In-vitro co-culture alveolar-capillary model system

Abstract

The invention provides a co-culture system of human alveolar epithelial cells and endothelial cells, which is used for researching interaction in an epithelial-endothelial barrier in lungs. It is indicated that infection with the highly pathogenic influenza A virus A / HK / 483 / 97 (H5N1) can significantly reduce alveolar fluid clearance (AFC) and increase protein permeability (APP). Administration of therapeutic extracellular vesicles (EV) after infection effectively restores AFC and APP, reduces viral replication, and enhances expression of antiviral genes. According to the invention, the treatment potential of EV is highlighted, and a valuable tool is provided for researching acute lung injury and improving a lung drug delivery technology.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 593,243 filed on October 26, 2023, and the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0002] The present invention utilizes primary cells isolated from human lung tissue seeded into a transwell system, thereby increasing the physiological relevance of the in vitro lung model and allowing the study of cellular interactions in the alveolar-capillary barrier outside of a clinical setting. Background Art

[0003] In severe respiratory diseases, vascular leakage and pulmonary edema accelerate the progression of the disease and may eventually lead to respiratory failure. Studies have shown that damage to the respiratory endothelium triggers cellular infiltration and fluid leakage into the alveolar space. On the other hand, the healthy respiratory epithelium provides a physical and immunological barrier against external pathogens, thereby protecting the underlying tissue from direct infection. However, in the diseased state, the integrity of the epithelium is compromised, which in turn leads to endothelial damage, allowing pathogens to enter the bloodstream. Therapeutic strategies to address these issues include restoring the integrity of the epithelium and endothelium through tight / adherence junctions and altering the cytoskeletal arrangement through specific signaling pathways.

[0004] With the advent of organ-on-a-chip (OOAC) culture systems, mechanical stretching can be applied to systems the size of an AA battery to better simulate the physiological movements of lung tissue during breathing. However, it has been reported that the responses of lung-on-a-chip still cannot fully reproduce the responses observed in native alveolar epithelial cells (AECs). In terms of drug testing, due to the extremely small size of microfluidic systems, surface effects may be more significant than volume effects, which may lead to more drug particles being adsorbed than expected, thereby affecting the fluid mixing effect at the intersection. In addition, special equipment may be required to enhance the reliability of the data when using OOAC model cancer environments or the human intestine.

[0005] Predictive in vitro modeling systems that accurately mimic human physiological conditions are critical to understanding the mechanisms driving disease pathogenesis outside of the clinical setting. Currently, in vitro models often consist of a single culture that may behave differently than what is observed in vivo due to the lack of microenvironmental interactions.

[0006] Furthermore, ethical issues surrounding animal models and their generally low predictive value for human disease further highlight the need for alternative approaches. Although immortalized cell lines (such as A549 cells derived from human lung cancer) have been widely used, the phenotypic differences they produce under in vitro culture conditions limit their ability to replicate key features of physiological epithelia.

[0007] There are many challenges in studying the physiological changes and associated mechanisms of acute lung injury caused by respiratory viruses outside of the clinical setting. Accurately modeling these physiologically relevant processes in vitro is critical to advancing drug screening efforts, especially for precision medicine. Developing more representative models can help drive the exploration of therapeutic interventions, thereby more accurately predicting clinical outcomes and ultimately improving the quality of care for patients. Summary of the invention

[0008] The present invention aims to enhance the physiological relevance of the lung model and thus establishes a co-culture model. The present invention discloses an in vitro co-culture lung injury format optimized for primary human AEC and human pulmonary microvascular endothelial cell (HMVEC) cultures for studying the mechanisms of pathogenesis and recovery of highly pathogenic influenza virus infection.

[0009] Human AECs and HMVECs were cultured in a collagen-coated transwell system. This lung injury model can be used to study changes in alveolar fluid clearance (AFC) and alveolar protein permeability (APP) after respiratory virus infection, both of which are important factors in the fatal development of acute respiratory distress syndrome (ARDS) caused by severe respiratory virus infection.

[0010] In a first aspect, the present invention provides an in vitro co-culture alveolar-capillary model system for studying acute lung injury induced by respiratory virus infection. The system comprises: a transwell system, wherein primary human alveolar epithelial cells are seeded on the apical chamber surface of the transwell system, and human pulmonary microvascular endothelial cells are seeded on the basolateral surface of the transwell system; and a device for measuring alveolar fluid clearance and alveolar protein permeability in infected cells. The in vitro co-culture alveolar-capillary model system simulates the alveolar-capillary barrier.

