A method for visually tracking human bronchial organoids infected with Mycoplasma pneumoniae

Abstract

This invention relates to the field of pathogen infection detection technology, specifically a method for visually tracking human bronchial organoids infected with Mycoplasma pneumoniae. It includes the following steps: (1) Constructing human bronchial organoids: culturing mature human bronchial organoids with cavities, the organoids comprising ciliated cells, basal cells, goblet cells, and Club cells, having a polarized three-dimensional structure with the apex facing outwards; (2) Mycoplasma pneumoniae culture and fluorescent labeling: Mycoplasma pneumoniae is amplified and cultured in MP complete medium, and then fluorescently labeled using CellTrace far-infrared cell staining solution; (3) Co-culture for infection: Mycoplasma pneumoniae fluorescently labeled in step (2) is co-cultured with the human bronchial organoids from step (1) for infection; (4) Live cell dynamic fluorescence tracking: Non-invasive in situ continuous imaging is performed on the human bronchial organoids infected by MP in step (3). This invention can achieve non-invasive dynamic tracking of the entire process of MP infection.

Description

Technical Field

[0001] This invention relates to the field of pathogenic microorganism infection detection technology, specifically a method for visually tracking Mycoplasma pneumoniae infection of human bronchial organoids. Background Technology

[0002] Mycoplasma pneumoniae (MP) is one of the main pathogens causing lower respiratory tract infections, bronchitis, and community-acquired pneumonia in children. It easily colonizes, adheres to, and proliferates in the airway epithelium, inducing airway inflammation and damage as well as related respiratory complications.

[0003] Current research on the pathogenesis, infection process, and drug screening of Mycoplasma pneumoniae largely relies on traditional two-dimensional cell culture models and in vivo animal models. Traditional two-dimensional cell culture models use monolayer cultured bronchial epithelial cells, which cannot simulate the three-dimensional tissue structure, intercellular interactions, polarity (apical-basal side), and mucociliary clearance function of the human bronchus, resulting in significant differences between the infection process and the actual in vivo situation. While experimental animal models have a certain degree of physiological systemicity, they suffer from species differences (MP host specificity), cannot perform real-time in-situ high-definition imaging, have long experimental cycles, high costs, strict ethical constraints, and are difficult to analyze the fine dynamic processes at the cellular level.

[0004] Current technologies typically use fluorescent labeling to study the infection process of Mycoplasma pneumoniae (MP). However, conventional genetic engineering (such as fluorescent protein labeling) requires genetic modification of Mycoplasma pneumoniae to achieve stable expression of fluorescent proteins. This process is technically challenging, time-consuming, and inefficient. Furthermore, the expression of exogenous genes may alter the growth characteristics, virulence, or infectivity of MP, affecting the reliability of the observation results. Conventional chemical fluorescent dye labeling generally suffers from rapid fluorescence quenching and high cytotoxicity, which can easily affect the normal metabolism, proliferation, and airway epithelial adhesion and colonization capabilities of Mycoplasma pneumoniae, making it difficult to meet the requirements for long-term in vivo dynamic tracking.

[0005] In terms of detection methods, the current mainstream detection methods for Mycoplasma pneumoniae mostly adopt techniques such as nucleic acid PCR detection, plate viable bacteria counting, protein immunoblotting, and tissue immunofluorescence. All of these require sample lysis, fixation, or inactivation, which are destructive, endpoint-based, and static detection methods. They can only obtain detection data at a single time point and cannot achieve non-invasive, continuous in situ observation on the same sample. It is difficult to dynamically track the complete temporal infection process of Mycoplasma pneumoniae adhering to, colonizing, proliferating, and spreading on the airway epithelial surface, and cannot fully restore the true infection evolution pattern. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a method for visually tracking human bronchial organoids infected with Mycoplasma pneumoniae. A biomimetic model of human bronchial organoids with complete cellular components and an "outward-facing" polarization structure is constructed. The method uses far-infrared fluorescent dye labeling, which is simple to operate, low in toxicity and resistant to quenching, and does not affect the activity of mycoplasma. It can achieve non-invasive dynamic tracking of the entire process of MP infection.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a method for visually tracking Mycoplasma pneumoniae infection of human bronchial organoids, comprising the following steps: (1) Construction of human bronchial organoids: mature human bronchial organoids with cavities were obtained by culturing, wherein the organoids contain ciliated cells, basal cells, goblet cells and Club cells, and have a polarized three-dimensional structure with the apex facing outward; (2) Culture and fluorescent labeling of Mycoplasma pneumoniae: After Mycoplasma pneumoniae was amplified and cultured in MP complete medium, the cultured Mycoplasma pneumoniae was fluorescently labeled using CellTrace far-infrared cell staining solution; (3) Co-culture for infection: The fluorescently labeled Mycoplasma pneumoniae from step (2) was co-cultured with the human bronchial organoids from step (1) for infection; (4) Live cell dynamic fluorescence tracking: Non-invasive in situ continuous imaging of human bronchial organoids after MP infection in step (3).

