Systems and compositions for viral proliferating respiratory organoids and methods thereof

Abstract

The present disclosure relates generally to compositions, methods, and systems of respiratory organoids that maintain proliferation of respiratory pathogens. Methods of mimicking a biological process in a human lung are also provided. Also provided are methods of screening for compounds affecting respiratory pathogens in respiratory organoid models.

Description

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 551,522, filed February 8, 2024, which is incorporated by reference in its entirety. 1. Technical Field

[0003] The present disclosure generally relates to systems, compositions, and methods for sustaining the proliferation of respiratory pathogens in respiratory organoids. Also provided are methods for simulating biological processes in the human lung. Also provided are methods for screening compounds that affect respiratory pathogens in respiratory organoid models. 2. Background Technology

[0004] Human rhinovirus (HRV) is a non-enveloped positive-strand RNA virus of the Picornaviridae family and the Enterovirus genus, divided into three species (HRV-A, HRV-B, and HRV-C). 1 HRV is the most common pathogen causing acute upper respiratory tract infections and is associated with lower respiratory tract infections and exacerbations of asthma and chronic lung diseases. HRV-A and HRV-B consist of approximately 100 serotypes and can be readily propagated in immortalized cell lines using intercellular adhesion molecule 1 (ICAM-1) and low-density lipoprotein receptor (LDLR) for cell entry. Since the identification of HRV-C in 2006, 2 By molecular methods, more than 60 different HRV-C sequences have been reported so far. 3 Unlike HRV-A and HRV-B, HRV-C is unable to infect and replicate in standard cell lines. During the past 20 years, significant efforts have been made to address this issue. Bochkov et al. first reported in vitro HRV-C infection in primary tissues from surgically resected sinonasal mucosa and nasal polyps, and they successfully propagated a single HRV-C15 strain using these tissues. However, viral growth showed significant variability, which may be related to tissues from different donors and the variable state of the tissues. 4 Using primary tissue for viral culture presents several challenges, including limited tissue availability, difficulties in standardization, and tissue degradation during experiments. 5 The same team subsequently identified CDHR3 as the cellular receptor for HRV-C. 6 , which is a milestone discovery in HRV-C research.

[0005] HRV-C infection and limited proliferation have also been reported in primary human airway epithelial cells. 7,8 Air-liquid interface cultures of primary human airway and nasal epithelial cells allow mucociliary differentiation into pseudostratified ciliated epithelium. These primary epithelial cells have been used to characterize respiratory viral infections, including COVID-19.9,10 However, the inherent limited proliferation capacity of primary epithelial cells greatly restricts their application for routine experiments, including virus isolation and serial virus passage. In addition, HRV-C is not the only non-culturable virus. Other respiratory tropic viruses, such as bocavirus, 11 and human coronavirus HKU1 12 , cannot infect immortalized cell lines other than isolated respiratory tissue and primary airway epithelial cells; these human viruses have yet to be cultured for comprehensive study. Therefore, robust and easily accessible culture systems are needed to propagate these unculturable viruses, which is the first step in understanding these pathogenic microorganisms. Overall, the lack of robust culture systems is a major obstacle to understanding the biology of HRV-C and other unculturable respiratory viruses, their interactions with human respiratory cells, and the development of antiviral strategies against these common respiratory pathogens.

[0006] Organoids, also known as "mini-organs," are three-dimensional (3D) cell clusters derived from stem cells (including embryonic stem cells, induced pluripotent stem cells, and organ-specific adult stem cells from primary tissues). They can self-renew and self-organize into complex functional structures that recapitulate the cellular diversity and physiological functions of the corresponding tissues and organs. 3. Summary of the Invention

[0007] We established the first human respiratory organoid culture system 13-19 Organoids are derived from adult stem cells in primary lung tissue with high efficiency and can be stably expanded for more than half a year. We have developed differentiation protocols to induce the maturation of long-term expandable organoids and generate mature airway organoids and alveolar organoids that faithfully mimic the natural airway and alveolar epithelium, respectively. 20 In addition to the non-invasive procedure to obtain easily accessible nasal cells for organoid derivation, nasal mucosal organoids more adequately mimic the upper respiratory epithelium than airway organoids. 17 In this two-phase organoid culture system 17,20 , expansion culture provides a stable and long-term source of expansion, while the differentiation protocol enables us to generate large numbers of physiologically active airway epithelial cells ( Figure 1A ). Thus, the respiratory organoid culture system allows us to reconstitute and propagate the entire human respiratory epithelium in culture plates with excellent efficiency and stability. These respiratory organoids have become a robust and popular tool for studying SARS-CoV-2 and other respiratory viruses. 16,17,21-23 .

