Microorganism-intestinal tract microsphere co-culture system based on intestinal tract bionic microspheres and construction method thereof

Abstract

The invention relates to a microorganism-intestinal tract microsphere co-culture system based on intestinal tract bionic microspheres and a construction method of the microorganism-intestinal tract microsphere co-culture system, and belongs to the technical field of biology. The device comprises a culture device, a magnetic suction device and a microorganism-intestinal microsphere co-culture solution, the microorganism-intestinal tract microsphere co-culture solution is used for researching the effect of microorganisms on intestinal tracts; the culture device is used for culturing microorganism-intestinal tract microspheres; the magnetic suction device is fixed on the surface of the culture device and is used for adsorbing microorganisms-intestinal microspheres. The suspension and shaking microorganism-intestinal microsphere co-culture method is developed by applying suspension and shaking to intestinal microspheres and microorganisms cultured by a pore plate. The method effectively simulates the dynamic interaction between the microorganisms and the intestinal epithelial barrier in a real intestinal microenvironment, not only promotes the specific adhesion and colonization of the microorganisms on the surfaces of the microspheres, but also can amplify the inflammation signal response generated by the induction of the microorganisms, and obviously improves the detection sensitivity.

Description

Technical Field

[0001] This invention relates to a microbial-intestinal microsphere co-culture system based on intestinal biomimetic microspheres and its construction method, specifically involving the in vitro simulation of the human intestinal-microbial ecosystem, belonging to the field of biotechnology. Background Technology

[0002] The gut microenvironment refers to the complex micro-ecosystem composed of gut microbiota, the intestinal mucosal barrier, and related chemical substances. Research on the gut microenvironment is crucial for maintaining health, drug development, and disease research. In the field of disease research, Crohn's disease (CD), a major entity in the inflammatory bowel disease (IBD) spectrum, is a chronic inflammatory disease of the gastrointestinal tract that can lead to intestinal damage and disability. Crohn's disease is now a global health problem, severely impacting human health in many countries. The etiology of CD is currently unclear and involves a complex interplay of multiple factors, resulting from the combined effects of genetics, environment, immunity, and gut microbiota. Numerous studies have shown that the etiology of CD is largely related to the interaction between the microbial community and specific microorganisms with the gut. Adhesive invasive Escherichia coli (AIEC) is a major candidate bacterial trigger for CD. AIEC is more frequently isolated from mucosal biopsies of CD patients, with an abundance more than three times that of healthy patients. The interaction between AIEC and the gut is extremely complex, and the key mechanisms remain unclear. Therefore, research into the interaction mechanism between AIEC and the intestinal barrier is of great significance in order to reveal the etiology of Crohn's disease caused by AIEC.

[0003] In the study of the pathogenesis and treatment strategies of Crohn's disease (CD), various in vivo and in vitro models have been widely developed and applied. However, existing models all have their own limitations: animal models are constrained by ethical restrictions and throughput limitations; intestinal organoids are costly, and their enclosed luminal structure makes convenient manipulation and real-time analysis difficult; static Transwell models are insufficient in reproducing the diversity of intestinal cells and the dynamic microenvironment; and intestinal microarrays based on polydimethylsiloxane (PDMS) suffer from problems such as small molecule adsorption and medium evaporation, affecting experimental stability and reproducibility. Currently, no reliable research platform can fully reproduce or accurately simulate the complex physiological structure and microenvironment of the human intestine.

[0004] Therefore, the development of a novel in vitro intestinal model is particularly urgent. This model should possess characteristics such as simple preparation process, controllable cost, and excellent biocompatibility. It should also be able to more completely simulate the structure and function of the intestinal barrier, fully reflect the interaction between microorganisms and the intestinal barrier, and even amplify interaction signals for research purposes, providing a more reliable and efficient technical platform for the study of intestinal inflammation, Crohn's disease, and other inflammatory bowel diseases (IBD).

[0005] Alginate hydrogel microspheres, as a safe and multifunctional biomaterial, have shown broad application prospects in materials science, pharmacy, and life sciences. They possess excellent biocompatibility and controllable degradation, exhibit mild gelation conditions, and are easily functionalized, leading to their widespread application across multiple disciplines. In recent years, by controlling the microstructure of the microsphere surface, it has been demonstrated that they can promote cell adhesion and support the construction of in vitro cell barrier models. Furthermore, the design and fabrication of multi-chamber hydrogel microspheres have further expanded the potential of this type of material in multifunctional integration and the construction of complex biomimetic structures, providing an important foundation for the development of next-generation biocompatible carriers and tissue engineering platforms. Summary of the Invention

[0006] The purpose of this invention is to overcome the above-mentioned shortcomings and provide a microbial-intestinal microsphere co-culture system based on intestinal biomimetic microspheres and its construction method, so as to solve the problem that existing intestinal models have failed to fully reproduce or accurately simulate the interaction between in vivo microorganisms and the intestinal barrier.

