Organ chip for simulating airway-alveolar continuous barrier function and application of organ chip in COPD model construction and drug evaluation

Abstract

The invention discloses a three-chamber integrated micro-fluidic chip for simulating an airway-alveolar continuous barrier function and an application of the three-chamber integrated micro-fluidic chip in COPD (chronic obstructive pulmonary disease) drug evaluation. The chip comprises an airway chamber, a pulmonary alveolus chamber, a blood vessel chamber and a porous PET (polyethylene terephthalate) membrane, and the continuous spatial hierarchy of'airway-pulmonary alveolus-qi and blood barrier-blood vessel 'is reconstructed through a'three-chamber-in-one' bionic structure. According to the invention, optimized culture systems for the airway epithelial cells and AT2 cells from COPD patients are respectively established, airway organoid and alveolar organoid with pathological characteristics are obtained, and co-culture is realized in the chip. The chip is provided with a micro sampling port, and effluent can be collected at different time points for dynamic monitoring of inflammatory factors. By combining multi-dimensional detection such as dead and living staining, ELISA (enzyme-linked immunosorbent assay), immunofluorescence and the like, an all-dimensional evaluation system from morphology to molecules and from intracellular to extracellular is constructed. The application of erdosteine intervention verification shows that the chip can effectively simulate airway-alveolar double-part damage characteristics (including shape shrinkage, cell death increase, inflammatory factor IL-6 increase, mucus factor MUC5B high secretion and the like) of a COPD patient, and can reliably evaluate the curative effect of a medicine. The invention provides an in-vitro platform with high bionic degree and high stability for COPD mechanism research, drug screening and personalized medical treatment, and has wide industrial application prospects in the fields of respiratory disease drug research and development, toxicity evaluation and precise medicine.

Description

Technical Field

[0001] This invention belongs to the field of microfluidic chip and organoid culture technology, specifically relating to a microfluidic organ-on-a-chip that integrates the co-culture of airway organoids and alveolar organoids, and its application in COPD model construction, drug screening and mechanism research. Background Technology

[0002] Chronic obstructive pulmonary disease (COPD) is a chronic respiratory disease characterized by airflow limitation, airway remodeling, and alveolar structural damage. Its pathological mechanisms include chronic inflammation, increased mucus secretion, impaired ciliary function, and extracellular matrix degradation. Existing research indicates that the occurrence of COPD is closely related to long-term inflammatory responses, hypermucus secretion, ciliary damage, and abnormal extracellular matrix degradation. Furthermore, the disease exhibits significant tissue heterogeneity and inter-patient variability, posing a significant challenge to constructing human in vitro models with stable pathological manifestations.

[0003] Currently, commonly used COPD models mainly include smoke-exposed mouse models and elastase-induced models. Although these animal models can reproduce some COPD pathological features, such as alveolar destruction and inflammatory infiltration, they are difficult to accurately simulate the three-dimensional anatomical structure of the human airway-alveolar interface and the long-term chronic course of the disease due to species differences and structural complexity.

[0004] In addition, animal models are difficult to reflect individual patient differences, which limits personalized drug screening and mechanism research. Although traditional two-dimensional cell culture technology is easy to operate, it lacks gas-liquid interface and three-dimensional cell-extracellular matrix interaction, and cannot simulate the complex intercellular communication and barrier function in COPD.

[0005] In recent years, lung organoid technology has been increasingly applied to lung disease research. Studies have shown that lung organoids can effectively reconstruct the polar structure and multicellular composition of lung epithelium, providing a new platform for simulating lung pathology. However, traditional organoid culture systems are static cultures, lacking key pathological factors such as vascularization, airflow stress, and the inflammatory microenvironment, making it difficult to fully simulate the chronic progression of COPD. More importantly, existing lung organoid models typically focus on a single epithelial structure (airway epithelium only or alveolar epithelium only), failing to simultaneously reconstruct both airway and alveolar structures, and lacking the conditions to simulate the synergistic effects of multiple cell types in the vascular endothelium-epithelial region.

