High-throughput in-vitro lung injury model system

Abstract

The present invention relates to a physiologically related high throughput in vitro human lung injury model for studying therapeutic interventions against severe respiratory pathogens. In this model, human alveolar epithelial cells are infected with highly pathogenic influenza A virus A / HK / 483 / 97 (H5N1), followed by treatment with extracellular vesicles (EV) 24 hours after infection. After 24 hours, the influenza A H5N1 infection obviously reduces alveolar fluid clearance (AFC) and increases alveolar permeability (APP). Administration of a therapeutic EV will restore AFC and APP in infected epithelial cells in vitro, reduce the expression of dysregulated pro-inflammatory cytokines, and enhance viral inhibited alveolar sodium and chlorine transporters. These findings demonstrate the potential therapeutic efficacy of EV in reducing epithelial injury caused by influenza A H5N1 and emphasize the applicability of high throughput lung models in screening respiratory disease treatments.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 593,242 filed on October 26, 2023, and the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0002] The present invention relates to the field of in vitro disease models and high throughput screening methods. Background Art

[0003] The airway epithelium in the human lung acts as a physical and immune barrier against external pathogens, protecting the underlying tissue from infection. The integrity of the airway epithelium is compromised in many respiratory diseases, a process that is difficult to study in the human lung outside of a clinical setting. Infection of the human lung with influenza virus often causes chronic damage to the alveolar epithelium, which maintains proper alveolar function, such as gas exchange. Tight junctions between alveolar cells form a robust epithelial layer that is able to regenerate after injury.

[0004] Although animal models are widely used in the study of acute lung injury, these models face challenges due to differences in lung anatomy and immune responses between species, which limits the translational value of research results. Ethical issues also continue to plague the use of live animals in drug research. Therefore, reliable and physiologically relevant in vitro models of human alveolar epithelium have become an important tool for rapid drug discovery, supporting preclinical research on the causes and treatments of respiratory diseases.

[0005] To understand the mechanisms driving disease development and progression, it is important to establish a predictive in vitro model system that can mimic human physiological conditions outside of the clinical setting. The use of submerged cultures of human lung epithelial cells as initial in vitro models has limited ability to replicate human lung physiology. The introduction of transwell systems that allow air-liquid or liquid-liquid interfaces has been able to better simulate the human respiratory environment. However, the use of immortalized cell lines, such as A549 cells derived from lung cancer, cannot reproduce key physiological features due to phenotypic differences in culture conditions. In addition, primary alveolar cells from non-diseased subjects are limited in supply and expensive.

[0006] In view of these limitations, there is a need for a scalable and reproducible in vitro model that can mimic human lung physiology and provide an effective platform for screening potential therapeutic interventions. The present invention addresses this need. Summary of the invention

[0007] The present invention aims to provide a high-throughput in vitro lung injury model optimized for culturing primary human alveolar epithelial cells (AECs). This model facilitates the study of the mechanisms involved in highly pathogenic influenza virus infection and enhances drug screening capabilities while reducing the need for donor cells and minimizing the time required.

[0008] According to one aspect of the present invention, the present invention provides a high-throughput in vitro lung injury model system. This system provides a more predictive and scalable in vitro model of human alveolar epithelium, thereby promoting the study of disease mechanisms and the development of therapeutic interventions. The system is used to screen a variety of candidate compounds for the treatment of respiratory infectious diseases. The system includes a plurality of transwell cell culture chambers (transwell inserts) in a 96-well system, a device for applying a plurality of candidate compounds to infected alveolar epithelial cells, and a device for measuring alveolar fluid clearance and alveolar protein permeability in infected alveolar epithelial cells. Each transwell cell culture chamber contains a monolayer formed by primary human alveolar epithelial cells, and the primary human alveolar epithelial cells are infected with one or more respiratory pathogens. The tight junction integrity of the alveolar epithelial cells is monitored by measuring the transepithelial electrical resistance (TEER). TEER maintains at least 2000Ω / cm 2 .