[0008] Given the respiratory tropism of HRV-C and the ability of nasal and airway organoids to accurately mimic the native epithelium in the human airway, we hypothesized that these respiratory organoids (hereafter referred to as airway organoids and nasal mucosal organoids) might be susceptible to HRV-C and maintain viral culture. We were prompted to develop an organoid-based culture system to reproducibly propagate HRV-C and characterize HRV-C infection and complex virus-host interactions in these respiratory organoids.

[0009] Provided is a composition for culturing respiratory microorganisms, the composition comprising: (i) a respiratory organoid selected from the group consisting of an airway organoid and a nasal mucosal organoid; and (ii) respiratory microorganisms, wherein the respiratory microorganisms maintain continuous proliferation in the respiratory organoid.

[0010] Provided are methods for culturing respiratory microorganisms, the methods comprising: (i) preparing a respiratory organoid, wherein the respiratory organoid is selected from the group consisting of an airway organoid and a nasal mucosal organoid; and (ii) infecting the respiratory organoid with a microorganism, wherein the microorganism maintains proliferation in the respiratory organoid.

[0011] Provided is a system for culturing respiratory microbes, the system comprising: (i) culturing lung tissue fragments in a culture medium comprising a JAK1 / JAK2 inhibitor or a TBK1 / IKKε inhibitor for a period of time sufficient for respiratory organoids to form; and (ii) maintaining continuously proliferating respiratory microbes in the respiratory organoids.

[0012] Here we present novel human respiratory organoids that can sustainably and reproducibly propagate human rhinovirus C.

[0013] Provided are organoid-based systems and compositions for reproducibly propagating previously unculturable HRV-C, enabling us to elucidate HRV-C infection and innate immunity in unprecedented ways. Organoid-based HRV-C infection models can be expanded for the development of antiviral strategies. In one embodiment, provided are pathways for propagating and studying other unculturable human and animal viruses.

[0014] Human airway organoid culture systems and compositions are provided to reconstitute and propagate the entire human airway epithelium in culture plates with excellent efficiency and stability. The key rationale for isolating and propagating non-culturable viruses from airway and nasal mucosal organoids lies in their high biological relevance to the native airway epithelium, a primary target for many viruses, including those that are non-culturable in standard cell lines.

[0015] Systems, compositions, and methods are provided by which previously unculturable or poorly culturable viruses can be reproducibly cultured and propagated.

[0016] Systems, compositions, and methods are provided for isolating and culturing biologically active airway and nasal mucosal organoids of previously unculturable human respiratory viruses.

[0017] Provided are airway organoids and methods that sustain continuous viral propagation with the aid of CYT387-mediated immunosuppression, providing nasal mucosal organoids that more closely mimic the upper airway without requiring any intervention to achieve this.

[0018] Also provided are systems, compositions, and methods that can be used as research tools to identify agents that are effective in treating respiratory diseases and infections. Also provided are compositions comprising respiratory organoid model cultures containing human respiratory organoids, including alveolar, airway, and nasal mucosal organoids. In certain embodiments, the organoids comprise epithelial cells, mesenchymal cells, and immune cells. Also provided are methods for infecting the composition with respiratory microorganisms and continuously propagating the microorganisms in culture. The methods further include screening for compounds that affect the microorganisms. The methods further include administering the compounds to the composition and determining whether there is a therapeutically effective response. In one embodiment, methods are provided for identifying therapeutic agents that are effective for treating respiratory diseases and infections caused by pathogens.

[0019] In one embodiment, an organoid chip is provided, which is a hybrid device in the form of a conventional semiconductor chip made by combining organoids and inorganic materials such as semiconductors or glass. By utilizing the unique functions of biomolecules to mimic the functions of living organisms, it has the characteristics of diagnosing infectious diseases, analyzing genes, and serving as a new functional device for new information processing. In certain embodiments, the organoid chip includes a biosensor that can detect and analyze various biochemical substances by compactly integrating sample pretreatment, biochemical reactions, detection, and data analysis, such as a laboratory on a chip with automatic analysis capabilities, and can be broadly defined.

[0020] Provided is a composition for culturing respiratory microorganisms, the composition comprising: (i) a respiratory organoid selected from the group consisting of an airway organoid and a nasal mucosal organoid; and (ii) respiratory microorganisms, wherein the respiratory microorganisms maintain continuous proliferation in the respiratory organoid.