[0006] Microfluidic organ-on-a-chip technology can more realistically simulate the in vivo tissue microenvironment by integrating physiologically relevant factors such as fluid shear forces, gas-liquid interfaces, and chemical gradients. However, existing lung-on-a-chip and organoid cultures struggle to simulate the multicellular and multi-structural damage characteristics of COPD, lacking an in vitro platform capable of simultaneously integrating patient-derived airway-alveolar organoids, multi-compartment co-culture, and vascularized dynamic perfusion. Therefore, developing an in vitro platform that can simultaneously integrate the airway-alveolar dual structure, vascularized microenvironment, and chronic inflammation simulation is of significant scientific importance and application value for COPD mechanism research and drug evaluation. Summary of the Invention

[0007] This invention aims to address the technical problems of existing COPD in vitro models, such as the inability to simultaneously simulate airway and alveolar dual-structure damage, lack of patient specificity, and vascularized dynamic microenvironment. It provides a three-chamber integrated microfluidic chip that simulates the continuous airway-alveolar barrier function and its application in COPD model construction and drug evaluation. It can achieve stable maintenance of organoids and monitoring of indicators such as inflammation and survival rate under micro-sample conditions, thereby providing a highly biomimetic and stable technical means for accurate modeling and clinical drug evaluation of respiratory diseases such as COPD.

[0008] To address the aforementioned technical problems, this invention provides a three-chamber integrated microfluidic chip that simulates the airway-alveolar continuous barrier function. The chip includes an airway chamber and an alveolar chamber physically connected by sidewalls, a vascular chamber located in the upper layer and separated from the lower layer by a porous membrane, and a microsampling port fluidly connected to the vascular chamber. The chip is entirely fabricated using PDMS, with the airway chamber open at the top to form a gas-liquid interface. The porous membrane is a PET membrane with a pore size of 5-8 μm, used to construct the air-blood barrier model. The microsampling port can collect effluent at different time points for dynamic monitoring of inflammatory factors.

[0009] The present invention also provides an expansion culture medium for culturing airway organoids derived from COPD patients. The culture medium is based on Advanced DMEM / F-12 and contains 10% FBS by volume and the following components: EGF 25-50 ng / mL, Insulin 10-50 μg / mL, Transferrin 5-25 μg / mL, Cholera Toxin 0.1-0.5 μg / mL, Retinoic Acid 50-100 nM, Noggin 20-50 μg / mL, R-Spondin 1 200-800 ng / mL, A8301 500-1000 nM, CHIR99021 3-10 μM, and Y-27632 5-10 μM. The synergistic mechanisms of these factors are as follows: Retinoic acid induces airway epithelial maturation and differentiation, promoting the formation of ciliated cells and mucus-secreting cells; EGF promotes basal cell proliferation; Y-27632 reduces stress-induced apoptosis; R-Spondin 1 and CHIR99021 jointly activate Wnt signaling to enhance organoid formation rate; A8301 inhibits TGF-β-mediated epithelial-mesenchymal transition; Noggin inhibits BMP signaling to promote ciliated cell differentiation; Insulin and Transferrin maintain energy metabolism and iron supply; and Cholera Toxin prolongs the organoid expansion cycle.

[0010] This invention also provides a differentiation culture medium for culturing alveolar organoids derived from COPD patients. The culture medium is based on Advanced DMEM / F-12 and contains the following components: FGF10 10-50 ng / mL, FGF7 25-50 ng / mL, SB431542 10-50 μM, and SPC 5-20 ng / mL. The synergistic mechanism of action of each factor is as follows: FGF10 maintains AT2 cell proliferation and self-renewal; FGF7 induces alveolar epithelial differentiation into secretory lineages, promoting the formation of vesicle-like structures containing SPC; SPC maintains the functional characteristics of AT2 cells; and SB431542 blocks epithelial-mesenchymal transition, maintaining the epithelial characteristics of alveolar epithelial cells.