[0007] The technical solution of the present invention is a microbial-intestinal microsphere co-culture system based on intestinal biomimetic microspheres, comprising a culture device, a magnetic adsorption device, and a microbial-intestinal microsphere co-culture solution; the microbial-intestinal microsphere co-culture solution is used to study the effects of microorganisms on the intestine; the culture device is used to culture microbial-intestinal microspheres; the magnetic adsorption device is fixed on the surface of the culture device for adsorbing microbial-intestinal microspheres; The intestinal microspheres are specifically double-chambered hydrogel microspheres with a rough, pitted surface, encapsulated by the intestinal barrier. One chamber contains M0 macrophages, and the other contains Fe3O4 nanoparticles.

[0008] Furthermore, the culture device specifically comprises a 96-well plate, a small shaker for placing the 96-well plate, and a corresponding incubator capable of accommodating the small shaker; the magnetic attraction device specifically comprises a magnet fixed to the surface of the well plate.

[0009] Furthermore, no more than 12 wells of microbial-intestinal microsphere co-culture medium are placed in the 96-well plate, and each well contains one microbial-intestinal microsphere; a small magnet is fixed above each well containing co-culture medium, and the spacing between the magnets is sufficient to prevent excessive attraction.

[0010] Another technical solution of the present invention, the method for constructing the microbial-intestinal microsphere co-culture system based on intestinal biomimetic microspheres, comprises the following steps: S1. Preparation of intestinal microspheres: First, prepare double-chamber hydrogel microspheres and change the microstructure of the microsphere surface by calcium ion aerosol; then use matrix gel to modify the surface of pitted microspheres; finally, incubate Caco-2 cells on the surface of pitted microspheres and obtain intestinal microspheres after a period of culture. S2. Construction of the microbial-intestinal microsphere co-culture system: The intestinal microspheres prepared in S1 were placed in a well plate, with each microsphere corresponding to one well. The culture medium for culturing the intestinal microspheres in the well plate was replaced with a microbial suspension containing the bacteria to be studied. The intestinal microspheres and microorganisms were co-cultured to obtain the microbial-intestinal microsphere co-culture system. The specific methods of co-cultivation are: direct cultivation, direct cultivation after magnetic attraction of the co-cultivation system, or cultivation by shaking after magnetic attraction of the co-cultivation system; S3. Quantitative analysis of microbial adhesion and inflammatory signals: Using the microbial-intestinal microsphere co-culture system constructed in S2, different culture conditions and bacterial species were changed to evaluate the effects of different culture methods and bacterial presence conditions on microbial adhesion to the intestinal barrier and the intensity of corresponding inflammatory signals.

[0011] Furthermore, the rough dual-chamber hydrogel microspheres described in step S1 are micron-sized hydrogel microspheres with two equal chambers, wherein M0 macrophages and Fe3O4 nanoparticles are loaded in the two chambers respectively; the rough dual-chamber hydrogel microspheres are obtained by changing the microstructure of the microsphere surface through calcium ion aerosol. The preparation steps are as follows: First, the surface of the rough microspheres is modified with matrix gel. Then, a uniform Caco-2 cell suspension is added to the droplet containing individual microspheres. After incubating upside down in a cell culture incubator for 3 hours, fresh culture medium is added and cultured for another 7 days to prepare intestinal microspheres.

[0012] Furthermore, in S2, magnetic attraction refers to suspending the intestinal microspheres above the bacterial suspension by using the magnetic force of a small magnet on the Fe3O4 in the microspheres; shaking culture refers to placing the well plate on a small track shaker, and then placing the entire system in an incubator for shaking culture.

[0013] Furthermore, the bacteria to be studied added to the co-culture system in S2 and the analytical process are as follows: Intestinal pathogens were assessed by altering different culture conditions to evaluate their impact on microbial adhesion to the intestinal barrier. Intestinal pathogens and probiotics: The intensity of inflammatory signals induced in a co-culture system was observed by adding probiotics.

[0014] The aforementioned enteric pathogens include, but are not limited to: Salmonella, pathogenic Escherichia coli, Staphylococcus aureus, Clostridium difficile, Listeria, Campylobacter jejuni, Vibrio parahaemolyticus, and / or Bacillus cereus.

[0015] The probiotics mentioned above include, but are not limited to: Bifidobacterium longum, Bifidobacterium animalis, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus plantarum and / or Ekkermansia myxotrophus.

[0016] Furthermore, the quantitative analysis method for the amount of microbial adhesion in S3 is as follows: by staining the intestinal pathogens or using genetic engineering methods to stably express fluorescence, the specific situation of intestinal pathogens adhering to the surface of intestinal microspheres can be observed.