[0008] Preferably, the process of cultivating mature human bronchial organoids with cavities in step (1) includes: taking airway tissue for enzymatic digestion; separating the digested tissue to obtain a single-cell suspension; resuspending the single-cell suspension in matrix gel and inoculating it; culturing it in bronchial organoid complete culture medium after solidification; passage it when the organoid diameter reaches 100~200 μm; and dissociating the matrix gel after the bronchial organoids grow cavities.

[0009] Preferably, the human bronchial organoids in step (1) are differentiated and mature, expressing the ciliated cell marker Ac-α-Tub, the basal cell marker P63, the goblet cell marker MUC5AC, and the Club cell marker CC10, and the cells are tightly connected to each other through the tight junction protein zonula occludens-1.

[0010] Preferably, the MP complete culture medium in step (2) contains 20% fetal bovine serum, 800 U / mL penicillin, 0.5% glucose, 0.2% sodium pyruvate and 1% phenol red.

[0011] Preferably, the culture conditions in step (2) are 37°C, 95% N2, and 5% CO2, and cultured for 5 to 7 days. When the culture medium turns from red to orange-yellow and there is no flocculent precipitate, the culture is expanded.

[0012] Preferably, the fluorescent labeling process in step (2) is as follows: collect Mycoplasma pneumoniae cells, resuspend them in PBS, add CellTrace far-infrared cell staining solution, and incubate in a water bath at 37°C for 45 minutes.

[0013] Preferably, the culture and infection conditions in step (3) are cultured in a CO2 environment at 37°C for 12 hours.

[0014] Preferably, step (4) is performed using a conventional fluorescence microscope or a laser confocal microscope. This allows for non-invasive, in-situ continuous imaging of the organoids to track the adhesion, proliferation, and spread of Mycoplasma pneumoniae.

[0015] Preferably, the method for visually tracking human bronchial organoids infected with Mycoplasma pneumoniae further includes: performing immunofluorescence staining and confocal localization observation on human bronchial organoids infected with Mycoplasma pneumoniae in step (3). This verifies the fluorescence stability and long-term dynamic tracking capability of CellTrace far-infrared dye.

[0016] Preferably, the method for visually tracking human bronchial organoids infected with Mycoplasma pneumoniae further includes: detecting the biological activity of MP in human bronchial organoids after MP infection in step (3). This verifies whether MP retains its infectious activity after fluorescent labeling.

[0017] Preferably, the method for visually tracking Mycoplasma pneumoniae infection in human bronchial organoids further includes: detecting the mRNA of MP in human bronchial organoids after MP infection in step (3). This quantifies the proliferation load of MP in organoids at the gene level and verifies the change in infection severity over time.

[0018] The beneficial effects of this invention are: The method for visually tracking Mycoplasma pneumoniae infection of human bronchial organoids provided by this invention includes steps such as constructing human bronchial organoids, culturing and fluorescently labeling Mycoplasma pneumoniae, co-culturing with infection, and dynamic fluorescence observation of live cells. It utilizes a specialized chemical dye for cell labeling, whose fluorescence can last for up to one month without quenching and has no adverse effect on the physiological activity of Mycoplasma pneumoniae (MP). Leveraging the advantages of this dye's ease of operation and high labeling efficiency, it effectively overcomes the inherent defects of traditional genetically engineered fluorescent protein labeling, which requires genetic modification of MP, has a long experimental cycle, high operational complexity, and low construction efficiency. It also avoids the technical drawbacks of conventional chemical dyes, such as high biotoxicity, rapid fluorescence quenching, and easy interference with MP's normal metabolism, proliferation, and airway adhesion and colonization. This invention achieves long-term stable fluorescence tracing without requiring gene editing of MP, enabling continuous, non-destructive, in-situ dynamic observation of the chronic, persistent MP infection process that can last for several weeks. It breaks through the limitation of traditional endpoint detection, which can only obtain a single time point, and can completely capture the entire process of MP infection occurrence, development, adhesion, and spread.

[0019] This invention constructs a human bronchial organoid with an outward-polarized apex structure as an infection research model. This organoid fully retains the three-dimensional structure, multiple functional cell components, and epithelial polarization physiological characteristics of natural bronchial tissue. Compared with the shortcomings of traditional culture models, such as simple structure and lack of tissue polarity and microenvironment simulation capabilities, it can highly biomimetically reproduce the physiological microenvironment of human bronchus and the natural infection pathway of Myxobolus leukemia (MP), which is more in line with the real pathological process of infection in the human body. The biomimicry of the model and the reliability of the experiment are greatly improved.