[0021] In one embodiment, the respiratory organoid is an airway organoid and is derived from lung tissue.

[0022] In one embodiment, the respiratory organoid is a nasal mucosal organoid and is derived from nasal epithelial cells.

[0023] In one embodiment, the respiratory microorganism is maintained for at least 3-6 passages and has genomic stability.

[0024] In one embodiment, the composition further comprises an immunosuppressive compound.

[0025] In one embodiment, the immunosuppressive compound interferes with the antimicrobial response in the respiratory organoid.

[0026] In one embodiment, the immunosuppressive compound is a JAK1 / JAK2 inhibitor or a TBK1 / IKKε inhibitor.

[0027] In one embodiment, the immunosuppressive compound is Momelotinib (CYT387), Amlexanox, BX795, MRT67307, Ruxolitinib, or a combination thereof.

[0028] In one embodiment, the respiratory microorganism is a respiratory virus or a respiratory bacteria.

[0029] In one embodiment, the respiratory virus is HRV-C, HRV-C3, HRV-C8, HRV-C11, HRV-C15, HRV-C45, influenza virus, adenovirus, human bocavirus, human coronavirus including SARS-CoV1 and SARS-CoV2, human metapneumovirus, human parainfluenza virus, human respiratory syncytial virus, or human rhinovirus.

[0030] In one embodiment, the respiratory bacterium is Mycobacterium tuberculosis, Streptococcus pneumoniae, Mycoplasma pneumoniae, Hemophilus influenzae, Chlamydophila pneumoniae, Chlamydia psittaci, Coxiella burnetii, Legionella pneumophila, Staphylococcus aureus, or Klebsiella pneumoniae.

[0031] In one embodiment, the airway organoid is a human airway organoid comprising human airway epithelial cells.

[0032] In one embodiment, the airway organoid comprises human lung epithelial cells.

[0033] In one embodiment, the airway organoid comprises human nasal epithelial cells.

[0034] In one embodiment, the airway organoids are derived from human epithelial stem cells.

[0035] In one embodiment, the airway organoids survive in culture for a period of at least 30 days.

[0036] Provided are methods for culturing respiratory microorganisms, the methods comprising: (i) preparing a respiratory organoid, wherein the respiratory organoid is selected from the group consisting of an airway organoid and a nasal mucosal organoid; and (ii) infecting the respiratory organoid with a microorganism, wherein the microorganism maintains proliferation in the respiratory organoid.

[0037] In one embodiment, the method further comprises the steps of isolating and culturing said respiratory microorganisms.

[0038] In one embodiment, the airway organoid is an airway organoid derived from lung tissue.

[0039] In one embodiment, the respiratory organoid is a nasal mucosal organoid derived from nasal epithelial cells.

[0040] In one embodiment, airway organoids are prepared by (a) providing a fragment of airway or nasal tissue; and (b) culturing the fragment of airway or nasal tissue in culture for a period of time sufficient to form the airway organoid.

[0041] In one embodiment, the culture medium further comprises an immunosuppressive compound.

[0042] In one embodiment, the immunosuppressive compound interferes with the antimicrobial response in the respiratory organoid.

[0043] In one embodiment, the immunosuppressive compound is a JAK1 / JAK2 inhibitor or a TBK1 / IKKε inhibitor.

[0044] In one embodiment, the immunosuppressive compound is molotinib (CYT387), amlexanob, BX795, MRT67307, ruxolitinib, or a combination thereof.

[0045] In one embodiment, the respiratory microorganism is maintained for at least 3-6 passages and has genomic stability.

[0046] In one embodiment, the respiratory microorganism is a respiratory virus or a respiratory bacteria.

[0047] In one embodiment, the respiratory virus is HRV-C, HRV-C3, HRV-C8, HRV-C11, HRV-C15, HRV-C45, influenza virus; adenovirus; human bocavirus; human coronavirus including SARS-CoV1 and SARS-CoV2; human metapneumovirus; human parainfluenza virus; human respiratory syncytial virus; and / or human rhinovirus or a combination thereof.

[0048] In one embodiment, the respiratory bacteria is Mycobacterium tuberculosis, Streptococcus pneumoniae, Mycoplasma pneumoniae, Haemophilus influenzae, Chlamydia pneumoniae; Chlamydia psittaci; Coxiella burnetii; Legionella pneumophila, Staphylococcus aureus; and / or Klebsiella pneumoniae.

