 As shown in particle size distribution diagram b, under these conditions, the average diameter of the microspheres produced is approximately 446 μm, and they have a small coefficient of variation (CV = 4.94%), indicating that these microspheres have good monodispersity, which is beneficial for uniform cell assembly and avoids biological quantitative errors between particle models.

[0051] Example 5: Fabrication and characterization of a biomimetic intestinal organ model based on dual-chamber hydrogel microspheres.

[0052] The intestinal biomimetic organ model, namely the preparation mechanism of intestinal microspheres, is as follows: Figure 4 As shown, the process specifically includes four steps: S1, preparation of the dual-chamber hydrogel microsphere microfluidic device; S2, preparation of roughened dual-chamber hydrogel microspheres with roughened surfaces and loaded with contents; specifically, Example 3; S3, modification of the microsphere surface matrix gel; and S4, construction of an intestinal barrier on the surface. The microspheres prepared in Example 3 were used to further prepare an intestinal biomimetic organ model.

[0053] S3. Modification of microsphere surface with matrix gel: The rough double-chamber hydrogel microspheres prepared in S2 were placed in a culture medium solution of 4-6 mM CaCl2 for swelling treatment, and then transferred to a matrix gel solution containing 0.5 mg / mL. The microspheres were then incubated at 37℃ and 5% CO2 for 2 hours, and were inverted or shaken slightly twice during the matrix gel incubation process.

[0054] S4. Surface construction of the intestinal barrier: To attach and culture Caco-2 cells on the surface of the microspheres, a uniform cell suspension (2×10⁻⁶) was prepared. 7 (cells / ml) was added to a droplet containing individual microspheres, and then incubated upside down in a cell culture incubator for 3 hours. After that, the culture dish was placed upright, fresh culture medium was added, and the culture was continued for 7 days. The entire system was cultured at 37 ℃ and 5% CO2 until Caco-2 cells proliferated and covered the surface of the microspheres, forming a barrier structure, thus preparing biomimetic intestinal organ microspheres with basic intestinal functions.

[0055] The culture medium is MEM medium (20% FBS) containing 5 mM CaCl2.

[0056] In the process of preparing intestinal microspheres, the diameter of the crater microspheres and the incubation time after adding Caco-2 cells have a significant impact on the formation of the intestinal barrier. After multiple experiments, the experimental conditions favorable for Caco-2 barrier formation are concentrated around a diameter of 400-800 μm and an incubation time of more than 3 hours.

[0057] like Figure 7 As shown, this illustrates successful and unsuccessful cases of intestinal microspheres adhering to Caco-2 cells under a bright-field microscope one day later. Successful microspheres will form a tightly connected intestinal barrier on their surface after a further 6 days of culture.

[0058] The failed cases exhibited two scenarios: 1. Insufficient cell adhesion prevented the formation of a tightly connected intestinal barrier after 6 days of culture; 2. Excessive cell adhesion resulted in thick cell clusters forming on the microsphere surface after only 1 day of culture following Caco-2 cell adhesion, failing to demonstrate the potential for creating a single-layer intestinal barrier in subsequent cultures. The constructed intestinal microspheres aim to mimic the single-layer cell barrier function of the human intestinal organ. Excessive cell quantity leading to multi-layered cell barriers hinders the subsequent application of the intestinal microspheres and does not fully simulate the actual physiological structure of the human intestinal organ.

[0059] This embodiment describes a series of characterizations of the successfully fabricated intestinal microspheres to demonstrate their basic intestinal organ functions. The cell-loaded microspheres were incubated for 25 minutes in Calcein AM / PI working staining solution under growth conditions. In a fluorescence field, live cells exhibited uniform green fluorescence, while dead cells produced red fluorescence. Cell viability was calculated using ImageJ software and defined as: .

[0060] like Figure 8 As shown, the cell viability of the successfully fabricated intestinal microspheres was characterized to demonstrate that they retain high cell viability after maturation for various applications. Cell viability on the surface of the intestinal microspheres was monitored from day 1 to day 7 of culture. Confocal images show that as the culture time increases, the cells on the surface of the microspheres continuously proliferate, ultimately covering the entire microsphere and forming a tightly connected intestinal barrier by day 7 of culture. Figure 8 a). Within 7 days of culture, the cells inside and outside the microspheres maintained high viability (external > 94%, internal > 85%). Figure 8 b).