[0017] The quantitative analysis method for microbial adhesion using fluorescence is as follows: Enteric pathogens are genetically engineered to stably express green fluorescent protein (GFP), and then cultured using a co-culture method. Fluorescent bacteria adhering to the surface of intestinal microspheres are observed using a fluorescence microscope, and the average gray value is used as an indicator to assess the amount of bacterial adhesion.

[0018] Furthermore, the quantitative analysis method for the inflammatory signals described in S3 is as follows: ELISA, Western Blot, or RT-qPCR methods are used to evaluate the corresponding inflammatory signal indicators in the co-culture system; specific indicators are as follows: 1. Detection indicators for the ELISA detection method: (1) Pro-inflammatory cytokines: TNF-α, IL-1β, IL-6, IL-17, IFN-γ; (2) Anti-inflammatory cytokines: IL-10, TGF-β; (3) Chemokines: IL-8, MCP-1.

[0019] 2. Detection metrics for the Western Blot method: (1) Apoptosis proteins: Caspase-3, Cleaved Caspase-3, Bax, Bcl-2; (2) Signaling pathway proteins: p-NF-κB p65, p-STAT3, p-p38 MAPK; (3) Tight junction proteins (to assess barrier function): Occludin, Claudin, ZO-1.

[0020] 3. Detection indicators for RT-qPCR detection method: (1) Inflammatory factor mRNA: Genes corresponding to the cytokines detected by the above ELISA.

[0021] (2) Antimicrobial peptide: Reg3γ,β-defensin.

[0022] (3) Transcription factors: NF-κB, STAT3.

[0023] When tumor necrosis factor-α (TNF-α) is detected using the ELISA method, the quantitative analysis method for the inflammatory signal is as follows: a microbial-intestinal microsphere co-culture system containing intestinal pathogens and probiotics is cultured, the supernatant of the culture medium is taken, and the content of pro-inflammatory cytokines in it is detected by ELISA as an indicator for evaluating the inflammatory signal in the co-culture system.

[0024] The third technical solution of the present invention is the application of a microbial-intestinal microsphere co-culture system based on intestinal biomimetic microspheres. By changing the types of bacterial suspensions added to the co-culture system, the interaction between different microorganisms and the intestinal barrier is realized, and the interaction between various microorganisms and intestinal organ microspheres is explored; or by using the detection methods of genes, proteins and metabolites, the various interactions between microorganisms and intestinal microspheres are studied.

[0025] The beneficial effects of this invention are as follows: Based on multifunctional intestinal microspheres, this invention develops a suspension-shaking microbial-intestinal microsphere co-culture method by applying suspension and shaking to intestinal microspheres and microorganisms cultured in well plates. This method effectively simulates the dynamic interaction between microorganisms and the intestinal epithelial barrier in the real intestinal microenvironment by applying controllable suspension and periodic shaking to the co-culture system in well plates. These culture conditions not only promote the specific adhesion and colonization of microorganisms on the surface of the microspheres but also amplify the inflammatory signal response induced by microorganisms, significantly improving detection sensitivity. This method, by constructing a biomimetic and functionally controllable dynamic co-culture system of microorganisms and intestinal microspheres, provides a novel in vitro model platform with high biological relevance, good reproducibility, and standardized operation for the study of the pathological mechanisms of intestinal diseases and the screening of therapeutic drugs.

[0026] Preservation of biological material samples: Adhesive invasive Escherichia coli AIEC LF82 was purchased from Shanghai Baosai Biotechnology Co., Ltd.

[0027] Lactobacillus rhamnosus LGG, a publicly disclosed strain, is classified as Lactobacillus rhamnosus GG and has the ATCC cell bank number ATCC 53103. Attached Figure Description

[0028] 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.

[0029] Figure 1This is a schematic diagram of the microbial-intestinal microsphere co-culture system prepared by the present invention.

[0030] Figure 2 This is a schematic diagram of the shaking + suspension culture method in the microbial-intestinal microsphere co-culture system prepared by the present invention.

[0031] Figure 3 This is a microscopic characterization result of the rough double-chamber hydrogel microspheres prepared in Example 1.

[0032] Figure 4 This is a graph showing the results of characterizing the cell viability of the intestinal microspheres prepared in Example 1.

[0033] Figure 5 This is a fluorescent image of GFP-LF82 adhering to intestinal microspheres in the microbial-intestinal microsphere co-culture system of Example 3.

[0034] Figure 6 This is a statistical graph of fluorescence signals of GFP-LF82 adhering in the microbial-intestinal microsphere co-culture system of Example 3.

[0035] Figure 7 This is a data graph of LF82-induced inflammatory signal (TNF-α) production in intestinal microspheres in Example 4. Detailed Implementation

[0036] 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.

[0037] This application provides a method for constructing a microbial-intestinal microsphere co-culture system based on intestinal biomimetic microspheres, which solves the problem that existing intestinal models have failed to fully reproduce or accurately simulate the interaction between in vivo microorganisms and the intestinal barrier.

[0038] 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.