[0020] The fluorescent dye used in this application is safe and low in toxicity. The labeling process and post-labeling do not affect the growth viability, adhesion ability and infection characteristics of MP. It allows operators to perform non-invasive in situ repeated observations on the same organoid sample at different infection time points. It can accurately locate the initial adhesion site of MP on the organoid surface and quantitatively characterize the dynamic change of infection degree over time. It avoids the experimental errors caused by individual differences in multiple batches of samples in traditional methods, and the observation data has stronger continuity, comparability and accuracy.

[0021] This invention establishes a complete and standardized operating procedure, from MP fluorescent labeling, organoid co-culture infection, and timed in vivo imaging to subsequent immunofluorescence verification, CCU cell viability detection, and RT-qPCR molecular quantitative detection. The procedures are standardized and controllable, with good experimental repeatability and stable and reliable results, facilitating method replication and cross-sectional data comparison between different laboratories. This invention not only systematically elucidates the spatiotemporal infection patterns of MP adhesion, colonization, and proliferation, but also supports in-depth research into fundamental scientific issues such as MP pathogenicity, host infection specificity, and immune escape mechanisms. Furthermore, it provides a stable, biomimetic, and continuously dynamic in vitro technology platform for screening clinical anti-MP candidate drugs, evaluating efficacy, and developing prevention and treatment strategies, possessing significant scientific research value and promising clinical application prospects. Attached Figure Description

[0022] Figure 1 Image showing the immunofluorescence identification results of human bronchial organoid cells.

[0023] Figure 2 This is a real-time live cell observation image under a standard fluorescence microscope (40x magnification).

[0024] Figure 3 This is a confocal microscope image of live cells (40x magnification).

[0025] Figure 4 This is a confocal microscopy image (20×) of human bronchial organoids infected with *M. bronchiseptica*, verified by immunofluorescence staining.

[0026] Figure 5 The image shows the results of MP activity detection using the CCU method.

[0027] Figure 6 This is a graph showing the mRNA expression levels of MP in human bronchial organoids at different time points after MP infection. Detailed Implementation

[0028] To enable those skilled in the art to better understand the technical solution of the invention, the invention will be further described in detail below with reference to specific embodiments.

[0029] Example 1: Culture, identification and preparation of human bronchial organoids 1. Culture and identification of human bronchial organoids Step 1: Clinical Sample Collection and Cryogenic Transport Children's airway tissues were collected via bronchoscopy, and the samples were stored in DMEM culture medium and transported to the laboratory at 4°C. This was done to maintain the in vitro viability of the tissue cells, prevent cell death and degradation, and ensure the quality of the original tissues for subsequent organoid construction.

[0030] Step 2: Tissue cleaning and enzymatic digestion Rinse the tissue twice with DPBS, transfer the tissue to 1 ml of digestion solution, and place it on a shaker at 37°C for 1 hour to digest.

[0031] Digestion solution preparation: 1 ml of AdDF+++ medium (Advanced DMEM / F12 medium containing 1× GlutaMAX, 10 mM HEPES and antibiotics) contains 400 U / ml collagenase I (Sigma, 9001-12-1), 0.25 mg / ml proteinase E (Sigma, P5147), 10 μM Y27632 (Selleck, S6390) and 10 U / ml DNAse I (Sigma, 10104159001).

[0032] Step 3: Filtration and centrifugation to obtain single-cell suspension Use a 1 ml pipette to repeatedly pipette the tissue suspension to break up any incompletely digested tissue fragments. Filter the tissue through a 40 μm cell filter to remove large tissue fragments and obtain uniform single cells or small cell clusters. Centrifuge at 200g for 3 min, discard the supernatant, and then wash the cells twice with 1 ml of AdDF+++ culture medium and centrifuge at 200g for 3 min to purify the cells.

[0033] Step 4: Matrigel resuspension and inoculation Cell pellets were resuspended in Matrigel (Corning, 356231) and seeded into 24-well plates at a ratio of 30 μl Matrigel to 5000 cells per well. This was to mimic the in vivo extracellular matrix microenvironment, providing a three-dimensional scaffold to support organoid formation and control cell density, ensuring sufficient space for organoid growth and formation efficiency.

[0034] Step 5: Add culture medium The Matrigel was allowed to stand at 37°C for 15 minutes to fully solidify and form a stable three-dimensional structure, preventing the cells from floating. 500 μl of complete bronchial organoid culture medium was added to each well, and the culture plate was placed under standard culture conditions (37°C, 5% CO2) for culture. The culture medium was changed every 4 days. When the organoid diameter reached 100~200 μm, it was passaged.

[0035] Complete culture medium for bronchial organoids consisted of AdDF+++, 1×B27 (Gibco, 0080085SA), 5 mM nicotinamide (Sigma, N0636), 1.25 mM N-acetylcysteine ​​(Sigma, A0737), 500 ng / ml R-spondin1 (R&D, 4645), 25 ng / ml recombinant human FGF7 (PeproTech, 450-61), 100 ng / ml recombinant human FGF10, 100 ng / ml recombinant human Noggin (R&D, 6057), 5 μM Y27632 (CST, 13624), 500 nMSB202190 (Selleck, S1077), and 500 nM A-8301 (Selleck, S8301).