[0049] In one embodiment, the airway organoid is a human airway organoid comprising human airway epithelial cells.

[0050] In one embodiment, the airway organoids survive in culture for a period of at least 30 days.

[0051] Provided is a system for culturing respiratory microbes, the system comprising: (i) culturing lung tissue fragments in a culture medium comprising a JAK1 / JAK2 inhibitor or a TBK1 / IKKε inhibitor for a period of time sufficient for respiratory organoids to form; and (ii) maintaining continuously proliferating respiratory microbes in the respiratory organoids. 4. Description of the Figures

[0052] The patent or application file contains at least one drawing executed in color. Copies of the patent or patent application publication with color drawing(s) will be provided upon request and payment of the necessary fee.

[0053] Figure 1A -C. Human airway organoids susceptible to HRV-C clinical samples. (A) Schematic diagram of human nasal mucosal organoids and airway organoid culture system. (B) Airway organoids were inoculated with 9 HRV-C+ nasopharyngeal aspirates (in 9 transwell inserts). At the indicated days post-infection (dpi), culture medium was harvested from infected airway organoids and used for viral load detection by RT-qPCR. A schematic diagram of the experimental procedure was created using Biorender.com. (C) Airway organoids (n=3) were inoculated with HRV-A1 and HRV-C3 at 100 viral gene copies / cell. Culture medium was harvested from the apical and basolateral compartments of infected airway organoids at the indicated dpi and used for viral load detection. Data represent the mean and SD of the indicated number (n) of biological replicates from a representative experiment performed three times independently. Statistical significance was determined using a two-tailed Student's t-test (in Figure C). **P<0.01, ***P<0.001.

[0054] Figure 2A -C. CYT387 can continuously proliferate HRV-C in human airway organoids. (A) Schematic diagram

[0055] The experimental procedure is summarized in Figure B. (B) At the first passage (P1), cells were cultured with CYT387 or DMSO.

[0056] After overnight pretreatment, airway organoids were inoculated with HRV-C3 (n=2). Culture medium was harvested from infected airway organoids at the specified hpi to detect viral replication. In the second passage (P2), 500L of P1 culture medium collected from CYT387- or DMSO-treated organoids (n=2) were inoculated. Culture medium was harvested at the specified hpi to detect viral replication. Starting from P3, airway organoids pretreated with CYT387 or DMSO were inoculated with culture medium collected from CYT387-treated organoids at 100 viral gene copies / cell (P3, n=2; P4 and P5, n=3). Culture medium was harvested at 96h.pi to detect viral replication. The viral loads in P1 and P2 culture media at 96h.pi were combined. Data represent the mean and SD of the indicated number (n) of biological replicates from a representative experiment. Statistical significance (P4 and P5) was determined using a two-tailed Student's t-test. **P < 0.01. ns, not significant. (C) At 24 h p.i., airway organoids were fixed and immunolabeled with α- and α-airway (green) and α-α-airway (red). Nuclei and actin filaments were counterstained with DAPI (blue) and phalloidin-647 (white), respectively. The experiment was performed three times independently with similar results. Scale bar, 10 μm.

[0057] Figure 3A-C. Human nasal mucosal organoids undergo continuous HRV-C proliferation. (A) Schematic diagram outlining the experimental procedures for panels b, c, and d. Nasal mucosal organoids pretreated with CYT387 or DMSO were inoculated with HRV-C3, C11, and C15 at 100 viral gene copies / cell and incubated in the presence of CYT387 or DMSO, respectively (n=3). CYT387- and DMSO-treated culture media served as inoculum for the next round of infection, during which CYT387 and DMSO treatments were continued. Culture media were harvested at 96 h p.i. to detect viral replication. (B) At 24 h p.i., nasal mucosal organoids inoculated with HRV-C3 were fixed and double-stained with α-species nasal organoids (green) and αα-species nasal mucosal organoids (red). Nuclei and actin filaments were counterstained with DAPI (blue) and phalloidin-647 (white), respectively. Scale bar, 10 μm. (C) TEM images of HRV-C3 viral particles in culture medium of infected nasal mucosal organoids. Arrows indicate viral particles (black) or empty capsids (white). Scale bar, 100 nm. Data represent the mean and SD of the indicated number (n) of biological replicates from a representative experiment. Statistical significance (in A) was determined using a two-tailed Student's t-test. *P<0.05, **P<0.01. ns not significant. Experiments in B and C were performed three times independently with similar results.