[0009] After a 24-hour incubation period, alveolar fluid clearance (AFC) was measured as a function of the concentration of fluorescently labeled dextran present in the apical chamber. Alveolar permeability (APP) was measured using fluorescently labeled dextran with a molecular weight of 70 kDa. Unidirectional flux from the apical chamber of the transwell to the basolateral chamber was tracked using fluorescently labeled dextran.

[0010] According to one aspect of the present invention, the respiratory tract infectious disease comprises a viral respiratory tract infection.

[0011] According to one aspect of the present invention, the one or more respiratory pathogens include influenza virus, respiratory syncytial virus (RSV), coronavirus, Middle East respiratory syndrome coronavirus (MERS-CoV), human parainfluenza virus (HPIV), human metapneumovirus (hMPV), adenovirus, rhinovirus and enterovirus.

[0012] According to one aspect of the present invention, the primary human alveolar epithelial cells are isolated from non-malignant lung tissue.

[0013] According to one aspect of the present invention, the primary human alveolar epithelial cells are cultured at 1×10 4 Up to 1×10 5 The cells were seeded at a density of 10 cells / mL in the transwell cell culture chamber.

[0014] According to one aspect of the present invention, the plurality of candidate compounds include nano-sized extracellular vesicles (EVs) derived from human mesenchymal stromal cells.

[0015] According to one aspect of the present invention, the present invention provides a method for high-throughput screening of multiple candidate compounds for treating respiratory infectious diseases, the method comprising: isolating primary human alveolar epithelial cells from non-malignant lung tissue; inoculating and culturing the primary human alveolar epithelial cells in multiple transwell cell culture chambers in a 96-well transwell system; infecting the primary human alveolar epithelial cells with one or more respiratory pathogens; treating the infected primary human alveolar epithelial cells with multiple candidate compounds; and evaluating the effects of the multiple candidate compounds on at least one physiological parameter comprising AFC and APP to screen therapeutic compounds.

[0016] According to one aspect of the present invention, the respiratory tract infectious disease comprises a viral respiratory tract infection.

[0017] According to one aspect of the present invention, the one or more respiratory pathogens include influenza virus, respiratory syncytial virus (RSV), coronavirus, Middle East respiratory syndrome coronavirus (MERS-CoV), human parainfluenza virus (HPIV), human metapneumovirus (hMPV), adenovirus, rhinovirus and enterovirus.

[0018] According to one aspect of the present invention, the primary human alveolar epithelial cells are isolated from non-malignant lung tissue.

[0019] According to one aspect of the present invention, the plurality of candidate compounds includes nano-sized EVs derived from human mesenchymal stromal cells.

[0020] According to one aspect of the invention, the step of measuring the effect of the therapeutic compound on at least one physiological parameter comprises measuring the expression of inflammatory cytokines to evaluate the therapeutic effect of the candidate compound.

[0021] According to one aspect of the invention, the step of measuring the effect of the therapeutic compound on at least one physiological parameter comprises monitoring the activity of one or more ion transporters selected from the group consisting of: an epithelial sodium channel (ENaC), a CFTR channel, and a Na / K-ATPase pump.

[0022] According to one aspect of the present invention, the primary human alveolar epithelial cells are cultured in the liquid-liquid interface within the 96-well transwell system.

[0023] According to one aspect of the present invention, the primary human alveolar epithelial cells are cultured at 1×10 4 Up to 1×105 The cells were seeded at a density of .

[0024] According to one aspect of the present invention, the primary human alveolar epithelial cells are infected with the one or more respiratory pathogens at a multiplicity of infection (MOI) in the range of 0.1 to 3.

[0025] According to one aspect of the present invention, the plurality of candidate compounds are present in a concentration of 1×10 7 Up to 1×10 9 EV particles were administered.

[0026] According to another aspect of the present invention, the method comprises generating in vitro alveolar epithelium that exhibits tight junction formation in a monolayer, thereby establishing an in vitro lung injury model in a high throughput format (96-well).

