Pulmonary fibrosis organ-like model and construction method and application thereof

Abstract

The invention relates to the technical field of organoid culture, in particular to a pulmonary fibrosis organoid model and a construction method and application thereof. The pulmonary fibrosis organ-like model is obtained by co-culturing lentivirus stably transfected alveolar epithelial cells, human embryonic lung fibroblasts MRC-5 and macrophages. According to the invention, macrophages are innovatively introduced, a ternary co-culture system of lentivirus stably transfected alveolar epithelial cells, human embryonic lung fibroblasts MRC-5 and macrophages is constructed, and a key'epithelial injury-immune infiltration-fibroblast activation 'pathological triangle in a pulmonary fibrosis pathogenesis process can be more comprehensively simulated; the finally obtained pulmonary fibrosis organ-like model provides an efficient tool for pulmonary fibrosis mechanism research and research, development and screening of anti-pulmonary fibrosis drugs.

Description

A lung fibrosis organoid model, its construction method and application Technical Field

[0001] This invention relates to the field of organoid culture technology, and in particular to a lung fibrosis organoid model, its construction method, and its application. Background Technology

[0002] Pulmonary fibrosis (PF) is a chronic, progressive interstitial lung disease caused by various etiologies. Idiopathic PF (IPF) is a common and representative form of chronic pulmonary fibrosis in clinical practice. Its pathological features mainly include alveolar epithelial damage, fibroblast foci formation, and excessive extracellular matrix deposition, ultimately leading to lung tissue destruction and loss of lung function. Currently, the pathogenesis of pulmonary fibrosis is not fully understood, and effective treatments are lacking. Therefore, constructing organ models that can simulate the pathological process of pulmonary fibrosis is of great significance for in-depth research into its pathogenesis and the development of new therapeutic drugs. Traditional pulmonary fibrosis research mainly relies on animal models and in vitro cell culture models. However, animal models exhibit species differences and cannot fully simulate the pathological process of human pulmonary fibrosis; while in vitro cell culture models lack tissue structure and intercellular interactions, making it difficult to accurately reflect the complex pathological changes of pulmonary fibrosis.

[0003] In recent years, the rise of organoid technology has provided new ideas and methods for pulmonary fibrosis research. Organoids are three-dimensional cell cultures that can mimic the structure and function of organs in vitro, possessing advantages such as intact tissue structure, diverse cell types, and the ability to simulate physiological and pathological processes in vivo. Therefore, constructing organoid models of pulmonary fibrosis has significant application value for in-depth research into the pathogenesis of pulmonary fibrosis and the development of new therapeutic drugs.

[0004] Chinese invention patent application CN118048297A discloses a method for constructing a human pulmonary fibrosis organoid model and its application. It uses human AEC II cells and pathological fibroblasts to co-culture in vitro to construct the pulmonary fibrosis organoid model, simulating the interaction between AEC II cells and pathological fibroblasts under long-term pathological conditions. However, it cannot comprehensively simulate the key pathological triangle of "epithelial damage-immune infiltration-fibroblast activation" in the pathogenesis of pulmonary fibrosis. Summary of the Invention

[0005] To address the aforementioned technical problems, the first aspect of this invention provides a method for constructing a lung fibrosis organoid model, which involves co-culturing lentivirus-transfected alveolar epithelial cells, human embryonic lung fibroblasts (MRC-5), and macrophages to obtain a lung fibrosis organoid model.

[0006] Organoid models of pulmonary fibrosis are often obtained using the traditional binary co-culture of epithelial cells and fibroblasts. However, this binary co-culture system cannot fully simulate the complex intercellular interactions and pathological changes during the pathogenesis of pulmonary fibrosis. This invention innovatively introduces macrophages to construct a ternary co-culture system of lentivirally transfected alveolar epithelial cells, human embryonic lung fibroblasts (MRC-5), and macrophages. Macrophages, as important components of the immune system, play a crucial role in the pathogenesis of pulmonary fibrosis. Their inclusion is essential for reproducing the inflammatory microenvironment, intercellular crosstalk, and the continuous progression of fibrosis.

[0007] In some embodiments, the lentivirus-stable alveolar epithelial cells are pSLenti-EF1a-EGFP-PGK-Puro lentivirus-stable alveolar epithelial cells.