[0011] This invention also provides a method for constructing an in vitro COPD disease model, comprising the following steps: First, using the aforementioned amplification culture medium and differentiation culture medium, airway organoids and alveolar organoids derived from COPD patients are obtained through 3D culture; second, HUVEC cells are plated on the vascular chamber of a microarray and cultured for 12 hours to form a vascular endothelial layer; then, airway organoids and alveolar organoids are seeded into the airway chamber and alveolar chamber of the microarray, respectively, with the top of the airway chamber open to form a gas-liquid interface; finally, they are co-cultured under dynamic perfusion conditions to obtain an in vitro disease model with the genetic background and pathological phenotype of COPD patients.

[0012] This invention also provides a method for drug screening and efficacy evaluation using the above-mentioned COPD in vitro disease model, comprising the following steps: first, applying candidate drugs to the chip; then, collecting effluent from the micro-sampling port at different time points (0h, 36h, 72h) to detect the dynamic changes of inflammatory factors IL-6, IL-8, TNF-α, and mucus secretion factor MUC5B; simultaneously, staining organoids within the chip for viability and quantitatively analyzing cell viability; and collecting organoids within the chip for qPCR detection to analyze the relative mRNA expression levels of ciliated cell markers FOXJ1 and MYB, secretory cell markers SCGB1A1, MUC5AC, and MUC5B, and basal cell markers P63 and KRT5; finally, comprehensively evaluating the efficacy of the candidate drugs by comparing the differences in the above indicators among the control group, COPD model group, and drug treatment group.

[0013] This invention also provides the application of the above-mentioned chips, culture media, construction methods or evaluation methods in in vitro modeling, mechanism research, drug screening and personalized medicine for chronic airway diseases such as COPD, asthma, and pulmonary fibrosis.

[0014] Compared with the prior art, the present invention has the following beneficial effects: Strong biomimetic structure: The "three-chamber integrated" microfluidic structure of this invention integrates the airway chamber, alveolar chamber and vascular chamber into the same platform through biomimetic spatial layout, and constructs an airway-alveolar-vascular co-culture environment that meets the needs of COPD research. This is beneficial for observing COPD-related changes in airway and alveolar epithelium and their interaction with the vascular microenvironment under in vitro conditions.

[0015] Targeted culture system: This invention is based on patient-derived airway epithelial cells and alveolar epithelial cells, which are cultured using organoid expansion culture medium and differentiation culture medium optimized for COPD tissue characteristics, respectively, to provide a suitable microenvironment for different epithelial lineages and enhance the feasibility and operability of patient-derived cells in in vitro culture.

[0016] Convenience of dynamic monitoring: With the chip's micro-sampling port, the effluent can be obtained in real time and non-invasively during the culture process for the detection of inflammation-related indicators, realizing the correlation analysis of "live cells-secretions" and providing a more convenient experimental method for COPD-related in vitro research and drug evaluation.

[0017] Comprehensive evaluation system: Through multi-dimensional evaluation of morphology, cell viability, gene expression and secretion detection, it can comprehensively reflect the functional changes of various cell types and the effects of drug intervention under the pathological state of COPD.

[0018] With broad application prospects, this invention provides an efficient and biomimetic technology platform for in vitro modeling, mechanism research, drug screening, and personalized medicine for chronic airway diseases such as COPD, asthma, and pulmonary fibrosis.

[0019] To facilitate understanding of this invention, the meanings of the English abbreviations used herein are explained as follows: COPD stands for Chronic Obstructive Pulmonary Disease; 3D stands for Three-Dimensional Culture; Advanced DMEM / F-12 is a culture medium containing various amino acids and glucose; FBS stands for Fetal Bovine Serum; PBS stands for Phosphate Buffered Solution; HEPES stands for Buffer Retinoic Acid; EGF stands for Epidermal Growth Factor; Y-27632 is an inhibitor of the ROCK signaling pathway; R-Spondin1 stands for R-Spondin 1; A8301 is an inhibitor of TGF-β receptor; CHIR99021 is an inhibitor of GSK-3; SB431542 is an inhibitor of the TGF-β signaling pathway; TGF-β stands for Transforming Growth Factor-β; Noggin stands for Noggin protein; Insulin stands for Insulin; Transferrin stands for Transferrin; Cholera Toxin is cholera toxin; FGF10 is fibroblast growth factor 10; FGF7 is fibroblast growth factor 7; FGFR2b is fibroblast growth factor receptor 2b; SPC is pulmonary surfactant protein C; AT2 is alveolar type II epithelial cells; AT1 is alveolar type I epithelial cells; HUVEC is human umbilical vein endothelial cells; PDMS is polydimethylsiloxane; PET is polyethylene terephthalate; ELISA is enzyme-linked immunosorbent assay; qPCR is real-time quantitative polymerase chain reaction. All components in the above culture media are commercially available and readily available from various manufacturers. Attached Figure Description