[0027] According to another aspect of the present invention, the method includes studying the mechanisms involved in influenza virus infection and screening for effective drugs for treatment.

[0028] The present invention increases the physiological relevance of in vitro acute lung injury models using primary cultures of cells isolated from human lung tissue seeded into a transwell system in a 96-well format and allows for rapid and high-throughput screening and identification of patient-specific drug treatments.

[0029] This high-throughput in vitro lung injury model facilitates the development of patient-specific therapies because primary human lung cells are derived from specific donors. This allows for tailored treatments for respiratory diseases to be fine-tuned based on the results of phenotypic drug screening in the in vitro lung injury model, facilitating translation into clinical applications and maximizing the chances of successful treatment.

[0030] In addition, the high-throughput format of the 96-well transwell system allows for simultaneous screening of larger scale drug / intervention candidates. This 96-well format allows for the use of a minimal number of primary human lung cells per transwell, thereby maximizing screening capacity. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Embodiments of the present invention will be described in more detail below with reference to the accompanying drawings, in which:

[0032] Figure 1A Schematic diagram showing an in vitro acute lung injury model that mimics human alveoli in a clinical setting using a confluent monolayer of AECs seeded into the top chamber of each transwell.

[0033] Figure 1B Shown is the daily monitoring of tight junction integrity in cells by measuring TEER using an epithelial resistance voltmeter (EVOM) machine.

[0034] Figure 1C TEER values ​​of AECs observed during the first six days of the experiment are depicted.

[0035] Figure 1D Shown is an increase in AFC and APP after infection with highly pathogenic influenza A virus (H5N1). Cytomix containing proinflammatory cytokines (IL-1β, TNF-α, and IFN-γ) was used as a positive control for induced injury. Data are mean ± SD of three experiments. *p≤0.05; **p≤0.01; ***p≤0.001; and ****p≤0.0001.

[0036] Figure 2A Shown are the AFCs of infected cells with or without EV treatment compared to untreated infected cells.

[0037] Figure 2B Shown are APP of infected cells with or without EV treatment compared to untreated infected cells.

[0038] Figure 2C The expression of different inflammatory cytokines in cells infected with H5N1 with or without EV treatment is shown. Data represent mean ± SD of three experiments. *p ≤ 0.05; **p ≤ 0.01; and ***p ≤ 0.001.

[0039] Figure 2D The activity of different ion transporters in cells infected with H5N1 with or without EV treatment is shown. Data represent mean ± SD of three experiments. *p ≤ 0.05; **p ≤ 0.01; and ***p ≤ 0.001. DETAILED DESCRIPTION

[0040] Physiological changes and their associated mechanisms in acute lung injury induced by emerging respiratory viruses are difficult to study outside of clinical settings. Therefore, it is important to accurately characterize these changes in a physiologically relevant in vitro setting in order to advance drug screening for precision and personalized medicine.

[0041] Therefore, the present invention provides a high-throughput in vitro lung injury model that replicates the physiological conditions of human alveolar epithelium using a 96-well transwell system seeded with AECs. This model facilitates rapid screening of therapeutic compounds while effectively reducing the use of primary cells. The present invention allows the study of respiratory pathogens such as highly pathogenic influenza viruses, as well as screening for potential therapeutic compounds. This system enables researchers to measure key physiological parameters, including AFC and APP, both of which are affected during acute lung injury.

[0042] One aspect of the present invention relates to isolating primary human alveolar epithelial cells from non-malignant lung tissue and culturing them, and then inoculating the primary human alveolar epithelial cells into a transwell system, in which the primary human alveolar epithelial cells form a monolayer with tight junction integrity to simulate human alveoli. After infection with respiratory pathogens, the monolayer exhibits a decrease in AFC and an increase in APP, thereby simulating the conditions observed in acute lung injury. AFC is evaluated by tracking the movement of fluorescently labeled dextran across the epithelial layer, while APP is evaluated based on the unidirectional flux of dextran from the apical chamber to the basolateral chamber. This allows real-time monitoring of the response of the lung epithelium to infection and treatment.