[0036] Step 6: Identification of organoid cell composition and structure Combination Figure 1 The immunofluorescence identification results (60×) were used to analyze the cell composition, cell connectivity and polar structure of the human bronchial organoid constructed in this application. Figure 1 A diagram shows the cell type identification of the human bronchial organoid. It can be seen that the airway organoid constructed in this application is composed of ciliated cells, basal cells, goblet cells, and Club cells. These cell types are identified by the expression of specific markers: ciliated cells express acetylated α-tubulin (Ac-α-Tub, Santa Cruz, sc-23950), confirming that the organoid possesses a ciliated structure and ciliary beating function; basal cells express P63 (Abcam, ab124762), suggesting the presence of bronchial epithelial stem cells / progenitor cells in the organoid, possessing self-renewal and differentiation capabilities; goblet cells express mucin-5AC (MUC5AC, Abcam, ab198294), indicating that the organoid can secrete mucus and possesses airway mucosal barrier function; Club cells express CC10 (Santa Cruz, sc-365992), proving that the organoid contains non-ciliated columnar secretory cells of the bronchus, consistent with the physiological cell composition of the human airway. The above demonstrates that the airway organoids constructed in this application highly simulate the cellular diversity of human bronchial epithelium, with all four key functional cells being normally differentiated and expressed, thus truly reflecting the cellular composition characteristics of airway tissues in vivo.

[0037] Figure 1Image B shows an immunofluorescence staining image (60×) of the tight junctions between cells in the human bronchial organoid constructed in this invention (where DAPI is for nuclear staining, ZO-1 is for labeling tight junction proteins, and Merge is a superimposed image of the fluorescence signals of ZO-1 and DAPI). As can be seen from the image, cells in the organoid are tightly connected by the tight junction protein zonula occludens-1 (ZO-1, Abcam, ab216880), with clear cell boundaries and tight junctions, forming a complete epithelial barrier structure. This indicates that stable tight junctions are formed between cells within the organoid, providing a basis for the establishment of an epithelial barrier structure and polarity consistent with that of the human bronchus.

[0038] Figure 1 C is an image identifying the polar structure of a human bronchial organoid (where DAPI is for nuclear staining, ZO-1 is for labeling tight junction proteins, Ac-α-Tub is for labeling ciliated cells, and Merge is a multi-fluorescence signal overlay image). The polarization direction of the organoid is determined by the relative positioning of Ac-α-Tub and ZO-1. As shown in the figure, Ac-α-Tub mainly covers the outer surface of the airway organoid, while ZO-1 is located below Ac-α-Tub, i.e., in the region below the apical membrane. This indicates that the human bronchial organoid constructed in this application exhibits a "apical-outward" polarization characteristic, with cilia exposed towards the outer side of the organoid, consistent with the orientation of cilia in the lumen of the human bronchus, which is more conducive to the adhesion, infection, and in-situ real-time observation of Mycoplasma pneumoniae (MP).

[0039] 2. Preparation of human bronchial organoids before infection Step 1: Collect mature organoids When the organoids grow into cavities with a diameter of about 200 μm, use a pipette to aspirate the culture medium and blow it to the bottom of the culture well. After eluting the organoids and Matrigel together, transfer them to a 1.5 mL EP tube, centrifuge at 200 g for 3 min, and discard the supernatant.

[0040] Step 2: Matrigel removal Add an equal volume of Cell Recovery (Corning, 354253) to the precipitate, mix by pipetting, place on ice for 30 min, then centrifuge at 200g for 3 min and discard the supernatant.

[0041] Step 3: Washing Add 1 ml of AdDF+++ culture medium to the precipitate obtained in step 2, mix well by pipetting, and divide into two tubes for later use. Centrifuge at 200g for 3 min for later use.

[0042] After processing according to this embodiment, human bronchial organoids with no matrix gel encapsulation, intact structure, good activity, and uniform grouping can be obtained, which can be directly used for subsequent MP labeling, infection co-culture, and dynamic visualization tracking experiments.

[0043] Example 2: Culture and far-infrared fluorescent labeling of Mycoplasma pneumoniae (MP) 1. Experimental Objective A stable MP resuscitation and amplification culture system was established, and CellTrace far-infrared fluorescent dye was used to safely, efficiently, and for a long time label MP, so as to obtain fluorescently labeled MP that can be used to infect human bronchial organoids without affecting virulence and activity.

[0044] 2. Operating Procedures (1) Recovery and expanded culture of MP Perform aseptic procedures in a biosafety cabinet. Take 1 ml of frozen MP bacterial suspension (ATCC) into a T25 culture flask and add 5 ml of MP complete medium (containing 20% ​​fetal bovine serum, 800 U / ml penicillin, 0.5% glucose, 0.2% sodium pyruvate, and 1% phenol red). Then, transfer the bacterial suspension to the culture flask and mix by pipetting. Incubate at 37°C, 95% N2, and 5% CO2 for 5-7 days. When the medium changes from red to orange-yellow and there is no flocculent precipitate, the culture can be expanded at a ratio of 1:5 (bacterial suspension: MP complete medium).