[0058] Figure 4A-C. Receptor blockade, antiviral inhibition, and viral titers in nasal mucosal organoids. (A) Airway (AwO) and nasal mucosal organoids (NsO) and their undifferentiated counterparts were immunostained with α-CDHR3 and isotype IgG controls and applied to flow cytometric analysis (n=2). Representative histograms are shown on the left. Nasal mucosal organoids were fixed and double-stained with α (green) and α (red). Confocal images of the front (top) and cross-section (bottom) are shown. Nuclei and actin filaments were counterstained with DAPI (blue) and phalloidin-647 (white), respectively. Scale bar, 10 μm. (B) Nasal mucosal organoids were treated with two 20-nuclear α-nuclear IgGs and autologous IgG for 2 hours (n=3), then inoculated with HRV-C3 and further incubated with the same antibodies. The culture medium was harvested at the specified hpi to detect viral replication. The results show the relative viral load in organoids treated with α-harvest culture medium relative to the relative viral load in organoids treated with IgG. Airway organoids (n=3) infected with HRV-A1 or HRV-C3 were treated with 1 μM Rupintrivir or 3 μM Itraconazol or DMSO. Culture medium was harvested from infected organoids at the indicated hpi and applied to the detection of viral replication by RT-qPCR. The results show the relative viral load in drug-treated organoids relative to the relative viral load in DMSO-treated organoids. (C) Nasal mucosal organoids were infected with HRV-C3 after 10-fold serial dilution (10 -1 ~10 -3 ). At 24h.pi, the organoid monolayer was fixed and the organoids were immunostained with α, and then imaged and analyzed. Representative high-content confocal images show VP3+ cells (green) in monolayers infected with serially diluted viruses. Cell nuclei were stained with DAPI (blue). Scale bar, 100μm. Three different areas in each organoid monolayer were randomly selected for counting VP3+ cells. The results show the average number of VP3+ cells at different dilutions. The data represent the mean and SD of biological replicates of the indicated number (n) from representative experiments. Statistical significance (in B) was determined using a two-tailed Student's t-test. *P<0.05, **P<0.01, ***P<0.001. ns not significant. The experiments in A and C were performed three times independently with similar results.

[0059] 4.1 Definition

[0060] As used herein, the term "infection" refers to any condition in which at least one cell of an organism (i.e., a subject) is infected by an infectious agent. As used herein, the term "infectious agent" refers to a foreign biological entity, i.e., a pathogen. Infectious agents include, but are not limited to, bacteria, viruses, protozoa, and fungi. An infectious disease is a condition caused by an infectious agent. Some infectious agents do not cause recognizable symptoms or disease under certain conditions, but may cause symptoms or disease under altered conditions.

[0061] The term "culture" or "culture system" means maintaining cells in an artificial in vitro environment. It is intended to encompass not only the culture of single cells, but also the culture of tissues or organs. A culture or culture system is one in which cells or tissues grow under culture conditions that promote prolonged tissue expansion with proliferation, multilineage differentiation, and recapitulation of cellular and tissue ultrastructure.

[0062] As used herein, the term "subject" refers to an individual (e.g., a human, animal, or other organism) to be treated by the methods and compositions of the present disclosure. Subjects include, but are not limited to, mammals (e.g., mice, apes, horses, cattle, pigs, dogs, cats, etc.), and include humans. The term "subject" generally refers to an individual who is to receive or has received a medical device of the present disclosure for treatment (e.g., coated with probiotic microorganisms and optionally one or more other agents) characterized by the presence of pathogenic bacteria or the expected possible exposure to pathogenic bacteria. The term subject can be a "non-human animal," including, but not limited to, vertebrates such as rodents, non-human primates, sheep, cattle, ruminants, lagomorphs, pigs, goats, horses, dogs, cats, ayes, etc.

[0063] As used herein, the term "pharmaceutical composition" refers to a pharmaceutically acceptable composition, wherein the composition comprises a pharmaceutically active agent and, in some embodiments, further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition can be a combination of a pharmaceutically active agent and a carrier.