[0011] In one embodiment, the primary human alveolar epithelial cells are cultured on collagen-coated transwell inserts to promote the formation of tight junctions.

[0012] In one embodiment, the primary human alveolar epithelial cells are isolated from non-malignant lung tissue.

[0013] In one embodiment, 5×10 4 Up to 2×105 The primary human alveolar epithelial cells were seeded at a cell density of 5×10 cells per well. 4 Up to 2×10 5 The human pulmonary microvascular endothelial cells were seeded at a cell density of 10 cells.

[0014] In one embodiment, the pore size of the transwell system is 0.4 μm.

[0015] In a second aspect, the present invention provides a method for studying acute lung injury induced by respiratory viral infection in vitro, the method comprising: isolating primary human alveolar epithelial cells and human microvascular endothelial cells; inoculating the primary human alveolar epithelial cells on the apical chamber surface of the transwell membrane, and inoculating the human microvascular endothelial cells on the basolateral surface of the transwell system to form an in vitro co-culture alveolar-capillary model system; infecting the in vitro co-culture alveolar-capillary model system with one or more respiratory viruses; and evaluating the effect of viral infection on at least one physiological parameter, the physiological parameters comprising AFC and APP.

[0016] In one embodiment, the primary human alveolar epithelial cells and the human microvascular endothelial cells are isolated using enzymatic digestion followed by density gradient centrifugation and magnetic bead separation.

[0017] In one embodiment, the primary human alveolar epithelial cells are cultured on collagen-coated transwell inserts to promote the formation of tight junctions.

[0018] In one embodiment, the primary human alveolar epithelial cells are isolated from non-malignant lung tissue.

[0019] In one embodiment, 5×10 4 Up to 2×10 5 The primary human alveolar epithelial cells were seeded at a cell density of 5×10 cells per well. 4 Up to 2×10 5 The human pulmonary microvascular endothelial cells were seeded at a cell density of 10 cells.

[0020] In one embodiment, the pore size of the transwell system is 0.4 μm.

[0021] In one embodiment, the one or more respiratory viruses are selected from the group consisting of coronavirus, respiratory syncytial virus, adenovirus, influenza virus, parainfluenza virus, rhinovirus, enterovirus, and herpes simplex virus.

[0022] In one embodiment, the in vitro co-culture alveolar-capillary model system is infected with influenza A virus subtype H5N1.

[0023] In another embodiment, the method further comprises adding a therapeutic agent after the viral infection to inhibit viral replication in the human alveolar epithelial cells and the human pulmonary microvascular endothelial cells.

[0024] In one embodiment, the therapeutic agent comprises a concentration of 1×10 7 Up to 1×10 9 The invention relates to an extracellular vesicle particle (EV) derived from human mesenchymal stromal cells.

[0025] In another embodiment, the method further comprises measuring transepithelial electrical resistance (TEER) as a metric to assess the integrity of the epithelial-endothelial barrier over time, and the TEER remains ≥ 800 Ω / cm 2 .

[0026] In one embodiment, the step of assessing the effect of viral infection on at least one physiological parameter further comprises measuring viral gene expression and antiviral gene expression in said primary human alveolar epithelial cells and said human microvascular endothelial cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Embodiments of the present invention are described in more detail below with reference to the accompanying drawings, in which:

[0028] Figure 1A Schematic diagram of an in vitro co-culture alveolar-capillary model system using a confluent monolayer of human AECs seeded on a collagen-coated apical chamber and human HMVECs seeded on the underside of each transwell membrane to mimic human alveoli and surrounding capillaries in a clinical setting;

[0029] Figure 1B demonstrated the assessment of tight junction integrity in cells by measuring daily TEER using an epithelial volt-ohmmeter (EVOM) machine;

[0030] Figure 1C Depicted are the TEER values ​​of the alveolar-capillary barrier observed during the first six-day period of the experiment;

[0031] Figure 1D It is shown that infection of AEC with highly pathogenic H5N1 resulted in a significant decrease in AFC and an increase in APP. Data represent mean ± SD from three experiments. *p≤0.05; **p≤0.01 and ***p≤0.001;