[0045] (2) Far-infrared fluorescent labeling of MP ① Take an appropriate amount of MP culture solution, centrifuge at 12000 rpm for 15 min, and discard the supernatant; ② Resuspend the precipitate in 200µl PBS solution, add 0.2µl CellTrace far-infrared cell staining solution (ThermoFisher, catalog number: C34572, staining solution concentration: 1 µM), mix well, and incubate in a water bath at 37℃ for 45 min; add 2mL MP complete culture medium to resuspend, centrifuge at 12000rpm for 15 min, and discard the supernatant to remove unbound dye; ③ Add complete bronchial organoid culture medium to adjust the required multiplicity of infection, resuspend the precipitate, and add it to one of the precipitates of the organoids prepared in Example 1. Mix well and then add to an ultra-low adsorption 48-well plate (selection, 11418). The control group was given an equal volume of complete bronchial organoid culture medium, without MP, and the rest of the operation was the same.

[0046] Example 3: Co-culture of Mycoplasma pneumoniae and human bronchial organoids for infection 1. Purpose of Implementation Fluorescently labeled MP and prepared human bronchial organoids were subjected to standardized infection, washing, re-embedding, and continued culture to establish a stable and reproducible MP-infected organoid model for subsequent dynamic observation and detection.

[0047] 2. Operating Procedures ① The sample culture plate prepared in Example 2 was placed in a CO2 incubator at 37°C and cultured for 12 hours to achieve co-culture infection of MP and organoids.

[0048] ② Transfer the suspension to a new EP tube, centrifuge at 200g for 3 minutes, discard the supernatant, and collect the post-infected organoids.

[0049] ③ Add 1 ml PBS to resuspend the precipitate, mix by pipetting and washing, centrifuge again at 200 g for 3 min, remove the supernatant, and wash away any unadhered and uninfected free MP.

[0050] ④ After mixing Matrigel with the samples from the infected / uninfected control group, add 40 μL / well to the bottom of the plate.

[0051] ⑤ After placing the plate in a 37°C incubator for 10 minutes, invert the plate and continue to place it for another 10 minutes to allow the Matrigel to solidify completely.

[0052] ⑥ Add 500µl of complete bronchial organoid culture medium to each well and place in a 37℃ cell culture incubator for further culture.

[0053] This embodiment successfully constructs a stable, uniform, and reproducible human bronchial organoid model infected with *MP*. The organoid has a complete three-dimensional structure and the *MP* infection process is controllable. It can be directly used for subsequent live cell dynamic fluorescence observation, fixation and staining, gene detection, and viability analysis.

[0054] Example 4: Dynamic fluorescence observation of live cells after infection 1. Experimental Objective We conducted non-invasive, continuous, in-situ dynamic observation of human bronchial organoids infected with Mycoplasma gondii, recording the adhesion, spread, and infection process of Mycoplasma gondii in real time.

[0055] 2. Operating Procedures Live-cell fluorescence imaging was performed on human bronchial organoids infected with *MP* obtained in Example 3 at 24 hpi, 48 hpi, and 72 hpi post-infection to track the entire process of *MP* adhesion, colonization, proliferation, and diffusion on the organoid surface in real time, objectively reflecting the spatiotemporal dynamics of infection. The observation equipment used was a conventional fluorescence microscope (40x magnification) and a laser confocal microscope (40x magnification), with excitation wavelengths of 633 / 635 nm. CellTrace far-infrared fluorescently labeled *MP* signals were specifically acquired to achieve long-term, stable, and visualized monitoring of the infection process.

[0056] 3. Experimental Conclusions Figure 2 This is a real-time live-cell observation image using a conventional fluorescence microscope (40x magnification). Conventional fluorescence microscopy can quickly and intuitively reflect the overall dynamic trend of *MP* infection, meeting the needs of batch, real-time monitoring. As shown in the figure, at 24 hpi post-infection, *MP* fluorescence signals initially appeared on the organoid surface, indicating that *MP* had completed initial adhesion. With the extension of infection time to 48-72 hpi, the fluorescence signal on the organoid surface gradually increased and its range continued to expand, indicating that *MP* continuously proliferated and spread on the organoid surface, and the degree of infection significantly worsened over time.