[0008] In some embodiments, the method for constructing pSLenti-EF1a-EGFP-PGK-Puro lentivirus-stable alveolar epithelial cells includes: S1, obtaining alveolar organoids; S2, digesting and sorting the alveolar organoids to obtain alveolar epithelial cells, culturing the alveolar epithelial cells until the cell confluence is 30%-50%, adding pSLenti-EF1a-EGFP-PGK-Puro lentivirus, and culturing for 5-9 days to obtain EGFP-labeled alveolar epithelial cells, which are pSLenti-EF1a-EGFP-PGK-Puro lentivirus-stable alveolar epithelial cells.

[0009] Optionally, S1 includes: S11, taking primary waste lung tissue from the operation, washing and disinfecting it with sterile PBS buffer, cutting it into 1-2 mm, adding collagenase for digestion, sorting it through a sieve, centrifuging, collecting the cell pellet, removing red blood cells by lysis, and sorting to obtain AT2 primary cells; S12, culturing the AT2 primary cells in reprogramming medium until the confluence is ≥80%, digesting and resuspending them, and culturing them for 2-3 days to form lung bud organoids; S13, placing the lung bud organoids in differentiation medium and continuing to culture them for 7 days to obtain alveolar organoids.

[0010] Optionally, the collagenase includes neutral protease II and type II collagenase.

[0011] Optionally, the mass ratio of neutral protease II to type II collagenase is (25-50):(50-75).

[0012] Optionally, in step S11, cells are stained with EPCAM and HT2-280 antibodies to sort out cells that are double-positive for EPCAM and HT2-280, thereby obtaining purified AT2 primary cells.

[0013] Optionally, the reprogrammed culture medium comprises Ham's F-12 basal medium, N2 nutrient supplement, B27 nutrient supplement, epithelial growth factor (EGF), fibroblast growth factor (bFGF), triiodothyronine, hydrocortisone, ROCK kinase inhibitor, Wnt signaling pathway activator, TGF-β signaling inhibitor, and MAPK inhibitor.

[0014] In some embodiments, the N2 nutrient supplement accounts for 1-3% of the volume of Ham's F-12 basal medium, and the B27 nutrient supplement accounts for 1-3% of the volume of Ham's F-12 basal medium.

[0015] In some embodiments, the reprogramming culture medium contains 20-30 ng / mL of epithelial growth factor (EGF); 40-60 ng / mL of fibroblast growth factor (bFGF); 1-3 μg / mL of triiodothyronine; 1-3 μg / mL of hydrocortisone; 8-15 μM of ROCK kinase inhibitor; 1-5 μM of Wnt signaling pathway activator; 1-3 μM of TGF-β signaling inhibitor; and 1-3 nM of MAPK inhibitor.

[0016] In some embodiments, the ROCK kinase inhibitor is the ROCK kinase inhibitor Y-27632.

[0017] In some embodiments, the Wnt signaling pathway activator is Wnt signaling pathway activator CHIR-99021.

[0018] In some embodiments, the TGF-β signaling inhibitor is TGF-β signaling inhibitor A8301.

[0019] In some embodiments, the MAPK inhibitor is the p38 MAPK inhibitor BIRB796.

[0020] In some embodiments, the differentiation medium comprises DMEM / F12 basal medium, dexamethasone, cAMP, and IBMX.

[0021] Optionally, the differentiation medium contains 25-35 μM dexamethasone, 0.1-0.5 mM cAMP, and 0.1-0.5 mM IBMX.

[0022] In terms of cell source, the core alveolar epithelial cells of this invention are derived from alveolar precursor-like cells expanded in vitro. Compared with the pluripotent stem cell (iPSCs / ESCs) differentiation system, this strategy does not require a complex multi-stage differentiation process, which greatly shortens the model construction cycle and reduces culture costs and technical barriers. More importantly, it effectively avoids the off-target differentiation risk and potential tumorigenicity inherent in pluripotent stem cells, making the model safer and the phenotype purer.

[0023] In some embodiments, the amount of pSLenti-EF1a-EGFP-PGK-Puro lentivirus added is obtained by Equation 1; Equation 1: Viral amount (µL) = (MOI × cell number) / viral titer (TU / mL) × 1000; where the MOI value is 40, and the cell number is 6 × 10 4 One count.

[0024] In some embodiments, the pSLenti-EF1a-EGFP-PGK-Puro lentivirus is diluted with basal complete medium before addition, and Polybrene is added to a final concentration of 4-8 µg / mL to obtain a virus-medium mixture.