[0032] Figure 2AShown that EV treatment of infected cells restored AFC across the barrier compared to untreated infected cells. Data represent mean ± SD from three experiments. *p ≤ 0.05; **p ≤ 0.01 and ***p ≤ 0.001;

[0033] Figure 2B Shown that EV treatment of infected cells reduced APP across the barrier compared to untreated infected cells. Data represent mean ± SD from three experiments. *p ≤ 0.05; **p ≤ 0.01 and ***p ≤ 0.001;

[0034] Figure 2C A(H5N1)-infected AEC and A(H5N1)-infected HMVEC show high expression of influenza virus M gene. EV treatment inhibits viral replication across the barrier. Data represent mean ± SD from three experiments. *p≤0.05; **p≤0.01 and ***p≤0.001; and

[0035] Figure 2D The expression of antiviral genes is shown, indicating that the addition of EVs to infected cells enhances the expression of antiviral genes in both AECs and HMVECs. Data represent mean ± SD from three experiments. *p≤0.05; **p≤0.01 and ***p≤0.001. DETAILED DESCRIPTION

[0036] Severe H5N1 influenza virus infection in humans results in significant mortality, which is primarily attributed to acute lung injury, for which few treatment options are currently available.

[0037] Therefore, the present invention utilizes primary human alveolar epithelial cells and human microvascular endothelial cells to establish a highly physiologically relevant preclinical model for studying respiratory pathogens in an in vitro setting.

[0038] Specifically, the present invention provides an in vitro co-culture alveolar-capillary model system for studying acute lung injury induced by respiratory virus infection. The system includes: a transwell system, in which primary human alveolar epithelial cells are seeded on the apical chamber surface of the transwell system, and human pulmonary microvascular endothelial cells are seeded on the basolateral surface of the transwell system; and a device for measuring the alveolar fluid clearance rate and alveolar protein permeability in infected cells. The in vitro co-culture alveolar-capillary model system simulates the alveolar-capillary barrier. The primary human alveolar epithelial cells are cultured on collagen-coated transwell inserts to promote the formation of tight junctions.

[0039] This model is superior to existing systems because it more accurately replicates the alveolar-capillary barrier of the human lung by culturing primary human AECs and HMVECs on a collagen-coated transwell system. The co-culture model facilitates patient-specific therapies because primary human lung cells are obtained from specific donors, which allows for tailored treatment of respiratory diseases based on fine-tuning of phenotypic drug screening in the lung model. This can be directly translated to the clinical setting, maximizing the likelihood of therapeutic success.

[0040] The co-culture alveolar-capillary model formed by human AECs and HMVECs has several significant advantages. First, it provides enhanced physiological relevance by recreating the barrier between alveoli and capillaries, capturing epithelial-endothelial interactions that are critical for studying lung diseases. This model produces more accurate results than traditional monoculture systems, especially when studying lung injury caused by infection.

[0041] Unlike traditional models that use immortalized cell lines (such as A549), this system uses primary cells isolated from human tissue, resulting in a more accurate physiological representation. The use of primary cells offers the potential for patient-specific therapies, as these cells can be derived from individual patients, enabling personalized treatment evaluation.

[0042] In one embodiment, the pore size of the transwell system is approximately 0.4 μm. This pore size was used to assess AFC and APP by measuring the rate of migration of FITC-dextran (70 kDa) from the apical chamber to the basolateral chamber of the transwell.

[0043] The transwell system of the present invention realizes a bilayer co-culture, in which AECs are seeded on the apical chamber surface and HMVECs are cultured on the basolateral surface. This simulates the natural structure and function of the human alveolar-capillary barrier. 4 Up to 2×10 5 Primary human alveolar epithelial cells were seeded at a cell density of 5 × 10 cells per well. 4 Up to 2×10 5 Human pulmonary microvascular endothelial cells were seeded at a cell density of 10 cells.

[0044] Preferably, 1×10 5 Primary human alveolar epithelial cells were seeded at a cell density of 1 × 10 cells per well. 5 Human pulmonary microvascular endothelial cells were seeded at a cell density of 10 cells.

[0045] The cell density required for HMVEC in OOAC is twice that of the present invention. The present transwell system appears to remain the mainstream choice for cell co-culture because it is easily accessible and does not require specialized equipment, while maintaining the physiological relevance required for accurate data acquisition.

[0046] A significant advantage of the present invention is that it can measure the integrity of the epithelial-endothelial barrier by monitoring TEER. This is an important numerical criterion because TEER values ​​within this range indicate that the cell layer has formed fully functional, complete and stable tight junctions, demonstrating good barrier function.