[0039] Example 1: Preparation of intestinal microspheres The preparation of intestinal microspheres includes four steps: preparation of dual-chamber hydrogel microspheres, modification of surface microstructure, matrix gel modification, and construction of intestinal barrier.

[0040] (1) Preparation of dual-chamber hydrogel microspheres: Microspheres containing M0 macrophages (5 × 10⁻⁶ cells) were prepared. 6A solution containing 1.25% alginate and Fe3O4 nanoparticles was injected into the left and right inlets of the microfluidic chip to prepare two-chamber microspheres loaded with M0 macrophages and Fe3O4 on the left and right sides, respectively.

[0041] 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.

[0042] (2) Changes in surface microstructure: On both sides of the path of the droplet, the CaCl2 aerosol injector begins to generate CaCl2 (1 M) mist. The generated alginate droplets, under the impact of the CaCl2 mist, form a pitted structure on the surface and then fall into the collection bath (100 mM CaCl2), forming rough microspheres.

[0043] (3) Matrix gel modification: The obtained microspheres were transferred to a solution containing matrix gel (0.5 mg / mL) for surface modification.

[0044] (4) Construction of the surface intestinal barrier: In order to attach and culture Caco-2 cells on the surface of microspheres, a uniform cell suspension (2×10⁻⁶) was prepared. 7 Cells / mL were added to droplets containing individual microspheres. After incubation in a cell culture incubator for a period of time, fresh culture medium was added. The cell-loaded microspheres were then incubated in MEM medium (20% FBS) containing 5 mM CaCl2 in culture dishes. The entire system was cultured at 37°C and 5% CO2 until Caco-2 cells proliferated and covered the surface of the microspheres, forming a barrier structure.

[0045] The specific preparation process and equipment used in this embodiment are the same as those disclosed in application number 2026102770598, invention title: A biomimetic intestinal organ model based on dual-chamber hydrogel microspheres and its construction method.

[0046] like Figure 3 As shown, the successfully prepared roughened double-chamber hydrogel microspheres exhibit good surface roughness. The overall morphology of the double-chamber crater microspheres loaded with M0 macrophages and Fe3O4 nanoparticles was revealed using bright-field microscopy.

[0047] 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. The successfully fabricated intestinal microspheres were then subjected to cell viability assays, specifically as follows: Figure 4 As shown in the confocal images, by day 7 of culture, the cells on the surface of the microspheres covered the entire microsphere and formed an intestinal barrier, proving that the intestinal cells still had high viability after seven days of culture.

[0048] Example 2: Stable green fluorescent expression of enteropathogenic bacteria In this embodiment, adhesive invasive Escherichia coli (AIEC LF82, Baosai Biotechnology) was used as the enteric pathogen. Genetic engineering methods were used to modify the AIEC LF82 bacteria to stably express green fluorescent protein (GFP). The specific method is as follows: (1) Preparation of high-efficiency AIEC LF82 competent cells: a. Remove LF82 glycerol bacteria from the -80℃ freezer, streak them onto LB agar plates (without antibiotics), and incubate them upside down in a 37℃ incubator for 12-16 hours. Pick a single LF82 colony from the LB agar plate, inoculate it into 10 mL of LB liquid medium, and incubate overnight at 37℃ with shaking until the mid-to-late logarithmic growth stage.

[0049] b. Inoculate the overnight cultured bacterial suspension at a ratio of 1:100 into an Erlenmeyer flask containing 100 mL of LB medium (without antibiotics), and incubate at 37°C with shaking for about 2-3 hours. During this period, measure OD600 at intervals until OD600 reaches 0.4-0.5.

[0050] c. Place the conical flask in ice water and cool rapidly for 10-15 minutes. Then, divide 100 mL of culture medium into two equal portions and transfer them to 50 mL centrifuge tubes. Centrifuge at 3000 r / min for 10 minutes at 4°C. Discard the supernatant, add 30 mL of pre-cooled 0.1 mol / L CaCl2 solution, mix gently, and incubate in ice water for 30 minutes. Centrifuge and resuspend again.

[0051] d. Remove from ice water, centrifuge at 3000 r / min for 10 min at 4℃, discard the supernatant and gently aspirate excess liquid with a pipette. Add 0.5 mL of 0.1 mol / L CaCl2 solution containing 10% glycerol, gently aspirate the liquid with a Pasteur tube, and splash it onto the wall to mix the precipitate. Place on ice. Quickly aliquot into 0.5 mL centrifuge tubes, adding 40 μL of suspension to each tube. Cap the tubes and freeze rapidly in liquid nitrogen, then transfer to -80℃ for storage.