[0025] The basic complete culture medium is DMEM / F12 basic culture medium.

[0026] In some embodiments, the culture in S2 includes: culturing in a 37°C, 5% CO2 incubator for 24 hours, then replacing the culture medium with fresh medium and continuing the culture; 48 hours after infection, screening with puromycin for 3-7 days to obtain pSLenti-EF1a-EGFP-PGK-Puro lentivirus stably transfected alveolar epithelial cells.

[0027] During the selection process using puromycin, a large number of uninfected negative control cells died and detached under a microscope, while successfully infected experimental cells continued to proliferate and form cell colonies. The selection was complete when the negative control cells were completely killed and no more cells in the experimental group died. After selection, the surviving cells were digested and collected, then seeded into larger culture dishes for scale-up culture.

[0028] In some embodiments, the method for culturing the human embryonic lung fibroblast MRC-5 cells includes: resuspending the MRC-5 cells in MEM medium containing fetal bovine serum and penicillin-streptomycin solution, and then passage culturing them until the cell confluence reaches 80%-90% to obtain human embryonic lung fibroblast MRC-5 cells.

[0029] In some embodiments, the volume percentage of fetal bovine serum in the MEM culture medium is 8-12%; the volume percentage of the penicillin-streptomycin solution is 1-5%.

[0030] In some embodiments, the passage culture includes: when the cells reach 80%-90% confluence, digesting them with a digestive solution for 1-2 minutes, adding complete culture medium to terminate the digestion, and pipetting them into a single-cell suspension; centrifuging the single-cell suspension and resuspending it in complete culture medium for continued culture.

[0031] In some embodiments, the digestive solution is a trypsin-EDTA digestive solution.

[0032] In some embodiments, the method for culturing macrophages includes: taking human monocytic leukemia cells in the logarithmic growth phase, resuspending them, adding phorbol ester for induction culture for 10-15 hours, replacing with RPMI-1640 complete medium and continuing culture for 6-8 hours to obtain macrophages.

[0033] In some embodiments, the human monocytic leukemia cells in the logarithmic growth phase are resuspended in RPMI-1640 complete medium without phorbol ester to obtain a cell suspension.

[0034] In some embodiments, the concentration of phorbol ester in the cell suspension is 100 ng / mL.

[0035] In some embodiments, the ratio of lentivirus-stabilized alveolar epithelial cells, human embryonic lung fibroblasts (MRC-5), and macrophages is 1:(2-3):(4-6). Examples include 1:2:4, 1:3:6, 1:2:6, etc., or any value within the range of 1:(2-3):(4-6).

[0036] In some embodiments, the co-culture includes: resuspending lentivirus-transfected alveolar epithelial cells, human embryonic lung fibroblasts (MRC-5), and macrophages in a co-culture medium, adding Matrigel for three-dimensional culture, adding a pulmonary fibrosis inducer for 3-4 days, and continuing culture to obtain a pulmonary fibrosis organoid model.

[0037] In some embodiments, the co-culture medium comprises DMEM / F12 basal medium, dexamethasone, cAMP, and IBMX.

[0038] Optionally, the co-culture medium contains 25-35 μM dexamethasone, 0.1-0.5 mM cAMP, and 0.1-0.5 mM IBMX.

[0039] In this invention, lentivirus-stable alveolar epithelial cells, MRC5 cells, and macrophages are co-cultured in a ratio of 1:(2-3):(4-6), and Matrigel is added to simulate the extracellular matrix to form a three-dimensional organoid structure. Then, pulmonary fibrosis is induced by bleomycin to fully reproduce the pathological process of "epithelial injury-immune infiltration-fibroblast activation".

[0040] A second aspect of the present invention provides a lung fibrosis organoid model, which is constructed by the above-described construction method.

[0041] The third aspect of this invention provides an application of a pulmonary fibrosis organoid model in the study of the mechanism of pulmonary fibrosis, the development and screening of anti-pulmonary fibrosis drugs.

[0042] Beneficial effects: This invention provides a method for constructing and applying an organoid model of pulmonary fibrosis, which has the following advantages: (1) This invention innovatively introduces macrophages to construct a ternary co-culture system of lentivirally stable alveolar epithelial cells, human embryonic lung fibroblasts MRC-5, and macrophages. As an important component of the immune system, macrophages play a key role in the pathogenesis of pulmonary fibrosis, and their addition is crucial for reproducing the inflammatory microenvironment, intercellular crosstalk, and the continuous progression of fibrosis.