[0061] This invention develops an intestinal organ microsphere with a dual-chamber hydrogel microsphere as its core scaffold by modifying and altering the multi-chamber internal structure and surface microstructure of alginate microspheres. The intestinal microsphere fabrication process is simple, quick, and low-cost, and the subsequent culturing and maturation time is significantly shortened compared to organoid models. Intestinal organ microspheres hold promise as a research platform for intestinal diseases, advancing research into treatment methods for various intestinal diseases and helping to address the treatment challenges posed by complex intestinal diseases. Intestinal microspheres can also be used to detect the efficacy of drugs for intestinal disease treatment, providing a novel and reliable platform for research into treatment strategies.

[0062] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0063] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A biomimetic intestinal organ model based on dual-chamber hydrogel microspheres, characterized in that: Specifically, it is a dual-chamber hydrogel microsphere with a rough, pitted surface that is encapsulated by the intestinal barrier; the dual-chamber hydrogel microsphere is a micron-sized microsphere with two equally spaced chambers, one chamber containing M0 macrophages and the other containing Fe3O4 nanoparticles.

2. A device for fabricating a biomimetic intestinal organ model based on dual-chamber hydrogel microspheres, characterized in that: It includes a microfluidic liquid supply system, a dispersion and cutting system, an aerosol spraying system, and a collection system. The above four systems are arranged in order from top to bottom. The liquid is introduced through the microfluidic liquid supply system, then the microspheres are cut through the dispersion and cutting system, the microspheres are modified through the aerosol spraying system during the falling process, and finally the dual-chamber hydrogel microspheres are collected through the collection system.

3. The apparatus for preparing an intestinal biomimetic organ model based on dual-chamber hydrogel microspheres as described in claim 2, characterized in that: The microfluidic liquid supply system includes a liquid supply pump, a fixing mechanism, a microfluidic chip, a first liquid inlet tube, and a second liquid inlet tube. The microfluidic chip is a dual-channel microfluidic chip, with the first and second liquid inlets located on the two side channels for sample introduction, and the middle channel located in the dual-chamber outlet for sample exit. The two sample inlets of the microfluidic chip are respectively connected to the first and second liquid inlet tubes, and both the first and second liquid inlet tubes are supplied with liquid by the liquid supply pump. The microfluidic chip is fixed by the fixing mechanism. The dispersion cutting system includes a conductive needle, a high-voltage DC power supply, and a conductive ring; one end of the conductive needle extends into the sample outlet of the microfluidic chip for drainage, and the conductive needle is perpendicular to the microfluidic chip; a conductive ring is set below the conductive needle, and both the conductive needle and the conductive ring are powered by the high-voltage DC power supply; The aerosol spraying system includes aerosol spraying mechanisms on both sides, both of which are located below the conductive ring. The intersection of the aerosol spraying mechanisms on both sides is located on the same vertical plane as the conductive needle. The collection system includes a collector located below the intersection of the two aerosol spraying mechanisms; Furthermore, the spray centers of the aerosol spraying mechanisms on both sides are located on the same horizontal plane, and the center of the conductive ring is on the same plane as the spray contact surface of both; the plane where the spray nozzle of the aerosol spraying mechanism is located is between the conductive ring and the collector.