[0043] In one embodiment, the model monitors TEER to assess tight junction integrity, ensuring that values ​​remain above 2000 Ω / cm 2 , which is an indicator of proper barrier function.

[0044] In one embodiment, AECs are isolated from non-malignant lung tissue.

[0045] In one embodiment, the seeding density of AEC cells ranges from 1×10 4 Up to 1×10 5 cells.

[0046] Preferably, the seeding density of AEC cells is 5×10 4 cells.

[0047] The primary human AECs are infected with one or more respiratory pathogens at a multiplicity of infection (MOI) ranging from 0.1 to 3. Respiratory pathogens can include H5N1 virus, respiratory syncytial virus (RSV), coronavirus, Middle East respiratory syndrome coronavirus (MERS-CoV), human parainfluenza virus (HPIV), human metapneumovirus (hMPV), adenovirus, rhinovirus, and enterovirus. These respiratory pathogens are used to infect alveolar epithelial cells.

[0048] Preferably, the respiratory pathogen is the highly pathogenic influenza A virus A / HK / 483 / 97 (H5N1).

[0049] In one embodiment, nano-sized EVs are used as therapeutic compounds. These EVs have been shown to reverse the deleterious effects of pathogens such as H5N1 by restoring AFC and reducing APP. EV therapy will modulate inflammatory responses and improve ion transporter function, such as epithelial sodium channels and CFTR channels, which are often downregulated during infection. In addition to EVs, other potential therapeutic agents can also be evaluated and used, including but not limited to therapeutic proteins, drug compounds and their derivatives.

[0050] The present invention also provides a method for high-throughput screening of multiple candidate compounds for treating respiratory infectious diseases. The method comprises: isolating primary human alveolar epithelial cells from non-malignant lung tissue; inoculating and culturing the primary human alveolar epithelial cells in multiple transwell cell culture chambers in a 96-well transwell system; infecting the primary human alveolar epithelial cells with one or more respiratory pathogens; treating the infected primary human alveolar epithelial cells with multiple candidate compounds; and evaluating the effects of the multiple candidate compounds on at least one physiological parameter including AFC and APP to screen therapeutic compounds.

[0051] In one embodiment, the step of measuring the effect of the therapeutic compound on at least one physiological parameter comprises measuring the expression of inflammatory cytokines. Inflammatory cytokines may include IFNβ, IL-1β, RANTES, IP-10, MCP-1, IL-6, IL-8, and TNFα.

[0052] In one embodiment, the step of measuring the effect of the therapeutic compound on at least one physiological parameter comprises monitoring the activity of one or more ion transporters. The ion transporters may include CFTR, αENaC, βENaC, γENaC, α1NaKATPase, β1NaKATPase, and β3NaKATPase.

[0053] The present invention offers several advantages over conventional lung injury models, including scalability, reproducibility, and potential for personalized therapy.

[0054] The 96-well transwell format allows for the simultaneous screening of multiple candidate compounds, thereby increasing the efficiency of the drug discovery process.

[0055] The present invention reduces the need for a large number of primary human cells by optimizing cell seeding density, thereby improving cost-effectiveness and accessibility. Using primary cells from specific donor sources, the model will promote patient-specific drug screening and increase the possibility of successful clinical transformation. Its high-throughput design maximizes the use of limited primary cell resources, enabling the parallel screening of a large number of therapeutic compounds. This method is particularly important for identifying effective treatments for respiratory diseases caused by emerging pathogens.

[0056] Examples

[0057] Example 1

[0058] Materials and methods

[0059] Virus

[0060] Influenza virus A / HK / 483 / 97 (H5N1) was used for infection. All influenza viruses were passaged in Madin-Darby canine kidney (MDCK) cells. The half tissue culture infectious dose (TCID 50 ) Virus titer was determined. All experiments were performed in a biosafety level 3 facility.