[0043] (2) The pulmonary fibrosis organoid model provided by the present invention can better simulate the spatial structure of tissues in vivo and the extracellular matrix environment. Compared with two-dimensional culture, it can generate more physiological and pathologically relevant research data.

[0044] (3) The pulmonary fibrosis organoid model provided by the present invention has excellent scalability, which facilitates high-throughput drug screening; in terms of operability, it allows for independent genetic or pharmacological manipulation of the three cellular components, thereby accurately elucidating their mechanisms.

[0045] (4) The pulmonary fibrosis organoid model provided by this invention successfully balances physiological relevance, construction efficiency, safety and research convenience, providing a powerful in vitro research platform for the study of the mechanism of pulmonary fibrosis and drug development. Attached Figure Description

[0046] Figure 1 shows bright-field and fluorescence micrographs of EGFP-labeled alveolar epithelial cells and alveolar organoids; A and B in the figure are alveolar epithelial cells, and C and D are alveolar organoids.

[0047] Figure 2 shows bright-field and fluorescence micrographs of alveolar organoids and pulmonary fibrosis organoids; AD in the figure represents alveolar organoids and EH represents pulmonary fibrosis organoids.

[0048] Figure 3 shows the results of RT-qPCR detection of fibrosis-related gene expression.

[0049] Figure 4 shows the distribution of the three cell types in organoids as revealed by immunohistochemistry; in the figure, A0 represents lentivirus-transfected alveolar epithelial cells, A0+MRC5 represents lentivirus-transfected alveolar epithelial cells and human embryonic lung fibroblasts MRC-5, A0+dTHP1 represents lentivirus-transfected alveolar epithelial cells and macrophages, and A0+MRC5+dTHP1 represents lentivirus-transfected alveolar epithelial cells, human embryonic lung fibroblasts MRC-5, and macrophages.

[0050] Figure 5 shows the localization and distribution of three cell types in pulmonary fibrosis organoids detected by immunofluorescence. Detailed Implementation

[0051] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention. Experimental methods not specifically specified in the embodiments were performed under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the solvent for the solutions involved in this invention is water; the room temperature is 25°C; and the raw materials, consumables, and equipment used are all commercially available.

[0052] The first aspect of this example provides a method for constructing a lung fibrosis organoid model, which uses lentivirus-transfected alveolar epithelial cells, human embryonic lung fibroblasts MRC-5, and macrophages to co-culture to obtain a lung fibrosis organoid model.

[0053] The lentivirus-stable alveolar epithelial cells were pSLenti-EF1a-EGFP-PGK-Puro lentivirus-stable alveolar epithelial cells.

[0054] The method for constructing stable alveolar epithelial cells from the pSLenti-EF1a-EGFP-PGK-Puro lentivirus includes: S1, obtaining alveolar organoids; S2, digesting and sorting the alveolar organoids to obtain alveolar epithelial cells, and counting the alveolar epithelial cells at a ratio of 5 × 10⁻⁶ cells / year. 4Cells / well were seeded at a density of 0.5 mL of complete culture medium per well, shaken well, and incubated at 37°C, 5% CO2 for 24 hours until cell confluence reached 30%-50%. The 24-well plate was removed from the incubator, and the old culture medium in each well was carefully aspirated. The cells were gently rinsed once with pre-warmed PBS to remove residual serum and antibiotics. A virus-culture medium mixture (250 µL / well) was added to each well. The plate was gently shaken to ensure even virus contact with the cells. After incubation at 37°C, 5% CO2 for 24 hours, the medium was replaced with fresh medium. Forty-eight hours after infection, cells were selected with puromycin for 5 days. Selection was complete when no more cell death was observed, yielding EGFP-labeled alveolar epithelial cells, which are pSLenti-EF1a-EGFP-PGK-Puro lentivirus stably transfected alveolar epithelial cells.