[0052] (2) DNA transformation of competent bacteria: Thaw the competent bacteria prepared in step (1) at -80℃ on ice for 10 min. Transfer 100 μL of competent cells to a new transformation tube. Add 0.1-10 ng (about 3-10 μL) of transformation DNA to the competent cells, mix gently, and place on ice for 30 min. After placing in a 42℃ water bath for 45 s, immediately place on ice for 1-2 min. Add 890 μL of SOC medium (preheated to 37℃) and incubate at 37℃ with shaking for 1 h. Spread an appropriate amount onto a plate and invert the plate in a 37℃ incubator overnight. Confirm the colonies and proceed to the next experiment.

[0053] Example 3 Construction and characterization of a microbial-gut microsphere co-culture system The construction mechanism of the microbial-gut microsphere co-culture system is as follows: Figure 1 As shown, the intestinal microspheres prepared in Example 1 were placed in a 96-well plate, with each microsphere corresponding to one well. The culture medium for culturing the intestinal microspheres in the plate was replaced with MEM medium containing bacteria (20% FBS, penicillin-streptomycin-free). Simultaneously, magnets were placed above the wells to attract the microspheres, keeping them suspended in the culture system and increasing the surface area for contact between the microspheres and microorganisms, thus facilitating adhesion of the intestinal microspheres. Due to the magnetic force between the magnets, the magnets should not be arranged too densely on the well plate cover; hot melt adhesive or double-sided tape can be used to fix the magnets. No more than 12 microbial-intestinal microsphere co-culture systems can be prepared from a single 96-well plate.

[0054] like Figure 2 As shown in the figure, in this embodiment, the prepared co-culture plate is placed on a small shaker for shaking culture. Shaking culture facilitates the active and dynamic distribution of microorganisms in each well of the co-culture system, and the fluid shear stress between the culture medium solution and the intestinal microspheres highly simulates the real human intestinal microenvironment.

[0055] In this embodiment, the adhesiolytic invasive *Escherichia coli* (AIEC LF82, Baosai Biotechnology) and *Lactobacillus rhamnosus* (LGG, ATCC53103), which stably express green fluorescence as described in Example 2, were used as example strains. The example strains were co-cultured with intestinal microspheres. The preparation process of the co-culture medium is as follows: It contains only intestinal microspheres, that is, a single intestinal microsphere is distributed in MEM medium (20% FBS, without penicillin-streptomycin); The culture medium contains intestinal microspheres and GFP-LF82. Specifically, a GFP-LF82 bacterial suspension with an OD600 value of approximately 0.7 is centrifuged to obtain the bacterial pellet. The bacteria are then resuspended in an equal volume of MEM medium (20% FBS, penicillin-streptomycin-free) to prepare a co-culture medium containing GFP-LF82, in which a single intestinal microsphere is distributed.

[0056] The culture medium contains intestinal microspheres and LGG+LF82. Specifically, GFP-LF82 bacterial suspension with an OD600 value of approximately 0.7 and LGG bacterial suspension are taken, centrifuged to obtain bacterial precipitate, the two bacterial precipitates are mixed, and the bacteria are resuspended in an equal volume of MEM medium (20% FBS, without penicillin-streptomycin) to prepare a co-culture medium containing GFP-LF82 and LGG, in which a single intestinal microsphere is distributed.

[0057] The GFP-LF82 or LGG+LF82 prepared in Example 2 were co-cultured with intestinal microspheres for a period of time. After they adhered to the surface of the microspheres, fluorescence images were obtained using a fluorescence microscope. The specific method was as follows: the bacterial and intestinal microsphere system was cultured using different culture methods. The culture methods were divided into four culture conditions: (1) control group: no suspension and shaking were applied; (2) suspension group: only suspension was applied, no shaking was applied; (3) shaking group: only shaking was applied, no suspension was applied; (4) suspension + shaking group: both suspension and shaking were applied.

[0058] Meanwhile, the bacterial species in the co-culture system were divided into two types: (1) GFP-LF82 group: only GFP-LF82 was added; (2) LGG+LF82 group: both LGG and GFP-LF82 were added. The purpose of setting up the LGG + LF82 experimental group was to observe whether LGG would affect the adhesion and invasion of AIEC LF82 to intestinal microspheres.

[0059] Subsequently, at 2 h, 4 h, and 6 h of culture time, the intestinal microspheres in the co-culture system were removed by pipette, placed in physiological saline, and images of the intestinal microspheres with GFP-LF82 attached were taken using a fluorescence microscope.

[0060] Fluorescence images of intestinal microspheres cultured under different conditions were collected, and the fluorescence intensity of GFP-LF82 on the surface of the intestinal microspheres was statistically analyzed using ImageJ software. Specifically, the fluorescence images were imported into ImageJ software, and the selection tool was used to select circular regions containing green fluorescence. The average gray value within these regions was measured, and the average gray value was used as an indicator to assess the amount of GFP-LF82 adhering to the surface of the intestinal microspheres.