[0047] In one embodiment, TEER is always maintained at 800Ω / cm 2 The above confirms the formation of strong tight junctions. When the TEER value exceeds 800Ω / cm 2 The system was able to adequately simulate the human alveolar-capillary interface, indicating that the integrity of the tight junctions between epithelial cells and endothelial cells was sufficient to resist external stimuli or pathogen invasion.

[0048] In addition, the present invention also provides a method for studying acute lung injury induced by respiratory virus infection in vitro, the method comprising: isolating primary human alveolar epithelial cells and human microvascular endothelial cells; inoculating the primary human alveolar epithelial cells on the apical chamber surface of the transwell membrane, and inoculating the human microvascular endothelial cells on the basolateral surface of the transwell system to form an in vitro co-culture alveolar-capillary model system; infecting the in vitro co-culture alveolar-capillary model system with one or more respiratory viruses; and evaluating the effect of viral infection on at least one physiological parameter, wherein the physiological parameters include AFC and APP.

[0049] In one embodiment, the one or more respiratory viruses may include coronavirus, respiratory syncytial virus, adenovirus, influenza virus, parainfluenza virus, rhinovirus, enterovirus, and herpes simplex virus.

[0050] Preferably, the one or more respiratory viruses is H5N1.

[0051] The H5N1 virus significantly impaired AFC and APP in co-culture, inducing high levels of influenza virus gene activity that disrupted tight junctions between cells. This also hampered the expression of antiviral genes required to mitigate viral damage to the barrier.

[0052] EVs derived from human mesenchymal stromal cells have been used as therapeutic agents. Following infection, administration of EVs has been shown to significantly restore AFC and reduce APP, thereby alleviating the damage caused by viral replication.

[0053] In addition, administration of EVs to epithelia infected with A(H5N1) virus can effectively reduce viral replication by enhancing the expression of antiviral genes (e.g., ISG15 and MX-1) in both epithelia and endothelium. This in turn improves AFC and APP, bringing them closer to normal levels.

[0054] The results demonstrate the therapeutic potential of EVs and highlight the usefulness of the in vitro co-culture system to examine epithelial-endothelial interactions in the pathogenesis of acute lung injury in patient-specific settings. Furthermore, it can be used to improve pulmonary drug delivery technologies that require a functional barrier.

[0055] Examples

[0056] Example 1

[0057] Materials and methods

[0058] Influenza A virus / HK / 483 / 97 (H5N1) was used for infection. All influenza viruses were passaged in Madin-Darby canine kidney (MDCK) cells. The median tissue culture infectious dose (TCID 50 ) Virus titers were determined. All experiments were performed in a biosafety level 3 facility.

[0059] Separation of AEC

[0060] Human AECs were isolated from non-malignant human lung tissues removed. In brief, visible bronchi were removed, and lung tissue was minced into fragments less than 0.5 mm. The minced tissue was washed three times with Hank's balanced salt solution (HBSS) (Gibco) and 0.7 mM sodium bicarbonate (Gibco) (pH 7.4), to remove most of macrophages and hemocytes. At 37°C in a shaking water bath, a combination of 0.5% trypsin (Gibco) and 4 U / mL elastase (Worthington Biochemical Corp.) was used to digest the tissue for 40 minutes.

[0061] Digestion was stopped by adding DMEM / F12 medium (Gibco) containing 40% FBS and 350U / mL DNase I (Sigma). Cell clumps were dispersed by repeated pipetting of the cell suspension for 10 minutes. A disposable cell strainer (mesh size 50 μm) (BD Bioscience) was used to remove undigested large tissue fragments. The single cell suspension was centrifuged and the pellet was resuspended in a 1:1 mixture of DMEM / F12 medium and small airway basal medium (Lonza), supplemented with 0.5 ng / mL epidermal growth factor, 500 ng / mL epinephrine, 10 μg / mL transferrin, 5 μg / mL insulin, 0.1 ng / mL retinoic acid, 6.5 ng / mL triiodothyronine, 0.5 μg / mL hydrocortisone, 30 μg / mL bovine pituitary extract, 0.5 mg / mL BSA, 5% FBS and 350 U / mL DNase I. The cell suspension was placed in a tissue culture flask and incubated for 90 minutes in a water-jacketed incubator at 37° C. at 5% CO 2. Non-adherent cells were layered over a discontinuous cold Percoll density gradient (density 1.089 and 1.040 g / mL) and centrifuged at 25×g for 20 minutes. The cell layer at the interface of the two gradients was collected and washed four times with HBSS to remove the Percoll fluid. The cell suspension was incubated with magnetic beads coated with anti-CD14 antibody (MACS CD14 microbeads) at room temperature for 20 minutes.