[0055] S1 includes: S11, taking primary waste lung tissue during surgery, washing and disinfecting it with sterile PBS buffer, cutting it into 1-2 mm, transferring it to a 15 mL centrifuge tube, adding collagenase and incubating at 37°C for 1 h to obtain a cell suspension; using a 70-micron cell filter with the assistance of sterile PBS buffer to screen and collect the filtrate and remove mucus and undigested tissue. Then, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, add erythrocyte lysis balancing solution (Beyotime C3702) to the obtained precipitate for resuspending, centrifuge again, and repeat the above process until no erythrocytes are observed in the cell pellet after centrifugation again, to complete the erythrocyte lysis and removal; use EPCAM and HT2-280 antibodies to stain the cells, sort the cells that are double positive for EPCAM and HT2-280, and obtain purified AT2 primary cells; S12, culture the AT2 primary cells in reprogramming medium until the confluence is ≥80%, digest with 0.25% trypsin digestion solution for 7 minutes, and resuspend in reprogramming medium containing 5% Matrigel (Corning, 354230), and form a three-dimensional structure after 2 days, which is the lung bud organoid; S13, place the lung bud organoid in differentiation medium and continue to culture for 7 days to obtain alveolar organoids.

[0056] The collagenase includes neutral protease II and type II collagenase in a mass ratio of 25:75.

[0057] The reprogrammed culture medium consisted of Ham's F-12 basal medium, and, by volume of Ham's F-12 basal medium, 1% N2 nutrient supplement and 2% B27 nutrient supplement; 20 ng / mL epithelial growth factor (EGF), 50 ng / mL fibroblast growth factor (bFGF), 2 μg / mL triiodothyronine (Tao Shu, T1653), 0.5 μg / mL hydrocortisone, 10 µM ROCK kinase inhibitor Y-27632, 3 µM Wnt signaling pathway activator CHIR-99021, 1 µM TGF-β signaling inhibitor A8301, and 1 nM p38 MAPK inhibitor BIRB796.

[0058] The differentiation medium contains DMEM / F12 basal medium, dexamethasone, cAMP, and IBMX.

[0059] The differentiation medium contained 30 μM dexamethasone, 0.1 mM cAMP, and 0.1 mM IBMX.

[0060] The method for preparing the virus-culture medium mixture is as follows: dilute pSLenti-EF1a-EGFP-PGK-Puro lentivirus with basal complete culture medium, and add Polybrene to a final concentration of 6µg / mL to obtain the virus-culture medium mixture.

[0061] The basic complete culture medium is DMEM / F12 basic culture medium.

[0062] The pSLenti-EF1a-EGFP-PGK-Puro lentivirus was obtained from Heyuan Biotechnology (Shanghai) Co., Ltd.

[0063] The method for culturing human embryonic lung fibroblasts MRC-5 includes: removing frozen MRC5 cells from a liquid nitrogen tank and immediately placing them in a 37°C water bath, gently shaking to thaw them completely within 1 minute. In a clean bench, transfer the thawed cell suspension to a 15 mL centrifuge tube containing 5 mL of preheated complete culture medium. Centrifuge at 1000 rpm for 5 minutes, carefully discard the supernatant, and remove any residual cryoprotectant DMSO. Gently resuspend the cell pellet in 1 mL of complete culture medium (MEM basal medium + 10% (v / v) fetal bovine serum + 1% (v / v) penicillin-streptomycin solution), agitate thoroughly, and transfer to a T25 cell culture flask containing an appropriate amount of complete culture medium. Gently agitate the culture flask to distribute the cells evenly and incubate statically in a 37°C, 5% CO2 saturated humidity incubator. After 24 hours, replace the culture medium with fresh medium to remove any non-adherent dead cells.

[0064] When the cell confluence reached 80%, the cells were passaged. The old culture medium was discarded, and the cells were gently rinsed twice with pre-warmed 1× PBS buffer. 0.25% trypsin-EDTA digestion solution was added to cover the cell layer, and the cells were incubated at 37°C for 2 minutes. Under an inverted microscope, when the intercellular spaces increased and the cells became more rounded, the digestion was immediately stopped by adding twice the volume of complete culture medium. The cells were gently agitated with a pipette to detach completely, forming a single-cell suspension. The single-cell suspension was collected in centrifuge tubes and centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The cells were resuspended in fresh complete culture medium, passaged in separate flasks at a 1:3 ratio, and fresh culture medium was added to the appropriate volume. The cells were then returned to the incubator for further culture to obtain human embryonic lung fibroblasts MRC-5.