[0064] As used herein, the terms "treatment", "treating" and the like refer to the administration of an agent or the performance of a procedure in order to obtain an effect in a subject, individual or patient. The effect may be preventative, i.e., the complete or partial prevention of a disease or its symptoms, and / or may be therapeutic, i.e., the achievement of a partial or complete cure of a disease and / or the symptoms of a disease. As used herein, "treatment" may include treating an infection in a mammal, particularly a human, and includes one or more of: (a) preventing a disease; (b) inhibiting a disease, i.e., arresting its development; and (c) alleviating a disease or its symptoms, i.e., causing regression of a disease or its symptoms. Treatment may also refer to any sign of successful treatment or improvement or prevention of a disease, including any objective or subjective parameter, such as alleviation; relief; relieving symptoms or making the patient more tolerant to the disease condition; slowing the rate of regression or decline; or making the endpoint of regression less debilitating. Treatment or improvement of symptoms may be based on objective or subjective parameters; including the results of a doctor's examination. The term "therapeutic effect" refers to the reduction, elimination or prevention of a disease, a symptom of a disease or a side effect of a disease in a subject.

[0065] As used herein, the term "pharmaceutically acceptable" refers to preparations approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia, other generally recognized pharmacopeia, and other formulations safe for use in animals, more particularly in humans and / or non-human mammals.

[0066] As used herein, the term "effective amount" refers to an amount of a composition (e.g., a probiotic microorganism) sufficient to achieve a beneficial or desired result. An effective amount can be administered in one or more administrations, uses, or dosages and is not intended to be limited to a particular formulation or route of administration. It is within the ability of those skilled in the art to relatively easily determine the effective amount of a probiotic microorganism or other therapeutic composition. 5. Detailed Description of the Invention

[0067] In the following description, the details of the present invention are set forth as preferred embodiments. Modifications, including additions and / or substitutions, may be made without departing from the scope and spirit of the present invention, as will be apparent to those skilled in the art. Specific details may be omitted so as not to obscure the present invention; however, this disclosure is prepared to enable those skilled in the art to practice the teachings herein without undue experimentation.

[0068] 5.1 Respiratory Organoids

[0069] The respiratory organoids used in the present disclosure refer to three-dimensional cell aggregates and may include organoids or cell clusters formed by suspension cell cultures. Organoids may also be referred to as small organ-like organs, organ analogs or organ-like organs. Organoids specifically contain one or more of the several cell types that make up an organ or tissue, and are able to reproduce the form and function of a tissue or organ. Organoids reproduce the cellular structure and ultrastructure of the sample from which they come. In addition, by using 3D culture to reaggregate and reorganize cells to make them similar to their living environment, the limitations of 2D cell lines cultured using 2D cultures can be overcome, and the physiological functions of living organisms can be similarly reproduced, which can be applied to disease modeling, drug screening, and the like.

[0070] Respiratory organoids useful in the present disclosure are artificial organs that exhibit, for example, respiratory (= airway) function. In one embodiment, the respiratory organoid can be one of an alveolar organoid, an airway organoid, and a nasal mucosal organoid, but is not limited thereto. In one embodiment, the organoid is an organoid derived from adult stem cells. In one embodiment, the organoid is derived from epithelial stem cells. In one embodiment, the organoid is an airway organoid. In one embodiment, the airway organoid is derived by inducing proximal differentiation of an organoid. In one embodiment, the organoid is derived from primary lung tissue.

[0071] In certain embodiments, organoids can be cultured until they reach a size of 115 μm (±6.4), but are not limited thereto. For example, the size of the organoids can be 100 μm to 130 μm, 100 μm to 128 μm, 100 μm to 126 μm, 100 μm to 125 μm, 100 μm to 124 μm, 100 μm to 121.4 μm, 103 μm to 130 μm, 103 μm to 128 μm, 103 μm to 126 μm, 103 μm to 125 μm, 103 μm to 124 μm, 103 μm to 121.4 μm, 106 μm to 130 μm, 106 μm to 128 μm, 106 μm to 126 μm, 106 μm to 125 μm, 106 μm to 124 μm, 106 μm to 121.4 μm, 108.9 μm to 130 μm, 108.9 μm to 128 μm, 108.9 μm to 126 μm, 108.9 μm to 125 μm, 108.9 μm to 124 μm, or 108.9 μm to 121.4 μm, but is not limited thereto.

[0072] Organoids prepared by the subject methods can be used for basic research, for example, to better understand the basis of disease and infection, and for drug discovery, for example, as reagents in screening, such as those described further below, and for diagnostic purposes. Organoids can also be used to evaluate the pharmacokinetics and pharmacodynamics of pharmaceutical agents, such as the ability of mammalian tissues to absorb active pharmaceutical agents, the cytotoxicity of pharmaceutical agents to primary mammalian tissues, etc. The immune components of these organoids can be used to evaluate vaccines, adjuvants, or to identify the effects of candidate therapeutic agents on immune responses associated with disease or infection.