[0020] Appendix Figure 1The diagram illustrates the organ-on-a-chip (OA-C) usage pattern, showcasing the overall structure and operational process. Four independent chip units are arranged side-by-side on each glass plate, with each chip containing two airway culture chambers and one alveolar culture chamber. This structural layout constructs a multi-chamber culture system with airway and alveolar partitioning characteristics in vitro, and its spatial structure design shows a high degree of similarity to the tissue hierarchy of the airway-alveolar partitioning in vivo.

[0021] Appendix Figure 2 The document analyzes the structure of organ-on-a-chip and presents a diagram of organoid culture, showcasing a multi-layered "three-chamber integrated" design. Each chip consists of four stacked layers, with two inlets / outlets on the upper and lower layers of each chip. The upper and lower layers are connected by a PET membrane, on which endothelial cells are cultured to construct blood vessel-like structures, thereby creating an interface environment within the chip that simulates the air-blood barrier.

[0022] Appendix Figure 3 : Isolation and culture process of lung organoids and identification results of cell lineage characteristics in control and COPD groups. A: Schematic diagram of lung organoid isolation and culture; B: Immunofluorescence identification of various markers in control and COPD groups (airway: Ac-tub / F-actin; MUC5AC / F-actin; alveoli: HOPX / Abca3). Ac-tub: ciliated cell marker; MUC5AC: goblet cell marker; F-actin: cytoskeleton structural marker; HOPX: alveolar type I epithelial cell marker; Abca3: alveolar type II epithelial cell marker; Scale bar: 100 µm.

[0023] Appendix Figure 4 Morphological changes and growth trends of lung organoids in vitro before transfer to the microarray. AB: Light microscopic images and diameter changes of airway organoids on days 2, 5, and 9 before transfer to the microarray, scale bar 100 µm; CD: Light microscopic images and diameter changes of alveolar organoids on days 2, 5, and 9 before transfer to the microarray, scale bar 150 µm.

[0024] Appendix Figure 5 Representative images of lung organoids cultured on a microarray from the control group, COPD group, and erdosteine-treated group. A: Airway organoids; B: Alveolar organoids; Scale bar: 100 µm.

[0025] Appendix Figure 6 qPCR was used to detect the relative mRNA expression levels of inflammation-related genes, secretion-related genes, and lung epithelium-specific marker genes in lung organoids from the control group, COPD group, and erdosteine-treated group. A: Inflammation-related genes IL-6 TNF-α , IL-8 Relative mRNA expression; B: Mucus secretion-related genes MUC5AC, MUC5BRelative mRNA expression; C: Basal cell-associated marker genes SCGB1A1 , P63, KRT5 Relative mRNA expression; D: Ciliary differentiation-related genes FOXJ1 , MYB The relative mRNA expression.

[0026] Appendix Figure 7 Cell survival of lung organoids under different treatment conditions. A: Staining map of live and dead cells in airway organoids; B: Quantitative statistical map of live and dead cells in airway organoids; C: Staining map of live and dead cells in alveolar organoids; D: Quantitative statistical map of live and dead cells in alveolar organoids; Scale bar: 100 µm.