[0065] The macrophage culture method includes: routinely culturing human monocytic leukemia cells (THP1) in RPMI-1640 complete medium, maintaining a cell density of 2 × 10⁻⁶ cells / year. 5 -1×10 6 Cells / mL. Passage every 2-3 days. During passage, collect the cell suspension, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, resuspend in fresh, pre-warmed complete medium, and dilute 1:3 in a new culture flask. Collect THP1 cells in logarithmic growth phase, count them, and centrifuge at 1000 rpm for 5 minutes. Resuspend the cells in PMA-free RPMI-1640 complete medium to obtain a cell suspension; add phorbol ester (PMA) to the cell suspension to a final concentration of 100 ng / mL. Seed the cells into culture dishes and incubate at 37°C, 5% CO2 for 12 hours. After induction, carefully aspirate the medium containing PMA. Gently wash adherent cells 1-2 times with pre-warmed 1× PBS to thoroughly remove PMA and non-adherent cells. Replace with fresh, PMA-free RPMI-1640 complete culture medium and continue to incubate statically for 6 hours to allow the cells to fully differentiate into resting macrophages (M0).

[0066] The co-culture process involved mixing lentivirally stabilized alveolar epithelial cells, human embryonic lung fibroblasts (MRC-5), and macrophages at a ratio of 1:2:4. The cell suspension was centrifuged at 1000 rpm for 5 minutes at 4°C, the supernatant was discarded, and the cells were resuspended in co-culture medium. 5% Matrigel was added, and the cells were incubated at 37°C in a 5% CO2 incubator. The co-culture medium was replaced with fresh medium every two days. After three days of co-culture, bleomycin (BLM), a pulmonary fibrosis inducing agent, was added at 10 μg / mL, and the cells were cultured for another three days to obtain a pulmonary fibrosis organoid model.

[0067] The co-culture medium contains DMEM / F12 basal medium, dexamethasone, cAMP, and IBMX.

[0068] The co-culture medium contained 30 μM dexamethasone, 0.1 mM cAMP, and 0.1 mM IBMX.

[0069] The second aspect of this example provides a lung fibrosis organoid model, which is constructed using the above-described method.

[0070] The third aspect of this example provides an application of pulmonary fibrosis organoid models in the study of the mechanisms of pulmonary fibrosis, the development and screening of anti-pulmonary fibrosis drugs.

[0071] Performance Test 1: Verification using pSLenti-EF1a-EGFP-PGK-Puro lentivirus to construct stable alveolar epithelial cells: The green fluorescence of EGFP was directly observed using an inverted fluorescence microscope to assess the infection efficiency (which should be close to 100%). The test results are shown in Figure 1.

[0072] As shown in Figure 1, the pSLenti-EF1a-EGFP-PGK-Puro lentivirus successfully infected alveolar epithelial cells with a fluorescence positivity rate close to 100%, indicating that alveolar epithelial cell lines stably expressing EGFP were obtained after puromycin screening. These stably expressed alveolar epithelial cells can be successfully used to construct three-dimensional alveolar organoids, and the EGFP marker persists in the organoids, which can be used for cell localization tracking in subsequent co-culture.

[0073] 2. Morphological observation: The formation and growth of organoids were observed using an inverted bright-field microscope and a fluorescence microscope. Successful organoids should exhibit regular three-dimensional spherical or cystic structures.

[0074] As shown in Figure 2, BF represents the bright field observation mode and GFP represents the green fluorescence observation mode. This figure confirms the successful construction of alveolar organoids and pulmonary fibrosis organoids, and the EGFP-labeled alveolar epithelial cells are stably present in the organoids, providing morphological evidence for the structural integrity of the model.

[0075] 3. RT-qPCR detection of inflammation and fibrosis-specific genes in pulmonary fibrosis organoids: Total RNA was extracted from pulmonary fibrosis organoids according to the Promega kit instructions. qPCR was performed using primers for detecting inflammation (IL6, IL8, TNFα), fibrosis (COL1A1, TGFb1), and DNA damage (SERP1NE1) to assess the fibrotic pathological features of the model.

[0076] The test results are shown in Figure 3. The vertical axis represents the relative expression level, and the horizontal axis represents the relevant detected genes. AO (alveolar organoid), PFO is the pulmonary fibrosis organoid, and BLM10 is the pulmonary fibrosis organoid model provided in the embodiments of this application.