[0062] After removing the beads using a magnet, cell viability was assessed by trypan blue dye exclusion. Alveolar epithelial cell suspensions were resuspended in SAGM (Lonza) supplemented with 1% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin and plated at 3×10 5 / cm 2 The cells were plated at a cell density of 100 μg / mL. The cells were maintained in a humidified atmosphere (5% CO2, 37°C) and the growth medium was changed daily starting 60 hours after the cells were plated. When the cell layer approached 75% confluence, the alveolar epithelial cells were dissociated with trypsin, detached into HBSS, and then seeded in transwell culture inserts as described below.

[0063] Isolation of HMVEC

[0064] HMVEC is isolated from human non-tumor lung tissue. In short, lung tissue is minced and digested with 1% type II collagenase (Thermo Fisher scientific), dispase (Thermo Fisher scientific) and DNase I (Sigma) under shaking for 1 hour at 37°C. Digestion is quenched with DMEM containing 10% FBS. The digestion mixture is filtered through a 70 μm cell strainer (BD Falcon), and then HMVEC is separated from other cell types (e.g., erythrocytes, granulocytes and other cell debris) using Ficoll-Paque Plus (GE Healthcare). After centrifugation for 20 minutes at 400 x g, all interphase cells containing HMVEC are taken out. The cells were incubated with biotinylated anti-CD31 antibody (Abcam) labeled streptavidin-labeled M-280 immunomagnetic beads (Dynabeads) (Thermo Fisher Scientific) at 4°C with gentle agitation for 30 minutes. After incubation, the bead-bound cells were washed 5 times with 1 ml of MACS buffer using a DynaMag5 magnet (Thermo Fisher Scientific). The bead-bound CD31-positive cells were resuspended in endothelial cell growth medium-2EGM-2 (Lonza) and plated on 0.2% gelatin-coated tissue culture flasks for culture. The cells were kept in a humidified atmosphere (5% CO2, 37°C) and the growth medium was replaced every two days. When the cell layer approached 75% confluence, the endothelial cells were dissociated with trypsin, detached into HBSS, and then seeded in transwell culture inserts as described below.

[0065] Statistical analysis

[0066] All in vitro experiments were performed in triplicate independently. Groups were compared using an unpaired two-tailed t-test. Differences were considered significant if p ≤ 0.05.

[0067] Example 2

[0068] Generation of an in vitro co-culture alveolar-capillary model

[0069] To investigate the effects of influenza virus on alveolar fluid clearance and protein permeability in human AECs and HMVECs, this example modifies a physiologically relevant 24-transwell in vitro acute lung injury model to create a co-culture model suitable for use in BSL-3 laboratories.

[0070] Rat tail collagen was first coated onto the apical chamber surface of a 24-well Costar Transwell insert (Corning) with a pore size of 0.4 μm and allowed to solidify. 5 HMVEC were seeded onto the basolateral surface of the transwell membrane and allowed to attach for 2 hours. Subsequently, 1 x 10 5 AECs were seeded onto the apical chamber surface of the membrane and allowed to attach overnight. The plated cells were maintained in a humidified atmosphere (5% CO2, 37°C) and the medium was refreshed every 48 hours. TEER was maintained ≥800Ω / cm 2 , indicating good tight junction integrity between cells.

[0071] Cytomix, a mixture of cytokines IL-1β, TNF-α, and IFN-γ (R&D Systems), was used as a positive control for lung injury at 50 ng / mL.

[0072] Example 3

[0073] Measurement of AFC and APP

[0074] The net alveolar fluid transport rate from the apical chamber of the transwell system to the basolateral chamber of the transwell system was measured 24 hours after infection.