[0027] Appendix Figure 8 : Collection methods of organ-on-a-chip effluent at different time points and their application in the detection of inflammatory factors and secreted proteins. A: Schematic diagram of supernatant collection methods (0 hours, 36 hours, 72 hours); B: ELISA detection of IL-6 inflammatory factor; C: Western blot assay detection of SCGB1A1 and MUC5B secreted factors. Detailed Implementation

[0028] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. This embodiment fully presents the entire process from chip fabrication, organoid culture, model construction to drug evaluation. Those skilled in the art should understand that these embodiments are only used to explain the present invention and are not intended to limit the scope of protection of the present invention.

[0029] 1. Fabrication of a three-chamber integrated microfluidic chip The microfluidic chip provided by this invention constructs an in vitro culture microenvironment that conforms to the spatial hierarchical characteristics of lung tissue through multi-layer structure design and material selection. The specific preparation steps are as follows: First, determine the required amount of PDMS based on the chip's design dimensions (dielectric layer channel height of 200 μm and organoid culture layer chamber height of 300 μm). Mix the base adhesive and curing agent at a weight ratio of 10:1 and manually stir for 5 minutes to ensure thorough mixing. Vacuum the mixed PDMS for 1 hour to remove air bubbles, allow it to stand, pour it into a mold, and vacuum it again for 1 hour until completely free of air bubbles. Place the mold in a vacuum drying oven and let it stand at 75°C for 1.5 hours to cure the PDMS.

[0030] After curing, the chip was cut and inlet and outlet ports were fabricated on the PDMS dielectric layer using a 1 mm punch. A PET film with a 5 μm pore size was cut to a suitable size and subjected to oxygen plasma bonding treatment with the dielectric layer (75 W, 20 seconds), followed by curing at 60°C for 30 minutes. Subsequently, the above dielectric layer chip and organoid culture layer chip were subjected to oxygen plasma bonding treatment under the same conditions and cured at 60°C for 30 minutes.

[0031] Using a 1 mm punch, holes were drilled at the entrance and exit of the organoid culture chamber, and the chips were bonded side-by-side to the glass slides. Each glass slide can hold four chips for parallel experimental operations. Each chip contains the first two airway culture wells and one alveolar culture well (see attached image). Figure 1 The fabricated chip has a three-layer structure: the upper layer has inlets and outlets for adding culture medium or drugs; the lower layer has an organoid inlet for injecting organoids; after the matrix gel solidifies, the lower inlet and outlet can be used to collect the effluent that seeps out from the upper layer; and the middle PET membrane is used for culturing HUVEC cells (see attached image). Figure 2 ).

[0032] To improve the hydrophilicity of PDMS, the chip was treated with oxygen plasma for 5 minutes, followed by rinsing the channels with 70% ethanol and washing three times with PBS. Twelve hours before organ grafting, a concentration of 4.5 × 10⁻⁶ ppm was added. 6 HUVEC cell suspension at a concentration of cells / mL was injected into the microarray vascular chamber through the culture medium inlet and incubated at 37°C for 3 hours to allow the cells to adhere to the wall. The cells were then cultured for another 12 hours to form a complete vascular endothelial layer.

[0033] 2. Construction of airway organoids derived from COPD patients Tracheal tissue obtained from COPD patients postoperatively was placed in Advanced DMEM / F12 medium containing 1% penicillin-dextrose antibody, and the connective tissue around the trachea was carefully cleaned with sharp forceps. The tissue was washed three times in 1.5 mL centrifuge tubes containing 200 μL of PBS containing 1% penicillin-dextrose antibody to remove residual connective tissue.

[0034] Open the trachea longitudinally with scissors and place it in a digestion solution containing 0.1% collagenase I and 0.05% DNase I. Digest at 37°C for 30 minutes. After digestion, use forceps to remove airway cartilage and other tissues, and filter them sequentially through 300 μm and 100 μm cell filters. Collect the filtrate, centrifuge at 800×g, 37°C for 5 minutes, and discard the supernatant. Add 1 mL of pre-warmed DMEM / F12 complete medium containing 5% FBS to wash the cell pellet, and centrifuge again at 800×g, 37°C for 5 minutes. Discard the supernatant and resuspend the cells in 1 mL of pre-warmed basal medium.