[0061] Isolation of human alveolar epithelial cells

[0062] Human alveolar epithelial cells were isolated from non-malignant human lung tissues removed. In short, visible bronchi were removed and lung tissue was minced into small pieces less than 0.5 mm in size. The minced tissue was washed three times with Hank's balanced salt solution (HBSS) (Gibco) and 0.7 mM sodium bicarbonate (Gibco) with pH 7.4 to remove most of the macrophages and hemocytes. In a 37°C shaking water bath, a combination of 0.5% trypsin (Gibco) and 4U / mL elastase (Worthington Biochemical Corp.) was used to digest tissue for 40 minutes at 37°C. Digestion was stopped by adding DMEM / F12 culture medium (Gibco) containing 40% FBS and 350U / mL DNase I (Sigma). Cell suspension was repeatedly drawn with a pipette for 10 minutes so that cell clumps were fully dispersed. Disposable cell strainer (gauze size 50 μm) (BD Bioscience) was used to remove large undigested 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 plated in a tissue culture flask and incubated in a 37°C water jacket incubator at 5% CO2 for 90 minutes. Non-adherent cells were layered on 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 (MACS CD14 MicroBeads) coated with anti-CD14 antibodies at room temperature for 20 minutes. After removing the beads using a magnet, cell viability was assessed using trypan blue exclusion. The alveolar epithelial cell suspension was resuspended in SAGM (Lonza) supplemented with 1% FBS, 100 U / mL penicillin and 100 μg / mL streptomycin and incubated at 3×10 5 Pieces / cm 2The 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, separated into HBSS, and then plated in transwell cell culture chambers as described below.

[0063] Example 2

[0064] In vitro model setup

[0065] To investigate the effects of one or more respiratory pathogens on AFC and APP in human alveolar epithelial cells, this example adapts a physiologically relevant 24-well in vitro acute lung injury model to a high-throughput 96-well Transwell format suitable for use in BSL-3 laboratories.

[0066] Figure 1A Describe the establishment of a high-throughput in vitro lung injury model. Primary human alveolar epithelial cells were isolated from resected non-malignant lung tissue. The isolation process involved enzymatic digestion of lung tissue using trypsin and elastase, followed by filtration and density gradient centrifugation to obtain a purified alveolar epithelial cell population. Afterwards, alveolar epithelial cells were plated at 5 × 10 per well. 4 The cells were plated at a density of 10 cells / mL on the top chamber of a 96-well Costar Transwell cell culture chamber (Corning) with a pore size of 0.4 μm. The microporous transwell membrane established a liquid-liquid interface similar to that of the human lung epithelium, and the plated cells were maintained in a humidified atmosphere (5% CO2, 37°C). The TEER was maintained at ≥2,000 Ω / cm 2 , indicating sufficient tight junction integrity between cells, ensuring that the monolayer closely mimics the alveolar barrier found in the human lung. In addition, 50 ng / mL Cytomix, a mixture of cytokines IL-1β, TNF-α, and IFN-γ (R&D Systems), was used as a positive control for lung injury.

[0067] The integrity of tight junctions between AECs was monitored daily using EVOM. TEER values ​​increased as tight junctions formed and decreased when the monolayer was damaged ( Figure 1B A continuous increase in TEER values ​​was observed throughout the 6 days of the experiment in the established in vitro lung model ( Figure 1C ).

[0068] Acute lung injury caused by H5N1 virus infection is a major cause of high mortality, and currently available treatment options are very limited. Key features of acute lung injury include impaired AFC and increased APP observed in damaged alveolar epithelium.