[0077] As can be seen from the figure, compared with the control group, the relative expression levels of inflammation-related genes (IL-6, IL-8, TNFα), fibrosis-related genes (COL1A1, TGFβ1), and DNA damage-related genes (SERPINE1) in the model provided in this application were significantly increased. This result confirms that after bleomycin induction, this organoid model successfully activated the inflammatory response, collagen deposition, and DNA damage response in the pathological process of pulmonary fibrosis, which is consistent with the molecular characteristics of pulmonary fibrosis in vivo, thus verifying the pathological relevance and effectiveness of this organoid model.

[0078] 4. Immunohistochemical detection of cell localization and distribution in pulmonary fibrosis organoids: The pulmonary fibrosis organoids obtained after co-culturing for 6 days according to the embodiments of this application were discarded, and cells were fixed with paraformaldehyde, embedded in paraffin, sectioned, and subjected to immunohistochemical detection.

[0079] The testing method is as follows: 1) Baking paraffin sections at 62℃ for 1 hour.

[0080] 2) Dewaxing: Immerse the tissue microarray in xylene for 20 minutes, replace the xylene, and immerse for another 20 minutes; 3) Hydration: a) Immerse in anhydrous ethanol for 1 minute; b) Immerse in anhydrous ethanol for 1 minute; c) Immerse in 95% ethanol for 1 minute; d) Immerse in 95% ethanol for 1 minute; e) Immerse in 70% ethanol for 1 minute; f) Rinse with tap water for 5 minutes; g) Rinse with distilled water; 4) Add one drop or 100µL of hydrogen peroxide blocking solution to each slide and incubate at room temperature for 10 minutes to block the activity of endogenous peroxidase.

[0081] 5) Wash with PBS 3 times, 5 min each time; 6) Heat antigen retrieval (1 mmol Tris-EDTA pH=9.0): a) Place the washed slides on a plastic rack and put them in the boiling antigen retrieval solution. Boil for 15 min, then keep warm for 15 min. Turn off the power and let them cool naturally.

[0082] 7) Wash 3 times with PBS, 5 min each time; 8) Remove PBS, add 1 drop or 100 µL of 5% BSA blocking solution to each slide, and incubate at room temperature for 20 min. 9) Remove serum, add 1 drop or 50 µL of primary antibody diluted with primary antibody dilution buffer to each slide, and incubate overnight at 4°C; 10) Remove the humidifier the next day, and incubate at room temperature for 1 h or at 37°C for 30 min; 11) Wash 3 times with PBS, 5 min each time; 12) Remove PBS, add 1 drop or 50 µL of labeled secondary antibody to each slide, and incubate at 37°C for 30 min; 13) Wash 3 times with PBS, 5 min each time; 14) Remove PBS, add 1 drop or 50 µL of labeled secondary antibody to each slide... Develop with diaminobenzidine (DAB), and monitor the staining degree under a microscope (stop staining immediately when yellow granules or flaky precipitates appear); 15) Rinse off residual DAB solution with tap water, counterstain with hematoxylin for 30 seconds, rinse with tap water for 5 minutes; 16) Differentiate with hydrochloric acid alcohol for 1 second, rinse with tap water for 10 minutes to return to blue; 17) Dehydration: a) Soak in 70% ethanol for 3 minutes; b) Soak in 95% ethanol for 3 minutes; c) Soak in 95% ethanol for 3 minutes; d) Soak in anhydrous ethanol for 3 minutes twice; 18) Clearing: Soak the tissue in xylene for 5 minutes three times; 19) Mounting: Mount with neutral resin and cover with a coverslip; 20) Microscopic examination: Observe under a microscope and record the results.

[0083] The test results are shown in Figure 4. As can be seen from the figure, alveolar epithelial cells, MRC-5 cells, and M0 macrophages form stable intercellular interactions in the organoid, proving that the model of this invention successfully simulates the "epithelium-mesenchyma-immunity" ternary spatial configuration in the in vivo pulmonary fibrosis microenvironment. This provides morphological evidence for the accurate simulation of the pathological triangle of "epithelial injury-immune infiltration-fibroblast activation."