[0035] Thaw the matrix gel overnight on ice at 4°C. Mix the matrix gel and cell suspension at a 1:1 ratio on ice. Seed the cells at a density of 800 cells per well in the center of a 96-well plate, ensuring the cells form a three-dimensional droplet structure in the matrix gel. Incubate the plate at 37°C for 30-60 minutes until the matrix gel solidifies. Then, slowly add 200 µL of preheated (37°C) airway organoid amplification medium along the well walls. The amplification medium is based on Advanced DMEM / F-12 and contains 10% FBS and the following components: EGF 25-50 ng / mL, Insulin 10-50 μg / mL, Transferrin 5-25 μg / mL, CholeraToxin 0.1-0.5 μg / mL, Retinoic Acid 50-100 nM, Noggin 20-50 μg / mL, R-Spondin 1200-800 ng / mL, A8301 500-1000 nM, CHIR99021 3-10 μM, and Y-27632 5-10 μM.

[0036] The culture medium was replaced with half of the culture medium every two days under 5% CO2 and 37℃ conditions for 9 days. Organoid morphology was observed by photographing on days 2, 5, and 9 (see attached image). Figure 4 (AB). As the culture time increases, the organoids gradually increase in size. When their diameter reaches more than 100 µm, the organoids are transferred to an organ-on-a-chip for further culture and processing.

[0037] 3. Construction of alveolar organoids derived from COPD patients Lung tissue obtained surgically from COPD patients was placed in Advanced DMEM / F12 medium containing 1% penicillin-dextrose antibody, and the surrounding tissue was cleaned. The cleaned tissue was placed in a sterile 1.5 mL centrifuge tube and washed three times with PBS containing 1% penicillin-dextrose antibody. The cleaned lung tissue was minced to less than 1 mm³ and placed in an enzyme solution containing 0.05% DNase I and 2 mg / mL Dispase II. It was digested at 37°C for 40 minutes, with repeated pipetting until the tissue was in suspension.

[0038] Digestion was terminated by adding an equal volume of DMEM / F12 medium containing 5% FBS. After filtering through a 100 μm filter, the cells were centrifuged at 800×g and 37°C for 5 minutes. The supernatant was slowly aspirated, and the cells were resuspended in 1 mL of DMEM / F12 complete medium containing 5% FBS. The cells were centrifuged again at 800×g and 37°C for 5 minutes. The supernatant was discarded, and the cells were resuspended in 1 mL of basal medium and transferred to a culture dish. The cells were incubated in a cell culture incubator for 2-3 hours, and fibroblasts were removed using the differential adhesion method. The non-adhered AT2 cells were collected, centrifuged at 800×g and 37°C for 5 minutes, resuspended in basal medium, and pre-cooled on ice.

[0039] Mix the matrix gel and cell resuspension at a 1:1 ratio and seed them into 96-well plates. After incubating at 37°C for 30-60 minutes to allow the matrix gel to solidify, add 200 µL of preheated alveolar organoid differentiation medium (37°C) along the well wall. The differentiation medium is based on Advanced DMEM / F-12 and contains the following components: FGF10 10-50 ng / mL, FGF7 25-50 ng / mL, SB431542 10-50 μM, and SPC 5-20 ng / mL.

[0040] The culture medium was replaced with half of the culture medium every two days under 5% CO2 and 37℃ conditions for 9 days. Organoid morphology was observed by photographing on days 2, 5, and 9 (see attached image). Figure 4 (CD). When the average diameter of the organoids reaches 100 µm or more, they are transferred to the chip.

[0041] 4. In-chip co-culture and COPD disease model construction The airway and alveolar organoids prepared above were transferred to a microarray for co-culture. The specific procedures are as follows: Select airway organoids measuring 100-200 μm. Aspirate the culture medium from the well plate, repeatedly blow and aspirate to break up the matrix gel, and transfer the organoid suspension to a 1.5 mL centrifuge tube. Incubate at 37°C for 10 minutes. Centrifuge at 800×g for 8 minutes, discard the supernatant, and resuspend the organoids in differentiation medium and matrix gel at a 1:1 ratio. Immediately inject the organoid suspension through the organoid inlet into the first two airway culture wells of the chip until the droplets fill the wells. Incubate the chip at 37°C and 5% CO2 for 30 minutes to allow the matrix gel to solidify (see attached diagram). Figure 5 ).