[0069] Measure the net alveolar fluid transport from the top chamber of the transwell cell culture chamber (containing an alveolar epithelial cell monolayer infected with influenza virus) to the basolateral chamber of the transwell 24 hours after infection. In this model, AECs are infected with influenza A virus at MOI 0.1 for 1 hour. Then, 130 μl of FITC-labeled dextran (Sigma) with a size of 70 kDa is added to the cells (final concentration 500 ng / μl). After 5 minutes, 100 μl samples are collected from the top chamber for initial FITC measurement, and then transferred back to the top chamber for overnight cultivation. After 24 hours of dextran cultivation, 100 μl is collected from the top chamber, and 100 μl is collected from the basolateral chamber for final FITC measurement. The fluorescence of each sample is measured at an excitation wavelength of 485 nanometers and an emission wavelength of 520 nanometers using a modulus fluorometer (FLUOstar OPTIMA, BMG Labtech). A standard curve of known FITC concentrations was constructed and used to calculate the FITC-dextran present in the transwell chamber at different time points. Net alveolar fluid transport across the epithelial monolayer was calculated as [1-(FITC concentration in initial apical sample / FITC concentration in final apical sample)] / 130 μl / 0.143 cm 2 After 24 hours of dextran incubation, a final basolateral reading was collected to measure protein permeability based on the unidirectional flux of fluorescently labeled dextran from the apical to the basolateral chamber of the transwell cell culture insert.

[0070] See also Figure 1D Compared with the mock group, AEC infection caused a significant decrease in AFC and an increase in APP at 24 hours post-infection. These effects were assessed using fluorescently labeled dextran to monitor trans-monolayer fluid transport and protein leakage. Cytomix served as a positive control for monolayer damage.

[0071] Candidate therapeutic compounds were introduced into infected cells as nanosized EVs at 500 ng / μl to evaluate their therapeutic potential. After infection, EVs derived from human mesenchymal stromal cells were added to the transwell system and their effects on AFC and APP were measured after a 24-hour incubation period. Baseline fluorescence readings were taken and dextran was incubated with the monolayer for 24 hours. Net alveolar fluid transport across the monolayer was calculated using the final fluorescence readings obtained from the apical and basolateral chambers. Alveolar protein permeability was measured by tracking the unidirectional flux of fluorescently labeled dextran from the apical to the basolateral chamber.

[0072] Administration of EVs restored AFC and reduced APP in infected cells, suggesting their therapeutic efficacy in treating lung injury caused by respiratory pathogens. Figure 2A-2B , AFC was significantly restored and APP was decreased after EV treatment compared with untreated infected cells.

[0073] In addition, AECs in transwells were infected with H5N1 influenza virus at an MOI of 2 to study gene expression of cytokines and ion transporters after viral infection and potential therapeutic treatment. The results showed that EV treatment reversed the expression of pro-inflammatory cytokines in cells infected with H5N1, such as Figure 2C As shown in the literature, the levels of pro-inflammatory cytokines such as IL-1β, TNF-α and IFN-β were reduced.

[0074] Additionally, the activity of key ion transporters including the epithelial sodium channel (ENaC), CFTR channel, and Na / K-ATPase pump were monitored to determine their role in fluid clearance and epithelial barrier function. Figure 2D , the results showed that pathogen-induced downregulation of these ion channels and pumps is a hallmark of lung injury, which can be reversed after therapeutic intervention with EVs.

[0075] It has been found that influenza virus infection impairs alveolar fluid transport by inhibiting epithelial sodium channel activity through the interaction of viral hemagglutinin and matrix proteins with epithelial sodium channels. Activation of proinflammatory pathways has also been shown to be responsible for the downregulation of ion transporters, which are responsible for carrier fluid transport across the alveolar epithelium. In fact, the present data also show that after infection with H5N1, increased levels of proinflammatory cytokines are associated with reduced expression of CFTR, sodium ENAC channels, and sodium and potassium Na / K-ATPase pumps, indicating that impaired AFC is associated with reduced activity of ion transporters in the alveolar epithelium.

[0076] In summary, the present invention establishes a physiologically relevant high-throughput in vitro lung injury model aimed at maximizing the efficiency of molecular screening for severe human influenza treatments. Using this model, it was demonstrated that highly pathogenic H5N1 virus significantly reduced AFC and APP in cultured human alveolar epithelial cells by promoting proinflammatory cytokine production and downregulating alveolar ion transporter activity. This pathology was alleviated by the addition of EVs in vitro, indicating its therapeutic potential for treating diseases caused by H5N1 infection.