[0073] 5.2 Culture

[0074] 5.2.1 Organoid Cultures and Compositions

[0075] Provided are culture systems, compositions, and methods for culturing organoids derived from adult stem cells. Cultures can be maintained for up to 5 days, up to 7 days, up to 10 days, up to 15 days, up to 21 days, up to 28 days, up to 100 days, up to 180 days, more than 6 months, or more than one year. In some embodiments, the tissue or stem cells are obtained from any mammalian species, such as humans, horses, cattle, pigs, dogs, cats, rodents, such as mice, rats, hamsters, primates, and the like.

[0076] In one embodiment, respiratory tissue can be obtained by any convenient method, such as by biopsy, during surgery, by needle, etc., and is generally obtained as sterile as possible. Nasal epithelial cells are obtained invasively from the nasal cavity of an individual using a nasal swab. In one embodiment, suitable culture media for respiratory organoids are known in the art and are also disclosed in Section 6.6.1 of this disclosure.

[0077] Organoid cultures are used to model respiratory pathologies, including, for example, infection, autoimmunity, cancer, and the like; and to screen therapeutic agents for treating such pathologies; for precision medicine; and for stem cell-based therapies. Screening assays are provided that can be used to determine complex responses to therapies, including but not limited to antiviral, anti-inflammatory, and immunotherapies.

[0078] 5.2.2 Infection and growth culture

[0079] In one embodiment, suitable culture media for respiratory organoids to promote infection and growth of respiratory microorganisms are disclosed in Section 6.6.1 of the present disclosure. In one embodiment, the organoid culture medium includes one or more drugs for treating myelofibrosis, interferon inhibitors, and interferon-stimulated gene expression through the JAK1 / JAK2 (kinases that mediate IFN signaling) and TBK1 / IKKε (kinases that mediate RLR and TLR signaling) pathways. In certain embodiments, the organoid culture medium comprises CYT387, Amlexanox, BX795, MRT67307, Ruxolitinib, or a combination thereof. In one embodiment, the organoid culture medium comprises CYT387.

[0080] In one embodiment, organoids prepared by the methods of the present disclosure can be used to propagate respiratory microorganisms. In one embodiment, the methods of the present disclosure maintain continuous passage of microorganisms. In one embodiment, the methods of the present disclosure can be used to propagate and maintain passage of respiratory microorganisms that are unculturable.

[0081] 5.3 Respiratory microorganisms

[0082] Respiratory microorganisms include viral, bacterial, and fungal infectious agents. Respiratory microorganisms include respiratory pathogens, such as viruses, bacteria, and protozoa that infect the lungs, particularly the lower respiratory tract. Pathogens of interest include, for example, influenza, rhinoviruses, adenoviruses, coronaviruses, including but not limited to SARS-CoV1, SARS-CoV2, MERS-CoV; and tuberculosis. Respiratory viruses cause numerous diseases in humans and have a significant impact on morbidity and mortality worldwide, primarily among children. Many human respiratory viruses are commonly circulated across all age groups and are thought to be adapted for efficient human-to-human transmission. Respiratory pathogens can be used to infect respiratory organoid cultures, allowing for the propagation and study of pathogens by monitoring and screening for compounds that can be used to treat respiratory diseases. Common respiratory viruses include, but are not limited to, influenza; ADV, adenovirus; HBoV, human bocavirus; HCoV, human coronaviruses, including SARS-CoV1 and SARS-CoV2; HMPV, human metapneumovirus; HPIV, human parainfluenza virus; HRSV, human respiratory syncytial virus; and HRV, human rhinovirus.

[0083] Influenza viruses. There are four types of influenza viruses: A, B, C, and D. Influenza A and B cause human infections every year during the epidemic season. Influenza A has several subtypes, based on the combination of hemagglutinin (H) and neuraminidase (N) proteins expressed on the surface of the virus. Influenza viruses have a segmented, negative-sense, single-stranded RNA genome. There are 18 different hemagglutinin subtypes and 11 different neuraminidase subtypes (H1-18 and N1-11). Influenza A viruses are characterized by H and N types, such as H1N1 and H3N2. Influenza B viruses are classified into lineages and strains. Influenza viruses replicate in the epithelial cell lining of the upper and lower respiratory tract. Disease during infection is primarily a result of inflammation and damage to the lungs caused by infection and death of epithelial cells, combined with inflammation caused by the immune system's response to the infection. Severe respiratory disease can be caused by multiple, nonexclusive mechanisms, including airway obstruction, loss of alveolar architecture, loss of lung epithelial integrity due to infection and death of epithelial cells, and degradation of the extracellular matrix that maintains lung architecture. In particular, infection of alveolar cells appears to contribute to severe symptoms because it impairs gas exchange and enables the virus to infect endothelial cells, which produce large amounts of proinflammatory cytokines. Pneumonia caused by influenza virus is characterized by high levels of viral replication in the lower respiratory tract, accompanied by a robust proinflammatory response.