[0042] Using the same method, alveolar organoids with a size of 100-150 μm were selected, processed, and slowly injected into the third alveolar culture well of the chip. After the matrix gel solidified, 1 mL pipette tips with the tips cut off were inserted into the four perfusion wells. The upper pipette tip was used to inject culture medium for dynamic perfusion culture, and the lower pipette tip was used to collect the effluent.

[0043] Experimental groups were set up as follows: control group, COPD model group (organoids derived from COPD patients), and erdostein administration group (organoids derived from COPD patients + 10 μM erdostein). After continuous culture for 72 hours, the effluent was collected from the lower pipette tip at 0, 36, and 72 hours after drug intervention and stored at -80℃ for later use. (See attached...) Figure 8 A).

[0044] 5. Model validation and drug efficacy evaluation 5.1 Immunofluorescence identification After culture, immunofluorescence staining was performed on the organoids within the microarray. In the control and COPD groups, the expression of the cytoskeleton (F-actin) and ciliated epithelial cell marker (Ac-tub) was observed in airway organoids; the expression of AT1 cell marker (HOPX) and AT2 cell marker (Abca3) was observed in alveolar organoids (see attached image). Figure 3 B). The results showed that MUC5AC expression was increased and Ac-tub expression was decreased in airway organoids in the COPD group; HOPX / Abca3 expression was decreased in alveolar organoids, and the abnormal expression of these markers was consistent with the clinical phenotype.

[0045] 5.2 Morphological evaluation Morphological observation was performed 72 hours after chip transfer (see attached). Figure 5 The results showed that lung organoids derived from COPD patients exhibited significant shrinkage, and this morphological abnormality was partially improved after treatment with erdosteine.

[0046] 5.3 Evaluation of organoid cell viability After culture, organoids within the chip were stained for viability and death, and cell viability was observed and quantitatively analyzed using fluorescence microscopy (see appendix). Figure 7 The results showed that the proportion of dead cells in organoids was significantly increased in the COPD group, and erdosteine ​​intervention could effectively inhibit cell death.

[0047] 5.4 Gene Expression Analysis RNA was extracted from organoids collected within the microarray and inflammatory factors were detected using qPCR. IL-6, IL-8, TNF-α ), mucus secretion-related genes ( MUC5AC, MUC5B ), basal cell markers ( SCGB1A1, P63, KRT5 ) and ciliated cell markers ( FOXJ1、MYB The relative mRNA expression levels of ) (see attached) Figure 6 The results showed that, compared with the control group, the COPD model group had significantly increased expression of inflammatory factors and mucus secretion-related genes, and decreased expression of basal cell markers and ciliated cell marker-related genes; most of the above indicators were significantly improved after erdosteine ​​intervention.

[0048] 5.5 Dynamic monitoring of secretions The concentration of IL-6 in the collected effluent was detected using an ELISA kit, and the secretion levels of SCGB1A1 and MUC5B were detected using Western blotting. (See attached image) Figure 8 The results showed that the secretion of IL-6 and MUC5B in the supernatant of the COPD group was significantly increased, while the secretion of SCGB1A1 was significantly decreased. Erdosteine ​​treatment could effectively improve the abnormal secretion of the above indicators.

[0049] In summary, lung organoids derived from COPD patients exhibit significant functional disorders in multiple key pathophysiological aspects, including morphological integrity, cell survival, inflammatory secretion, and mucus hypersecretion. Erdosteine ​​demonstrated a broad-based ameliorative effect on these functional abnormalities. These results fully validate that the chip model constructed in this invention can effectively simulate the airway-alveolar dual-site damage characteristics of COPD patients and can be reliably used for drug efficacy evaluation, proving the reliability and practicality of this platform in simulating disease phenotypes and drug evaluation.