[0061] Specific observation results are as follows: Figure 5 and Figure 6 As shown in the image, fluorescence microscopy images revealed that both suspension and shaking cultivation methods were beneficial for LF82 adhesion, while the amount of LF82 adhering in the control group was extremely low. Figure 5 and Figure 6As shown, with only GFP-LF82 added, the control group showed almost no green fluorescence on the surface of the intestinal microspheres; the suspension group showed a small amount of green fluorescence; while in the shaking group and the suspension + shaking group, the green fluorescence signal on the surface of the intestinal microspheres increased significantly. This indicates that both suspension and shaking culture methods are beneficial for LF82 adhesion, with shaking having a greater impact on GFP-LF82 adhesion. Simultaneous application of suspension and shaking culture methods is the most favorable culture condition for GFP-LF82 adhesion to intestinal microspheres.

[0062] like Figure 5 and Figure 6 As shown in the fluorescence microscopy images, the addition of LGG effectively inhibited the adhesion of GFP-LF82. Figure 5 and Figure 6 As shown, after adding LGG, the green fluorescence signal on the surface of intestinal microspheres cultured using different methods was lower than that of the experimental group with only GFP-LF82 added. However, despite this, the experimental data after adding LGG still demonstrate the promoting effect of the suspension + shaking culture method on GFP-LF82 adhesion. That is, after adding LGG, the green fluorescence signal on the surface of intestinal microspheres cultured using the suspension + shaking method was still higher than that of the experimental groups with only suspension or only shaking.

[0063] Example 4: The Influence of Specific Microorganisms on Intestinal Microspheres and the Amplification of Inflammatory Signals This embodiment uses AIEC LF82 as an example strain and employs a microbial-intestinal microsphere co-culture system, causing LF82 to interact with intestinal microspheres and inducing an inflammatory response. Simultaneously, LGG is used to protect the intestinal microspheres and alleviate inflammatory damage.

[0064] In this embodiment, the co-culture time of intestinal microspheres and AIEC LF82 was fixed at 4 hours. Prolonged culture time can easily lead to decreased activity or death of cells on the surface of the intestinal microspheres due to bacterial invasion. The purpose of this embodiment is to observe the secretion of pro-inflammatory cytokines during the adhesion and invasion of intestinal cells by AIEC LF82; therefore, cell death should be minimized to avoid affecting data acquisition.

[0065] In this embodiment, an enzyme-linked immunosorbent assay (ELISA) kit was used to measure the level of tumor necrosis factor-α (TNF-α) in the supernatant of a microbial-intestinal microsphere co-culture system.

[0066] The specific method for the ELISA experiment is as follows: (1) Sample collection and storage: Transfer the culture medium in the co-culture system to a sterile centrifuge tube, centrifuge at 1000 ×g for 10 min at 4℃, and then aliquot the supernatant into small EP tubes and store at -20℃ or below to avoid repeated freeze-thaw cycles.

[0067] (2) Reagent preparation: Bring reagents to room temperature. 30 minutes before the experiment, place the kit and test samples at room temperature. If crystals appear in the concentrated washing solution, place it in a 37°C bath until all crystals dissolve. Prepare the washing solution: Calculate the volume of the diluted washing solution to be used beforehand, then dilute 20× concentrated washing solution with deionized water to 1× working solution. Perform serial dilution of standards: Before opening the container, ensure all lyophilized standards are at the bottom. Add 0.9 mL of SR1 standard / sample diluent to the lyophilized standards (concentration 1000 pg / mL). Let stand for 10-30 minutes until completely dissolved, then gently mix thoroughly before dilution. Perform 2-fold dilutions at the following concentrations: 1000, 500, 250, 125, 62.5, 31.25, and 15.62 pg / mL. 1000 pg / mL is the highest concentration of the standard curve. The SR1 standard / sample diluent is used as well 0 (0 pg / mL) of the standard curve. Prepare the biotinylated antibody working solution by diluting 100× antibody concentrate to 1× working solution with SR2 biotinylated antibody diluent (mix thoroughly before dilution) and adding it to the reaction wells within 30 min. Prepare the enzyme conjugate working solution by diluting 40× concentrated enzyme conjugate to 1× working solution with SR3 enzyme conjugate diluent (mix thoroughly before dilution) and using it within 30 min.

[0068] (3) Detection steps: Set up standard wells, 0 wells, and sample wells respectively. Soak the microplate, add 300 μL of 1× washing buffer, let it stand for 30 seconds, discard the washing buffer, and pat the microplate dry on absorbent paper. Repeat twice. Add standard: Add 100 μL of 2-fold serially diluted standard to the standard wells. Add 100 μL of standard / sample dilution to the 0 wells. Add sample: Add 100 μL of the sample to be tested to the sample wells. Seal the plate with a new sealing film and incubate at 37 ℃ for 90 min. Subsequent washing: Discard the liquid, add 300 μL of washing buffer to each well and wash the plate 4 times. Pat the plate dry on absorbent paper after each wash. Add biotinylated detection antibody: Add 100 μL of biotinylated antibody working solution to the reaction wells. Seal the plate with a new sealing film and incubate at 37 ℃ for 60 min. Wash again, following the same washing steps as before. Add enzyme conjugate: Add 100 μL of enzyme conjugate working solution to each well. Seal the plate using a new sealing membrane and incubate at 37 ℃ for 30 min. Wash again, following the same washing procedure as before. Add substrate for color development: Add 100 μL of TMB substrate to each well, and incubate at 37 ℃ for 15 min in the dark. Add stop solution: Add 50 μL of stop solution to each well, following the same order as the substrate addition. Detection and reading: Within 5 min, perform dual-wavelength detection using a microplate reader, measuring the OD values ​​at the maximum absorption wavelength of 450 nm and the reference wavelength of 630 nm.