[0077] The foregoing description of the present invention has been presented for purposes of illustration and description only and 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.

[0078] The embodiments were 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.

[0079] definition

[0080] Throughout this specification, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers. It should also be noted that in this disclosure and especially in the claims and / or paragraphs, terms such as "comprises", "comprised", "comprising", etc. may have the meanings ascribed to them in U.S. patent law; for example, they allow for elements not expressly stated, but exclude elements found in the prior art or that affect the basic or novel characteristics of the invention.

[0081] Furthermore, throughout the present 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 group of integers but not the exclusion of any other integer or group of integers.

[0082] References to "one embodiment", "an embodiment", "an example embodiment", etc. in this specification indicate that the described embodiment may include specific features, structures, or characteristics, but each embodiment may not necessarily include the specific features, structures, or characteristics. In addition, these phrases do not necessarily refer to the same embodiment. In addition, when a specific feature, structure, or characteristic is described in conjunction with an embodiment, it should be understood that whether or not explicitly described, it is within the knowledge of a technician in this field to affect this feature, structure, or characteristic in conjunction with other embodiments.

[0083] As used herein, the terms "approximately", "basically", "substantially" and "about" are used to describe and explain minor variations. When used in conjunction with an event or situation, the term 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 range, the term "about" generally means within the range of ±10%, ±5%, ±1% or ±0.5% of the given value or range. The range may be indicated herein from one endpoint to another or between two endpoints. Unless otherwise stated, all ranges disclosed in this disclosure include the endpoints. When referring to "substantially" the same numerical value or characteristic, the term may refer to a value within the range of ±10%, ±5%, ±1% or ±0.5% of the average value of the value.

[0084] In the preparation methods described herein, except for the cases where the chronological order or the order of operations is explicitly described, the steps can be performed in any order without departing from the principles of the present invention. The effect of describing in the claims that a step is performed first and then a number of other steps are performed subsequently should mean that the first step is performed before any of the other steps, but the other steps can be performed in any suitable sequence unless the sequence is further described in the other steps. For example, the claim elements 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 steps B, C and D can be performed in any order between step A and step E, and the order still belongs to the text of the method claimed for protection. A given step or a subset of steps can also be repeated. In addition, unless the clear claim language describes that the specified steps are performed separately, the steps can be performed simultaneously.

[0085] "Transepithelial electrical resistance (TEER)" is a quantitative measurement of the electrical resistance across a cell monolayer 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 weaker junctions and increased permeability. In in vitro models, TEER is often used to monitor the health and integrity of a cell monolayer over time.

[0086] "Respiratory infectious diseases" are diseases caused by pathogens that infect the respiratory system, which includes the nose, throat, sinuses, airways, and lungs. These diseases are usually spread through droplets produced by coughing, sneezing, talking, or direct contact with contaminated surfaces. Depending on the pathogen and the individual's immune response, the severity of these diseases can range from a mild cold to life-threatening conditions.

[0087] Other definitions of selected terms used herein can be found within the detailed description of the invention and are applicable to the entire text. Unless otherwise defined, all other technical terms used herein have the same meaning as those generally understood by those skilled in the art to which the invention belongs.

[0088] References: The disclosures of the following references are incorporated by reference 1. Chan MC, Kuok DI, Leung CY, et al. Human mesenchymal stromal cells reduce influenza AH5N1-associated acute lung injury in vitro and in vivo. Proc Natl Acad Sci US A. 2016; 113(13): 3621-3626. doi: 10.1073 / pnas.1601911113 2. Loy H, Kuok DIT, Hui KPY, et al. Therapeutic Implications of Human Umbilical Cord Mesenchymal Stromal Cells in Attenuating Influenza A(H5N1)Virus-Associated Acute Lung Injury [published correction appears in J Infect Dis. 2019 Jan 7; 219(2): 339]. J Infect Dis. 2019; 219(2): 186-196. doi: 10.1093 / infdis / jiy478.