[0050] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A three-chamber integrated microfluidic chip that simulates the airway-alveolar continuous barrier function, characterized in that, include: Airway chambers are used to culture airway organoids, with an open top forming a gas-liquid interface; Alveolar chambers, used for culturing alveolar organoids, are physically connected to the sidewalls of the airway chambers to form a continuous epithelial barrier structure; The vascular chamber, located in the upper layer, is separated from the lower airway chamber and alveolar chamber by a porous membrane, and is lined with endothelial cells to simulate blood vessels. A porous membrane, located between the vascular chamber and the lower culture chamber, is used to facilitate the exchange of signals and substances and to construct a gas-blood barrier. The micro-sampling port is in fluid communication with the blood vessel chamber and is used to collect the outflow fluid.

2. The chip according to claim 1, characterized in that, The airway chambers and alveolar chambers share the same vascular chamber and porous membrane, enabling parallel culture and collaborative analysis of airway and alveolar organoids.

3. An expansion culture medium for culturing airway organoids derived from COPD patients, characterized in that, The culture medium includes the basal medium Advanced DMEM / F-12, 10% FBS, and the following components: Epidermal growth factor (EGF) 25-50 ng / mL; Insulin 10-50 μg / mL; Transferrin 5-25 μg / mL; Cholera toxin 0.1-0.5 μg / mL; Retinoic acid 50-100 nM; Noggin 20-50 μg / mL; R-Spondin 1 200-800 ng / mL; A8301 500-1000 nM; CHIR99021 3-10 μM; Y-27632 5-10 μM.

4. A differentiation culture medium for culturing alveolar organoids derived from COPD patients, characterized in that, The culture medium includes a basal medium and the following components: fibroblast growth factor 10 (FGF10) 10-50 ng / mL; fibroblast growth factor 7 (FGF7) 25-50 ng / mL; SB431542 10-50 μM; surfactant protein C (SPC) 5-20 ng / mL.

5. A method for constructing COPD airway organoids based on the amplification culture medium described in claim 3, characterized in that, Includes the following steps: Obtain airway tissue from COPD patients and obtain airway epithelial cells through enzymatic digestion; The cells were mixed with matrix gel and cultured in 3D embedding, and the amplification medium described in claim 3 was added. Cultured until airway organoids with characteristics of ciliated cells, secretory cells, and basal cells are formed.

6. A method for constructing COPD alveolar organoids based on the differentiation culture medium of claim 4, characterized in that, Includes the following steps: Lung tissue from COPD patients was obtained and cell suspension was obtained through enzymatic digestion. Fibroblasts were removed to obtain AT2 cells; AT2 cells were mixed with matrix gel and cultured in 3D embedding, and the differentiation medium described in claim 4 was added. Cultured until alveolar organoids with AT2 cell characteristics and expressing HOPX / Abca3 were formed.

7. An in vitro COPD disease model constructed based on any one of claims 1-6, characterized in that, include: COPD patient-derived airway organoids, alveolar organoids, and vascular endothelial layer co-cultured on a chip; The chip operates under dynamic perfusion and gas-liquid interface conditions to form an in vitro disease model with a genetic background of COPD patients.

8. A method for drug screening and efficacy evaluation using the COPD in vitro disease model as described in claim 7, characterized in that, Includes the following steps: Drug intervention: Applying candidate drugs to the chip for cultivation; Dynamic monitoring of effluent: Collect effluent to detect dynamic changes in inflammatory factors and mucus secretion factors; Cell viability assessment: Organoids within the microarray were stained for viability and deadness, and cell viability was quantitatively analyzed; Gene expression analysis: Collect organoids within the microarray for gene expression detection; Efficacy evaluation: The efficacy of the candidate drug was comprehensively evaluated by comparing the differences in the above indicators among the control group, COPD model group and drug treatment group.

9. The use of the chip of claim 1 or 2, the culture medium of claim 3 or 4, the culture method of claim 5 or 6, the COPD in vitro disease model of claim 7, or the drug evaluation method of claim 8 in the preparation of a kit or device for chronic airway disease research or drug screening.

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