[0069] (4) Result Calculation: Measure the OD values ​​at wavelengths of 450 nm and 630 nm, and subtract the OD value at 630 nm from the OD value at 450 nm. Calculate the average OD value of the standards and samples: subtract the OD value of well 0 from the OD value of each standard and sample. Plot a standard curve using Excel software with the standard concentration as the x-axis and the absorbance OD value as the y-axis. The concentration of human TNF-α in the sample can be calculated from the standard curve using the corresponding OD value.

[0070] like Figure 7As shown, TNF-α secretion in the supernatant of intestinal microsphere culture systems co-cultured with LF82 and LGG + LF82 using different culture methods (control group, suspension group, shaking group, suspension + shaking group) was measured using ELISA. Compared with the untreated control group, LF82 invasion led to greater TNF-α secretion from intestinal microspheres, demonstrating that the adhesion and invasion of AIEC LF82 to intestinal microspheres can induce an inflammatory response in intestinal cells. Simultaneously, the suspension + shaking group showed greater TNF-α production than other groups. Combined with the effect of culture method on AIEC LF82 adhesion in Example 2, the adhesion of AIEC LF82 and the production of TNF-α from intestinal microspheres can be correlated. That is, the suspension + shaking culture method promotes the adhesion of AIEC LF82 to intestinal microspheres; the increased adhesion induces the production of more pro-inflammatory cytokines by intestinal microspheres, leading to a more severe inflammatory response.

[0071] like Figure 7 As shown, the addition of LGG effectively reduced the secretion of pro-inflammatory cytokines (TNF-α). Co-culturing with LGG significantly reduced the production of TNF-α in intestinal microspheres, demonstrating that LGG, in addition to inhibiting AIEC LF82 adhesion, also possesses anti-inflammatory effects, alleviating the inflammatory response of intestinal microspheres.

[0072] This invention, based on multifunctional intestinal microspheres, develops a suspension-shaking microbe-intestinal microsphere co-culture method by applying suspension and shaking to intestinal microspheres and microorganisms cultured in well plates. This method effectively simulates the dynamic interaction between microorganisms and the intestinal epithelial barrier in the real intestinal microenvironment by applying controlled suspension and periodic shaking to the co-culture system in the well plate. These culture conditions not only promote the specific adhesion and colonization of microorganisms on the microsphere surface but also amplify the inflammatory signal response induced by microorganisms, significantly improving detection sensitivity. This method, by constructing a biomimetic and functionally controllable dynamic co-culture system of microorganisms and intestinal microspheres, provides a novel in vitro model platform with high biological relevance, good reproducibility, and standardized operation for the study of the pathological mechanisms of intestinal diseases (such as Crohn's disease) and the screening of therapeutic drugs.

[0073] 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.

[0074] 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 microbial-intestinal microsphere co-culture system based on intestinal biomimetic microspheres, characterized in that: The system includes a culture device, a magnetic adsorption device, and a microbial-intestinal microsphere co-culture medium; the microbial-intestinal microsphere co-culture medium is used to study the effects of microorganisms on the intestine; the culture device is used to culture the microbial-intestinal microspheres; and the magnetic adsorption device is fixed to the surface of the culture device to adsorb the microbial-intestinal microspheres. The intestinal microspheres are specifically double-chambered hydrogel microspheres with a rough, pitted surface, encapsulated by the intestinal barrier. One chamber contains M0 macrophages, and the other contains Fe3O4 nanoparticles.

2. The microbial-intestinal microsphere co-culture system based on intestinal biomimetic microspheres as described in claim 1, characterized in that: The culture device specifically comprises a 96-well plate, a small shaker for placing the 96-well plate, and a corresponding incubator capable of accommodating the small shaker; the magnetic attraction device specifically comprises a magnet fixed to the surface of the well plate.

3. The microbial-intestinal microsphere co-culture system based on intestinal biomimetic microspheres as described in claim 2, characterized in that: The 96-well plate contains no more than 12 wells of microbial-intestinal microsphere co-culture medium, and each well contains one microbial-intestinal microsphere. A small magnet is fixed above each well containing co-culture medium, and the spacing between the magnets is sufficient to prevent excessive attraction.