[0075] AECs were inoculated with the corresponding influenza A virus at a multiplicity of infection (MOI) of 0.1 for 1 hour. Then, 200 μl of FITC-labeled dextran (Sigma) of 70 kDa was added to the cells (final concentration of 500 ng / μl). After 5 minutes, 100 μl of sample was collected from the apical chamber for initial FITC measurement and then transferred back to the apical chamber for overnight incubation. After 24 hours of dextran incubation, 100 μl was collected from the apical chamber and 100 μl was collected from the basolateral chamber for final FITC measurement. The fluorescence of each sample was measured at an excitation wavelength of 485 nm and an emission wavelength of 520 nm by a modulus fluorometer (FLUOstar OPTIMA, BMG Labtech). A standard curve of known FITC concentrations was constructed for calculating the FITC-dextran present in the transwell chamber at different time points. Net alveolar fluid transport across the epithelial monolayer was measured as: [1–(FITC concentration in initial apical sample / FITC concentration in final apical sample)] / 200 μl / 0.33 cm 2 / 24 hours

[0076] A final basolateral reading was collected after 24 h of dextran incubation to measure protein permeability based on the unidirectional flux of fluorescently labeled dextran from the apical to the basolateral chamber of the transwell system.

[0077] Figures 1A-1D Describes the development of an in vitro alveolar-capillary model system. Monolayers of primary human AECs and HMVECs were grown on the apical and basolateral chamber surfaces of transwell membranes (rat tail collagen coated), respectively. This device serves as a physiologically relevant model of the alveolar-capillary interface of the human lung, enabling the study of fluid transport across the epithelial-endothelial barrier in vitro ( Figure 1A ).

[0078] The integrity of tight junctions between cells is monitored daily using the EVOM machine. Figure 1B TEER increases when tight junctions form, while monolayer damage results in a decrease in TEER. Prior to experiments conducted on established in vitro model systems, TEER increased steadily over a 6-day period. Figure 1C , TEER values ​​from co-cultures of AEC and HMVEC were consistently higher than the TEER values ​​of either cell type cultured alone.

[0079] Figure 1D It was shown that H5N1 significantly decreased AFC and increased APP at 24 hours post infection compared to the mock group.Pro-inflammatory cytokine mixture [Cytomix (IL-1β, TNF-α and IFN-γ)] was used as a positive control for monolayer injury.

[0080] Example 4

[0081] Nanosized EVs prevent H5N1 virus damage to AFC and APP by inhibiting viral replication in AEC and HMVEC

[0082] In this example, AECs in the apical chamber were infected with influenza A(H5N1) virus at a multiplicity of infection (MOI) of 0.1 to monitor AFC and APP. Additionally, an MOI of 2 was used to study the expression of viral and antiviral genes in both AECs and HMVECs after infection.

[0083] Figures 2A-2D The antiviral activity of nanoscale therapeutic EVs was characterized, which helped prevent A(H5N1) virus-induced damage to alveolar epithelial-endothelial AFC and APP. First, AECs in transwells were infected with A(H5N1) influenza virus. The results showed that the addition of EVs to A(H5N1) infected cells restored AFC ( Figure 2A ) and reduced APP( Figure 2B ).

[0084] like Figure 2C As shown, EV treatment suppressed the elevated expression of the viral M gene following A(H5N1) infection in AEC. Although AEC are the first to be exposed to influenza virus, the underlying HMVEC are also susceptible to infection with A(H5N1). This resulted in a pattern of viral gene expression in HMVEC that was similar to that in the epithelium, albeit at lower levels. This suppression was associated with an increase in the expression of the antiviral genes ISG15 and MX-1 induced by EV treatment ( Figure 2D An interesting observation was the differential expression of IL-29 between epithelial cells and endothelial cells, suggesting that when influenza virus invades the outside of epithelial cells, the endothelial cells act as a secondary defense barrier to prevent the virus from entering the bloodstream.

[0085] In conclusion, the present invention established a physiologically relevant in vitro alveolar-capillary lung injury model to study the pathogenesis of severe human influenza through epithelial-endothelial cell interactions. By using this model, it was shown that highly pathogenic H5N1 virus significantly impairs AFC and APP in the epithelial-endothelial barrier by promoting viral replication, thereby increasing virus-induced damage to barrier integrity. Addition of EVs to virus-infected AECs in vitro suppressed this pathology, suggesting their therapeutic potential in treating diseases caused by H5N1 infection and avoiding the development of more severe complications.

[0086] The donor-specific nature of the primary cells used also provides a platform for studying patient-specific influenza virus-induced pathogenesis and customized therapies that can be easily translated to the clinical setting.