[0057] Figure 3 The image shows a confocal microscopy observation of live cells (40x magnification). As can be seen, confocal imaging more clearly reveals the fine distribution and localization of *MP* on the organoid surface, with sharp fluorescence signal boundaries and a clean background. At 24 hpi, *MP* appears as scattered dots on the outer surface of the organoid; at 48 hpi, the area and brightness of the fluorescence signal are significantly increased; and at 72 hpi, the fluorescence signal is continuously distributed over a large area, covering most of the organoid surface, consistent with the trend observed under ordinary fluorescence microscopy. The confocal results further confirm that *MP* mainly acts on the outer ciliated surface of the organoid, which highly matches the "apex-outward" polarization structure of the organoid constructed in this invention, and can realistically simulate the infection path of *MP* within the human bronchial lumen.

[0058] Both imaging methods confirmed that the CellTrace far-infrared fluorescent labeling used in this invention is stable, durable, and without significant quenching, enabling continuous dynamic visualization and tracking of the MP infection process. Within 24-72 hpi after infection, MP exhibits a typical infection pattern of time-dependent adhesion, proliferation, and spread on the surface of human bronchial organoids, indicating that this method can stably and reliably reproduce the real infection process of MP in the respiratory epithelium.

[0059] Example 5: Fixation and Immunofluorescence Staining Identification of Post-Infection Samples 1. Purpose of Implementation The samples at the infection endpoint were fixed and stained to verify the accuracy of the fluorescent labeling, locate the site of MP infection, and corroborate the results with in vivo observation.

[0060] 2. Operating Procedures ① Sample fixation: At the end of the infection time, the culture medium was aspirated, and PFA was added for fixation at room temperature for 30 min; after rinsing twice with PBS, the sample was stored at 4℃.

[0061] ② Blocking: Add 200 μL of goat serum to each well and block at room temperature for 1 hour. Discard the blocking solution.

[0062] ③ Primary antibody incubation: Add 50 μL of MP primary antibody (abcam, ab20704, 1:100) prepared with goat serum to each well, incubate overnight at 4°C, discard the primary antibody, and wash 5 times with PBS.

[0063] ④ Secondary antibody incubation: Add 200 μL of FITC-labeled secondary antibody diluted 1:800 to each well, incubate at room temperature in the dark for 1 h, and wash 5 times with PBS.

[0064] ⑤ Nuclear staining: Add 200 μL of DAPI (Beyotime, C1002) diluted 1:1000 to each well, incubate in the dark for 10 min, and wash thoroughly with PBS.

[0065] ⑥ Confocal microscopy imaging: Observe and photograph under a confocal microscope.

[0066] 3. Experimental Conclusions Figure 4 The image shows a confocal microscope (20×) observation of human bronchial organoids infected with *Polygonum multiflorum* (MP) after fixation and immunofluorescence staining verification. DAPI represents nuclear fluorescence staining; MP (Rb 488) represents MP signal labeled with MP-specific primary antibody combined with FITC fluorescent secondary antibody; MP (647) represents MP signal directly labeled with CellTrace far-infrared fluorescence; and Merge represents a superimposed image of multichannel fluorescence signals from DAPI, MP (647), and MP (Rb 488). The image shows that at 24 hpi post-infection, the MP antibody fluorescence signal was weak, appearing as scattered dots on the organoid surface, corresponding to the early adhesion stage of MP. As the infection time increased to 72 hpi, the MP antibody fluorescence signal significantly increased, and the distribution range expanded markedly, forming a continuous sheet-like coverage of the organoid surface. This indicates that MP continuously proliferated and spread on the organoid surface, and the degree of infection increased in a time-dependent manner, reflecting the spatiotemporal variation of the fluorescence signal. Furthermore, the CellTrace far-infrared labeling signal highly overlaps with the staining signal of MP-specific antibodies, and the Merge plot shows a perfect match in fluorescence location, proving that the CellTrace far-infrared dye used in this invention is accurate and highly specific. The fluorescence signal truly reflects the infection location and distribution of MP, with no false positives or non-specific binding. Moreover, the MP antibody fluorescence is mainly distributed on the outer surface of the organoid, which is highly consistent with the "apex-outward" polarization structure of the bronchial organoid constructed in this invention, further demonstrating that this model can realistically simulate the infection process of MP in the ciliated epithelium inside the human bronchial lumen.

[0067] After 15 days of storage, the samples were validated by immunofluorescence (IF) staining using an Abcam-derived MP-specific antibody at a 1:100 ratio. The results showed that the MP antibody labeling signal highly overlapped with the CellTrace far-infrared dye labeling signal, and the antibody staining results showed higher consistency and better matching with the previous live cell dynamic observation process. Furthermore, strong and stable fluorescence signals were still observed after 15 days of sample storage, indicating that the CellTrace far-infrared dye used in this invention has high fluorescence stability and is not easily quenched, making it convenient for storage and long-term observation.

[0068] Example 6 MP Viability Detection (CCU Method) 1. Experimental Objective To verify whether MP retains its infectious activity after fluorescent labeling, and to ensure that the observation results are true and valid.

[0069] 2. Operating Procedures ① Serial dilution: Take 10 enzyme-free and sterile EP tubes and label them; add 900 μl of MP complete culture medium to each tube, aspirate 100 μl of the test solution into the first EP tube, mix well, aspirate 30 μl of the test solution into the second EP tube, and so on, continuously diluting 10 times.