[0084] 5. Immunofluorescence detection of the localization and distribution of three cell types in pulmonary fibrosis organoids: Pulmonary fibrosis organoid samples were paraffin-embedded and sectioned to a thickness of 4-5 µm. Dewaxing and hydration were then performed: treatment was carried out sequentially with xylene and graded ethanol, followed by washing with PBS. Antigen retrieval was then performed, typically using sodium citrate buffer (pH 6.0) for heat-induced retrieval, followed by natural cooling and washing again with PBS. Blocking was then performed using PBS solution containing 1% BSA and 0.1% Triton X-100 at room temperature for 1 hour. After blocking, a mixture of primary antibody working solutions (for Pro-SFTPC, α-SMA, and MAC387) in appropriate proportions was added dropwise, and incubation was carried out overnight in a humidified chamber at 4°C. The next day, the primary antibody was recovered, and after thorough washing with PBS, the corresponding fluorescently labeled secondary antibody mixture was added dropwise under light-protected conditions and incubated at room temperature for 1.5 hours. After another wash with PBS in the dark, nuclear staining was performed using DAPI for approximately 7 minutes. Finally, excess DAPI was washed away with PBS, and the slides were mounted with anti-fluorescence quenching mounting medium. The prepared slides were stored at 4°C in the dark and observed and images were acquired using a fluorescence microscope or confocal microscope to analyze the localization and distribution of the three cell markers in the organoids. The test results are shown in Figure 5.

[0085] As can be seen from the figure, alveolar epithelial cells, MRC-5 cells, and M0 macrophages form a close spatial distribution relationship in the pulmonary fibrosis organoids, further verifying that the multi-cell co-culture system can simulate the cellular microenvironment of pulmonary fibrosis in vivo.

Claims

1. A method for constructing a lung fibrosis organoid model, characterized in that, A lung fibrosis organoid model was obtained by co-culturing lentivirus-stable alveolar epithelial cells, human embryonic lung fibroblasts (MRC-5), and macrophages.

2. The method for constructing a lung fibrosis organoid model according to claim 1, characterized in that, The lentivirus-stable alveolar epithelial cells were pSLenti-EF1a-EGFP-PGK-Puro lentivirus-stable alveolar epithelial cells.

3. The method for constructing a lung fibrosis organoid model according to claim 2, characterized in that, The method for constructing pSLenti-EF1a-EGFP-PGK-Puro lentivirus-stable alveolar epithelial cells includes: S1, obtaining alveolar organoids; S2, digesting and sorting the alveolar organoids to obtain alveolar epithelial cells, culturing the alveolar epithelial cells until the cell confluence is 30%-50%, adding pSLenti-EF1a-EGFP-PGK-Puro lentivirus and culturing for 5-9 days to obtain EGFP-labeled alveolar epithelial cells, which are pSLenti-EF1a-EGFP-PGK-Puro lentivirus-stable alveolar epithelial cells.

4. The method for constructing a lung fibrosis organoid model according to claim 1, characterized in that, The method for culturing human embryonic lung fibroblasts MRC-5 includes: resuspending MRC-5 cells in MEM medium containing fetal bovine serum and penicillin-streptomycin dual antibiotic solution, and then passage culturing them until the cell confluence reaches 80%-90% to obtain human embryonic lung fibroblasts MRC-5.

5. The method for constructing a lung fibrosis organoid model according to claim 1, characterized in that, The method for culturing macrophages includes: taking human monocytic leukemia cells in the logarithmic growth phase, resuspending them, adding phorbol ester to induce culture for 10-15 hours, then replacing with RPMI-1640 complete medium and continuing culture for 6-8 hours to obtain macrophages.

6. The method for constructing a lung fibrosis organoid model according to claim 1, characterized in that, The ratio of the number of lentivirus-stable alveolar epithelial cells, human embryonic lung fibroblasts MRC-5, and macrophages was 1:(2-3):(4-6).

7. The method for constructing a lung fibrosis organoid model according to claim 1, characterized in that, The co-culture process includes: resuspending lentivirus-transfected alveolar epithelial cells, human embryonic lung fibroblasts (MRC-5), and macrophages in a co-culture medium, adding Matrigel for three-dimensional culture, adding a pulmonary fibrosis inducing agent after 3-4 days of culture, and continuing culture to obtain a pulmonary fibrosis organoid model.

8. The method for constructing a lung fibrosis organoid model according to claim 1, characterized in that, The co-culture medium contains DMEM / F12 basal medium, dexamethasone, cAMP, and IBMX.

9. A lung fibrosis organoid model, characterized in that, It is constructed by the construction method described in any one of claims 1-8.

10. The application of a pulmonary fibrosis organoid model according to claim 9 in the study of the mechanism of pulmonary fibrosis, the development and screening of anti-pulmonary fibrosis drugs.

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