Claims

1. A high-throughput in vitro lung injury model system for screening multiple candidate compounds for treating respiratory infectious diseases, characterized in that: The system comprises: A plurality of transwell cell culture chambers in a 96-well system, wherein each of the transwell cell culture chambers contains a monolayer of primary human alveolar epithelial cells, and the primary human alveolar epithelial cells are infected with one or more respiratory pathogens; a device for applying the plurality of candidate compounds to infected alveolar epithelial cells; as well as A device for measuring alveolar fluid clearance and alveolar protein permeability in infected alveolar epithelial cells.

2. The high-throughput in vitro lung injury model system according to claim 1, wherein the respiratory infectious disease comprises a viral respiratory infection.

3. A high-throughput in vitro lung injury model system according to claim 1, wherein the one or more respiratory pathogens comprise influenza virus, respiratory syncytial virus (RSV), coronavirus, Middle East respiratory syndrome coronavirus (MERS-CoV), human parainfluenza virus (HPIV), human metapneumovirus (hMPV), adenovirus, rhinovirus and enterovirus.

4. The high-throughput in vitro lung injury model system according to claim 1, wherein the primary human alveolar epithelial cells are isolated from non-malignant lung tissue.

5. The high-throughput in vitro lung injury model system according to claim 1, wherein the primary human alveolar epithelial cells are cultured at 1×10 4 Up to 1×10 5 The cells were seeded at a density of 10 cells / mL in the top chamber of the transwell cell culture chamber.

6. The high-throughput in vitro lung injury model system of claim 1, wherein the plurality of candidate compounds comprise nanosized extracellular vesicles derived from human mesenchymal stromal cells.

7. A method for high-throughput screening of multiple candidate compounds for treating respiratory tract infectious diseases, characterized in that: The method comprises: Primary human alveolar epithelial cells were isolated from non-malignant lung tissue; Inoculating and culturing the primary human alveolar epithelial cells in multiple transwell cell culture chambers in a 96-well transwell system, wherein each of the transwell cell culture chambers contains a monolayer formed by the primary human alveolar epithelial cells; infecting the primary human alveolar epithelial cells with one or more respiratory pathogens; Treating infected primary human alveolar epithelial cells with a variety of candidate compounds; as well as The plurality of candidate compounds are evaluated for their effects on at least one physiological parameter comprising alveolar fluid clearance and alveolar protein permeability to screen for therapeutic compounds.

8. The method of claim 7, wherein the respiratory infectious disease comprises a viral respiratory infection.

9. The method of claim 7, wherein the one or more respiratory pathogens comprise influenza virus, respiratory syncytial virus (RSV), coronavirus, Middle East respiratory syndrome coronavirus (MERS-CoV), human parainfluenza virus (HPIV), human metapneumovirus (hMPV), adenovirus, rhinovirus, and enterovirus.

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

11. The method of claim 7, wherein the plurality of candidate compounds comprises nanosized extracellular vesicles derived from human mesenchymal stromal cells.

12. The method of claim 7, wherein the step of measuring the effect of the therapeutic compound on at least one physiological parameter comprises measuring the expression of inflammatory cytokines.

13. The method of claim 7, wherein the step of measuring the effect of the therapeutic compound on at least one physiological parameter comprises monitoring the activity of one or more ion transporters selected from the group consisting of: an epithelial sodium channel (ENaC), a CFTR channel, and a Na / K-ATPase pump.

14. The method according to claim 7, wherein the primary human alveolar epithelial cells are cultured in a liquid-liquid interface within the 96-well transwell system.

15. The method according to claim 7, wherein the primary human alveolar epithelial cells are cultured at 1×10 4 Up to 1×10 5 The cells were seeded at a density of .

16. The method of claim 7, wherein the primary human alveolar epithelial cells are infected with the one or more respiratory pathogens at a multiplicity of infection (MOI) in the range of 0.1 to 3.

17. The method according to claim 7, wherein the plurality of candidate compounds are present at 1×10 7 Up to 1×10 9 The extracellular vesicle particles were administered at a density of .

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