[0070] ②Inoculation and culture: Add 300 μl of dilution solution to each well of the 96-well plate, ensuring three replicates. After labeling, incubate in an incubator at 37℃, 95% N2, and 5% CO2 for 7 days.

[0071] ③ Result determination: The highest dilution factor at which a color change occurs is the concentration of the tested bacterial solution.

[0072] (3) Experimental conclusions Figure 5 The results are from the CCU assay. As shown in the figure, after serial dilution and incubation, the MP bacterial solution caused a characteristic color change in the culture medium. Based on the highest dilution factor, the concentration of MP after labeling could reach 5 × 10⁻⁶. 7 CCU / mL. This indicates that the dye is non-toxic, does not affect MP activity or infectivity, and can be safely used for long-term dynamic tracking of MP infection.

[0073] Example 7: Detection of MP mRNA (RT) qPCR) 1. Experimental Objective Quantifying the proliferation load of MP in organoids at the gene level reflects the change in infection degree over time.

[0074] 2. Operating Procedures (1) Extraction of total RNA (all consumables used were enzyme-free): ① At the end of the infection time, use a pipette to aspirate the culture medium and blow off the bottom organoids and matrix gel. Transfer the mixture to a 1.5ml EP tube and centrifuge at 200g for 3min. Remove the supernatant, add an equal volume of cell recovery (Corning, 354253), mix well by pipetting, place on ice for 30min, and centrifuge at 200g for 3min.

[0075] ② Remove the supernatant, add 1 mL of Trizol reagent and repeatedly pipette. Add an appropriate amount of sterile, enzyme-free steel ball to each EP tube, and use the MagNA Lyser fully automated tissue homogenizer to break up the tissue. Let it stand at room temperature for 5 minutes.

[0076] ③ Add 200µL of chloroform to the EP tube, rotate at maximum speed (Vortex) for 15s until the liquid turns pink, and leave at room temperature for 3 minutes until clear separation is visible.

[0077] ④ Cool the centrifuge to 4°C beforehand, then place the EP tube in the centrifuge and centrifuge (12000 g, 15 min). After centrifugation, three distinct layers will be observed: a colorless aqueous phase, a white protein layer, and a pink organic layer, from top to bottom. RNA is mainly in the colorless aqueous phase. Collect the upper layer using a 200 µL pipette, being careful to operate slowly and gently to avoid aspirating liquid from other layers.

[0078] ⑤ Take 4 μl of Glycogen (Thermo, 10814010) and mix well. Then add 100 μl of ammonium acetate (Beyotime, ST472) and mix well. Take 500 µL of isopropanol and add it to the EP tube. Rotate at the maximum speed (Vortex) for 15 s until the liquid is fully mixed. Then place it at room temperature for 10 min.

[0079] ⑥ Place the EP tube in a centrifuge and centrifuge (4℃, 12000 g, 10 min). After centrifugation, gently discard the supernatant, place the EP tube upside down on clean filter paper, and let it air dry in a clean bench for about 10 min.

[0080] ⑦ Add 1 mL of 80% ethanol (prepared with DEPC water) to the EP tube and centrifuge at the maximum speed (Vortex) until the precipitate floats up. Centrifuge the EP tube in a centrifuge (4℃, 7500g, 10min). After centrifugation, gently discard the supernatant, invert the EP tube onto clean filter paper, and let it air dry in a clean bench for about 10min.

[0081] ⑧ Observe the size of the precipitate, and thoroughly dissolve it by blowing it with a certain amount of DEPC water until it is dissolved. Then transfer it to a 200µL PCR tube for storage.

[0082] ⑨ RNA quality measurement: Use Nanodrop 2000, add DEPC water to zero the sample until the DEPC water value is within ±1, then measure each sample three times, take the average value and record it. Store RNA at -80℃.

[0083] ⑩ RNA quality: If A260 / A280 = 1.80~2.0, RT-qPCR can be performed.

[0084] (2) Reverse transcription to synthesize cDNA (all consumables used are enzyme-free) ① Genomic DNA removal: Select qualified RNA and add a total volume of 16µL of reaction system to a sterile 200µL PCR tube, including DEPC water, 4µL 4×g DNA wiper Mix and 1µg RNA. After short-term incubation, place the tube in a PCR instrument and react at 42℃ for 2min.

[0085] ② Reverse transcription reaction: Add 4 µL of 5×HiScript II qRT SuperMixII to the tube from step ① after the reaction is complete. After momentary centrifugation, place it in a PCR instrument and incubate at 50℃ for 15 min, then at 85℃ for 5 s.

[0086] (3) RT-qPCR detection of target gene expression (the tip and EP tube used were sterile and enzyme-free): ① All primers were synthesized by Sangon Biotech (Table 1). Before opening the EP tubes according to the instructions, the primers were centrifuged and diluted with DEPC water (concentration 10 µM / L).