4. A method for constructing an intestinal biomimetic organ model based on dual-compartment hydrogel microspheres, characterized by: The steps are as follows: S1. Preparation of the dual-chamber hydrogel microsphere microfluidic device: The dual-chamber hydrogel microsphere microfluidic device of claim 2 is used; a hole is drilled at the intersection of the two channels, and a conductive needle is inserted into the fluid convergence outlet to form a 3D microfluidic system; finally, the positive terminal of the DC high voltage power supply is connected to the conductive needle, and the negative terminal is connected to the conductive ring to obtain the dual-chamber hydrogel microsphere microfluidic device. S2. Preparation of roughened double-chamber hydrogel microspheres with rough surfaces and loaded with contents: Alginate solutions containing M0 macrophages and alginate solutions containing Fe3O4 nanoparticles were injected into the left and right channel inlets of a microfluidic chip, respectively. Dual-chamber hydrogel microspheres loaded with contents on both sides were prepared using a dual-chamber hydrogel microsphere microfluidic device prepared in S1. During the falling of the microspheres, calcium ion aerosols were sprayed on both sides through an aerosol spraying component, causing the alginate and calcium ions to cross-link, changing the surface microstructure of the dual-chamber hydrogel microspheres, forming a pitted structure on the surface, and obtaining rough dual-chamber hydrogel microspheres. S3, Microsphere surface matrix modification: The rough double-chamber hydrogel microspheres prepared by S2 were placed in a 4-6 mM CaCl2 culture medium solution for swelling treatment, and then transferred to a culture medium solution containing matrix gel for surface modification to obtain matrix gel-rough double-chamber hydrogel microspheres. S4. Surface construction of intestinal barrier: The prepared matrix gel-rough dual-chamber hydrogel microspheres were dropped onto the bottom of a culture dish to form droplets containing individual microspheres. A uniform Caco-2 cell suspension was added to the droplets containing individual microspheres. After incubating upside down in a cell culture incubator for a period of time, the culture dish was placed upright, fresh culture medium was added, and culture was continued for 7 days to obtain intestinal microspheres, which is an intestinal biomimetic organ model based on dual-chamber hydrogel microspheres.

5. The method for constructing an intestinal biomimetic organ model based on dual-chamber hydrogel microspheres as described in claim 2, characterized in that: in S2: the M0 macrophages and the alginate solution containing Fe3O4 nanoparticles are specifically a mixture of M0 macrophages / Fe3O4 nanoparticles and a 1%-2% volume ratio of alginate solution at a volume ratio of 1:9; the flow rate of the alginate aqueous solution in the dual-channel is 2500 μL / h, and the voltage of the high-voltage DC power supply is 5500 V; Preferably, the volume-to-mass ratio of the alginate solution is 1.25%.

6. The method for constructing an intestinal biomimetic organ model based on dual-chamber hydrogel microspheres as described in claim 2, characterized in that the calcium ion aerosol sprayed in S2 is specifically a 1M calcium chloride aqueous solution, and the spraying speed should be greater than 1.7 m / s.

7. The method for constructing an intestinal biomimetic organ model based on dual-chamber hydrogel microspheres as described in claim 2, characterized in that the specific method for surface modification in S3 is as follows: the microspheres obtained in S2 are placed in a 0.5 mg / mL matrix gel solution, and then incubated at 37°C and 5% CO2 for 2 hours, and the microspheres are inverted or slightly shaken 1-3 times during the matrix gel incubation process; Preferably, the swelling time of the microspheres in S3 is greater than 1 hour.

8. The method for constructing a biomimetic intestinal organ model based on dual-chamber hydrogel microspheres as described in claim 2, characterized in that the specific method for preparing the intestinal microspheres in S4 is as follows: adding a uniform Caco-2 cell suspension to a droplet containing individual microspheres, then incubating upside down in a cell culture incubator for 2-4 hours, then placing the culture dish upright, adding fresh culture medium, and continuing to culture for 6-8 days to prepare biomimetic intestinal organ microspheres with basic intestinal functions; the culture medium used in the culture of the intestinal microspheres is MEM medium containing 4-6 mM CaCl2 and 20% FBS.

9. The application of the intestinal biomimetic organ model based on dual-chamber hydrogel microspheres as described in claim 1, characterized in that: The applied research for drug testing specifically involves culturing drugs with intestinal biomimetic organ models, observing the metabolic effects of the intestinal biomimetic organ models on drugs, and elucidating the mechanism of action of drugs metabolized in the intestine.

10. The application of the intestinal biomimetic organ model based on dual-chamber hydrogel microspheres as described in claim 1, characterized in that: The research focuses on the interaction between microorganisms and the gut; specifically, bacteria can be co-cultured with a gut biomimetic organ model, and the interaction between microorganisms and the gut biomimetic organ model can be studied by using methods for detecting genes, proteins, and metabolites.

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