[0087] The foregoing description of the present invention has been provided for the purpose of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed. Many modifications and variations will be apparent to those skilled in the art.

[0088] The embodiment was chosen and described in order to best explain the principles of the invention and its practical application, thereby enabling others skilled in the art to understand the invention for various embodiments and with various modifications as are suited to the particular use contemplated.

[0089] definition

[0090] Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising" should be understood to imply the inclusion of the stated integer or groups of integers, but not the exclusion of any other integer or groups of integers. It should also be noted that in this disclosure and particularly in the claims and / or paragraphs, terms such as "comprises", "comprised", "comprising" and the like may have the meaning ascribed to them in U.S. patent law, such as to allow for elements not expressly recited, but to exclude elements found in the prior art or elements that affect the basic or novel characteristics of the invention.

[0091] Furthermore, throughout the specification and claims, unless the context requires otherwise, the word "include" or variations such as "includes" or "including", will be understood to imply the inclusion of a stated integer or groups of integers but not the exclusion of any other integers or groups of integers.

[0092] References in this specification to "one embodiment", "an embodiment", "example embodiment", etc. indicate that the described embodiment may include a particular feature, structure, or characteristic, but not every embodiment necessarily includes the particular feature, structure, or characteristic. In addition, such phrases do not necessarily refer to the same embodiment. Further, when a particular feature, structure, or characteristic is described in conjunction with an embodiment, whether or not explicitly described, it is considered to be within the knowledge of those skilled in the art to affect such feature, structure, or characteristic in conjunction with other embodiments.

[0093] As used herein, the terms "approximately," "basically," "substantially," and "about" are used to describe and explain small variations. When used in conjunction with an event or situation, the terms may refer to situations where the event or situation occurs precisely as well as situations where the event or situation occurs approximately. As used herein with respect to a given value or a given range, the term "approximately" generally refers to within a range of ±10%, ±5%, ±1%, or ±0.5% of the given value or given range. Ranges may be indicated herein as from one endpoint to another or between two endpoints. Unless otherwise specified, all ranges disclosed in this disclosure include the endpoints. When referring to "substantially" the same numerical value or characteristic, the term may refer to numerical values ​​within ±10%, ±5%, ±1%, or ±0.5% of the average of the numerical values.

[0094] In the preparation method described herein, except for the case of explicitly describing the time or operation sequence, the steps can be performed in any order without departing from the principles of the present invention. The description of performing a certain step first and then performing several other steps subsequently in a certain claim should be considered to mean that the first step is performed between any other steps, but the other steps can be performed in any suitable order unless the order is further described in other steps. For example, the claim element describing "step A, step B, step C, step D and step E" should be interpreted as meaning that step A is performed first and step E is performed last, and step B, step C and step D can be performed in any order between step A and step E, and the order still falls within the literal scope of the process claimed for protection. A given step or a subset of steps can also be repeated. In addition, unless the clear claim language describes the execution of the specified steps separately, the specified steps can be performed simultaneously.

[0095] "Transepithelial electrical resistance (TEER)" is a quantitative measurement of the electrical resistance across a monolayer of cells and is commonly used to assess the integrity and permeability of tight junctions in epithelial or endothelial cell cultures. TEER provides a direct indicator of the strength of the barrier function formed by the cells. Higher TEER values ​​indicate stronger tight junctions and lower permeability, reflecting a more intact and functional epithelial or endothelial barrier, while lower TEER values ​​indicate weakened junctions and increased permeability. In in vitro models, TEER is often used to monitor the health and integrity of a cell monolayer over time.

[0096] "Extracellular vesicles (EVs)" refer to nanoscale particles secreted by cells that facilitate intercellular communication and can have therapeutic effects by transferring biomolecules between cells.

[0097] "Alveolar fluid clearance (AFC)" refers to the process by which fluid is actively removed from the alveolar spaces (the small air sacs in the lungs) into the lung interstitium and ultimately absorbed into the bloodstream or lymphatic system. Proper AFC function is essential to prevent pulmonary edema (excess fluid in the lungs).

[0098] "Alveolar protein permeability (APP)" describes the ability of proteins, particularly plasma proteins, to cross the alveolar-capillary barrier (composed of the alveolar epithelium and capillary endothelium). An increase in APP may indicate damage to this barrier or increased permeability, leading to leakage of proteins and fluid into the alveolar space, which may impair lung function and lead to conditions such as acute respiratory distress syndrome (ARDS).