4. The method for constructing the microbial-intestinal microsphere co-culture system based on intestinal biomimetic microspheres as described in claim 1, characterized in that: The steps are as follows: S1. Preparation of intestinal microspheres: First, prepare double-chamber hydrogel microspheres and change the microstructure of the microsphere surface by calcium ion aerosol; then use matrix gel to modify the surface of pitted microspheres; finally, incubate Caco-2 cells on the surface of pitted microspheres and obtain intestinal microspheres after a period of culture. S2. Construction of the microbial-intestinal microsphere co-culture system: The intestinal microspheres prepared in S1 were placed in a well plate, with each microsphere corresponding to one well. The culture medium for culturing the intestinal microspheres in the well plate was replaced with a microbial suspension containing the bacteria to be studied. The intestinal microspheres and microorganisms were co-cultured to obtain the microbial-intestinal microsphere co-culture system. The specific methods of co-cultivation are: direct cultivation, direct cultivation after magnetic attraction of the co-cultivation system, or cultivation by shaking after magnetic attraction of the co-cultivation system; S3. Quantitative analysis of microbial adhesion and inflammatory signals: Using the microbial-intestinal microsphere co-culture system constructed in S2, different culture conditions and bacterial species were changed to evaluate the effects of different culture methods and bacterial presence conditions on microbial adhesion to the intestinal barrier and the intensity of corresponding inflammatory signals.

5. The method for constructing a microbial-intestinal microsphere co-culture system based on intestinal biomimetic microspheres as described in claim 4, characterized in that: The rough dual-chamber hydrogel microspheres described in step S1 are micron-sized hydrogel microspheres with two equal chambers, each loaded with M0 macrophages and Fe3O4 nanoparticles respectively; the surface microstructure of the microspheres is altered by calcium ion aerosol to obtain the rough dual-chamber hydrogel microspheres. The preparation steps are as follows: First, the surface of the rough microspheres is modified with matrix gel. Then, a uniform Caco-2 cell suspension is added to the droplet containing individual microspheres. After incubating upside down in a cell culture incubator for 3 hours, fresh culture medium is added and cultured for another 7 days to prepare intestinal microspheres.

6. The method for constructing a microbial-intestinal microsphere co-culture system based on intestinal biomimetic microspheres as described in claim 4, characterized in that: S2 Magnetic attraction refers to using a small magnet to suspend the intestinal microspheres above the bacterial suspension by applying the magnetic force of Fe3O4 in the microspheres; shaking culture refers to placing the well plate on a small track shaker, and then placing the entire system in an incubator for shaking culture.

7. The method for constructing a microbial-intestinal microsphere co-culture system based on intestinal biomimetic microspheres as described in claim 4, characterized in that S2 The bacteria to be studied added to the co-culture system and the analytical process are as follows: Intestinal pathogens were assessed by altering different culture conditions to evaluate their impact on microbial adhesion to the intestinal barrier. Intestinal pathogens and probiotics: The intensity of inflammatory signals induced in a co-culture system was observed by adding probiotics.

8. The method for constructing a microbial-intestinal microsphere co-culture system based on intestinal biomimetic microspheres as described in claim 7, characterized in that: The quantitative analysis method for the amount of microbial adhesion in S3 is as follows: by staining the intestinal pathogens or using genetic engineering methods to make them stably express fluorescence, the specific situation of intestinal pathogens adhering to the surface of intestinal microspheres can be observed. The quantitative analysis method for inflammatory signals described in S3 is as follows: ELISA, Western Blot, or RT-qPCR are used to evaluate the corresponding inflammatory signal indicators in the co-culture system.

9. The method for constructing a microbial-intestinal microsphere co-culture system based on intestinal biomimetic microspheres as described in claim 8, characterized in that: The quantitative analysis method for microbial adhesion described in S3 is as follows: the bacteria under study are modified by genetic engineering to stably express green fluorescent protein GFP, and after co-culture, the fluorescent bacteria adhering to the surface of intestinal microspheres are observed by fluorescence microscopy, and the average gray value is used as an indicator to evaluate the bacterial adhesion amount. The quantitative analysis method for inflammatory signals described in S3 is as follows: a microbial-intestinal microsphere co-culture system containing intestinal pathogens and probiotics is cultured, the supernatant of the culture medium is taken, and the content of pro-inflammatory cytokines in it is detected by ELISA as an indicator for evaluating inflammatory signals in the co-culture system.

10. The application of a microbial-intestinal microsphere co-culture system based on intestinal biomimetic microspheres, characterized by: By changing the types of bacterial suspensions added to the co-culture system, the interactions between different microorganisms and the intestinal barrier can be realized, and the interactions between various microorganisms and intestinal organ microspheres can be explored; or by using methods for detecting genes, proteins, and metabolites, various interactions between microorganisms and intestinal microspheres can be studied.

Images