[0087] Table 1. Primer sequences

[0088] ② Add a total volume of 10µL of reaction system to the PCR reaction tube, including 2×ChamQ UniversalSYBR qPCR Master Mix (5µL), 10µM front primer (0.2µL), 10µM back primer (0.2µL), cDNA (X µL) and DEPC water (X µL).

[0089] ③ Set up the PCR reaction program: pre-denaturation (95℃ for 30s); cycle reaction (95℃ denaturation for 10s, 60℃ extension for 30s, for a total of 40 cycles); melting curve (95℃ denaturation for 15s, 60℃ annealing for 60s, 95℃ for 15s).

[0090] ④ After spotting, briefly centrifuge and place on the heating plate of the RT-qPCR instrument. Record the CT value after completion. △△ Ct analysis results.

[0091] 3. Experimental Conclusions Using RT qPCR was used to quantify the mRNA expression level of *MP* in human bronchial organoids at different time points after *MP* infection. The results of *MP* RNA expression are shown in [Figure 1]. Figure 6 The results showed that, compared with the blank control group, the expression level of MP mRNA in organoids was significantly increased at 24 h, 48 h, and 72 h post-infection, and showed a significant increasing trend with the extension of infection time. This indicates that MP can stably colonize and continuously proliferate in the human bronchial organoid model constructed in this invention. This result verifies at the molecular level that this model can realistically simulate the natural infection process of MP in human bronchi. It is corroborated by the results of live cell dynamic observation and immunofluorescence staining, further proving the reliability and scientific validity of this method.

[0092] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for visually tracking Mycoplasma pneumoniae infection of human bronchial organoids, characterized in that, Includes the following steps: (1) Construction of human bronchial organoids: mature human bronchial organoids with cavities were obtained by culturing, wherein the organoids contain ciliated cells, basal cells, goblet cells and Club cells, and have a polarized three-dimensional structure with the apex facing outward; (2) Culture and fluorescent labeling of Mycoplasma pneumoniae: After Mycoplasma pneumoniae was amplified and cultured in MP complete medium, the cultured Mycoplasma pneumoniae was fluorescently labeled using CellTrace far-infrared cell staining solution; (3) Co-culture for infection: The fluorescently labeled Mycoplasma pneumoniae from step (2) was co-cultured with the human bronchial organoids from step (1) for infection; (4) Live cell dynamic fluorescence tracking: Non-invasive in situ continuous imaging of human bronchial organoids after MP infection in step (3).

2. The method according to claim 1, characterized in that, The process of cultivating mature human bronchial organoids with cavities in step (1) includes: taking airway tissue for enzymatic digestion; separating the digested tissue to obtain a single-cell suspension; resuspending the single-cell suspension in matrix gel and inoculating it; culturing it in bronchial organoid complete culture medium after solidification; passage it when the organoid diameter reaches 100~200 μm; and dissociating the matrix gel after the bronchial organoids grow cavities.

3. The method according to claim 1, characterized in that, In step (1), the human bronchial organoids differentiate and mature, expressing ciliated cell marker Ac-α-Tub, basal cell marker P63, goblet cell marker MUC5AC, and Club cell marker CC10, and the cells are tightly connected to each other through the tight junction protein zonula occludens-1.

4. The method according to claim 1, characterized in that, In step (2), the MP complete culture medium contains 20% fetal bovine serum, 800 U / mL penicillin, 0.5% glucose, 0.2% sodium pyruvate and 1% phenol red.

5. The method according to claim 1, characterized in that, The culture conditions in step (2) are 37℃, 95% N2, and 5% CO2. Culture for 5 to 7 days, and when the culture medium turns from red to orange-yellow and there is no flocculent precipitate, expand the culture.

6. The method according to claim 1, characterized in that, The fluorescent labeling process in step (2) is as follows: collect Mycoplasma pneumoniae cells, resuspend them in PBS, add CellTrace far-infrared cell staining solution, and incubate in a water bath at 37°C for 45 minutes.

7. The method according to claim 1, characterized in that, The culture and infection conditions in step (3) are to culture in a CO2 environment at 37°C for 12 hours.

8. The method according to any one of claims 1 to 7, characterized in that, The method for visually tracking human bronchial organoids infected with Mycoplasma pneumoniae further includes: performing immunofluorescence staining and confocal localization observation on human bronchial organoids infected with MP in step (3).

9. The method according to any one of claims 1 to 7, characterized in that, The method for visually tracking human bronchial organoids infected with Mycoplasma pneumoniae further includes: detecting the biological activity of MP in human bronchial organoids after MP infection in step (3).

10. The method according to any one of claims 1 to 7, characterized in that, The method for visually tracking human bronchial organoids infected with Mycoplasma pneumoniae further includes: detecting the mRNA of MP in human bronchial organoids after MP infection in step (3).

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