[0099] Other definitions of selected terms used herein may be present in the detailed description of the present invention and are applicable throughout the text. Unless otherwise defined, all other technical terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present invention belongs.

Claims

1. An in vitro co-culture alveolar-capillary model system for studying acute lung injury induced by respiratory virus infection, characterized in that: The in vitro co-culture alveolar-capillary model system comprises: a transwell system, wherein primary human alveolar epithelial cells are seeded onto the apical surface of the transwell system and human lung microvascular endothelial cells are seeded onto the basolateral surface of the transwell system; as well as A device for measuring alveolar fluid clearance and alveolar protein permeability in infected cells, The in vitro co-culture alveolar-capillary model system simulates the alveolar-capillary barrier.

2. The in vitro co-culture alveolar-capillary model system according to claim 1, wherein the primary human alveolar epithelial cells are cultured on collagen-coated transwell inserts to promote the formation of tight junctions.

3. The in vitro co-culture alveolar-capillary model system according to claim 1, wherein the primary human alveolar epithelial cells are isolated from non-malignant lung tissue.

4. The in vitro co-culture alveolar-capillary model system according to claim 1, wherein 5×10 4 Up to 2×10 5 The primary human alveolar epithelial cells were seeded at a cell density of 10 cells.

5. The in vitro co-culture alveolar-capillary model system according to claim 1, wherein 5×10 4 Up to 2×10 5 The human pulmonary microvascular endothelial cells were seeded at a cell density of 10 cells. The in vitro co-culture alveolar-capillary model system according to claim 1 , wherein the pore size of the transwell system is 0.4 μm.

7. A method for studying acute lung injury induced by respiratory viral infection in vitro, the method comprising: Isolation of primary human alveolar epithelial cells and human microvascular endothelial cells; The primary human alveolar epithelial cells are seeded on the apical surface of the transwell membrane, and the human microvascular endothelial cells are seeded on the basolateral surface of the transwell system to form an in vitro co-culture alveolar-capillary model system; Infecting the in vitro co-cultured alveolar-capillary model system with one or more respiratory viruses; The effect of viral infection on at least one physiological parameter is assessed, wherein the at least one physiological parameter comprises alveolar fluid clearance and alveolar protein permeability.

8. The method of claim 7, wherein the primary human alveolar epithelial cells and the human microvascular endothelial cells are separated using enzymatic digestion followed by density gradient centrifugation and magnetic bead separation.

9. The method of claim 7, wherein the primary human alveolar epithelial cells are cultured on collagen-coated transwell inserts to promote the formation of tight junctions.

10. The method of claim 7, wherein the primary human alveolar epithelial cells are isolated from non-malignant lung tissue.

11. The method according to claim 7, wherein each hole has 5×10 4 Up to 2×10 5 The primary human alveolar epithelial cells were seeded at a cell density of 10 cells.

12. The method according to claim 7, wherein each hole has 5×10 4 Up to 2×10 5 The human pulmonary microvascular endothelial cells were seeded at a cell density of 10 cells.

13. The method of claim 7, wherein the pore size of the transwell system is 0.4 μm.

14. The method of claim 7, wherein the one or more respiratory viruses are selected from the group consisting of coronavirus, respiratory syncytial virus, adenovirus, influenza virus, parainfluenza virus, rhinovirus, enterovirus, and herpes simplex virus.

15. The method of claim 14, wherein the in vitro co-culture alveolar-capillary model system is infected with influenza A virus subtype H5N1.

16. The method of claim 7, wherein the method further comprises adding a therapeutic agent after the viral infection to inhibit viral replication in the human alveolar epithelial cells and the human pulmonary microvascular endothelial cells.

17. The method of claim 16, wherein the therapeutic agent comprises 1×10 7 Up to 1×10 9 Extracellular vesicle particles.

18. The method of claim 17, wherein the extracellular vesicles are derived from human mesenchymal stromal cells.

19. The method of claim 7, further comprising measuring transepithelial electrical resistance (TEER) as an indicator for assessing the integrity of the epithelial-endothelial barrier over time, and the TEER remains ≥ 800 Ω / cm 2 .

20. The method of claim 7, wherein the step of assessing the effect of viral infection on at least one physiological parameter further comprises measuring viral gene expression and antiviral gene expression in the primary human alveolar epithelial cells and the human microvascular endothelial cells.

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