[0084] Human respiratory syncytial virus (HRSV) virions are heterogeneous in size and shape and consist of a helical nucleocapsid containing negative-sense single-stranded RNA tightly associated with a nucleoprotein (N). Two HRSV groups, A and B, were originally distinguished based on antigenic differences in the attachment glycoprotein G. HRSV is the single most common cause of lower respiratory tract infection (LRTI) in children worldwide. HRSV replicates in the respiratory epithelium, reaches high titers in nasal secretions, and results in viral shedding for up to 3 weeks after the end of symptoms. Cell-to-cell spread leads to HRSV involvement throughout the respiratory tree, reaching the bronchioles 1–3 days after symptom onset, where it induces ciliated cell necrosis, syncytia formation, peribronchiolar inflammation with abundant lymphocytes and macrophages, and impaired secretion clearance, resulting in the small airway obstruction and lung hyperinflation typical of bronchiolitis. HRSV pneumonia is associated with interstitial mononuclear infiltrates, eosinophilic cytoplasmic inclusions in epithelial cells, and multinucleated giant cells, often coexisting with bronchiolitis. HRSV disease is particularly severe in young children, whose immature airways are unable to compensate for virus-induced damage.

[0085] Human parainfluenza viruses (HPIVs) are common causes of LRTI in infants and children worldwide. HPIVs share structural and biological characteristics with HRSVs and are distributed in two genera of the Paramyxoviridae family. HPIVs are antigenically classified into types 1–4, with HPIV-4 having subtypes A and B. The virions are pleomorphic, with single-stranded negative-sense RNA and a diameter ranging from 150 to 200 nm. The virus does not persist in the environment for long periods of time and is primarily transmitted via large droplets and fomites. HPIVs replicate in ciliated cells, causing cytolysis of the respiratory mucosa. Infection begins in the upper respiratory tract and spreads downward along the respiratory tree. Croup syndrome primarily involves the larynx and trachea, with extensive involvement of the lower respiratory tree that can be present in tracheobronchitis, bronchopneumonia, and bronchiolitis. Similar to what occurs with HRSV, the amplified inflammatory response induced by viral infection of epithelial cells leads to mononuclear interstitial infiltrates, epithelial necrosis, inflammatory exudates into the alveoli, and hyaline membrane formation in the lungs. In cases of croup, mononuclear inflammatory cell infiltration may be seen in the subglottic area.

[0086] Human metapneumovirus (HMPV) is a common cause of community-acquired ARI in children and adults worldwide. HMPV particles are enveloped, polymorphic, spherical or filamentous particles with a diameter of approximately 209 nm. Like other paramyxoviruses, HMPV has a negative-sense, single-stranded RNA genome, and viral replication occurs in a gradient manner. Little is known about the specific pathogenesis of HMPV. Animal studies have shown destruction of the respiratory epithelium, epithelial cell desquamation, and inflammatory infiltrates in the lungs. In pathological studies of humans with underlying diseases and HMPV infection, the main findings are acute and organizing lung injury, diffuse alveolar damage, desquamated epithelial cells with eosinophilic cytoplasmic inclusions, multinucleated giant cells, histiocytes, and hyaline membrane formation.

[0087] Human rhinovirus (HRV) is the most common respiratory pathogen in humans and is the most commonly detected virus in samples from patients with the common cold. HRV is a small, non-enveloped positive-strand RNA virus in the genus Rhinovirus of the family Picornaviridae, distributed in two species, A (75 serotypes) and B (25 serotypes). HRV replication is limited to the respiratory epithelium, occurring in the scattered ciliated cells of the nose and the non-ciliated cells of the nasopharynx, and this tropism seems to be the result of receptor availability. Infection of a limited number of cells triggers the nuclear translocation of NF-κB and the gene expression of cytokines, chemokines and inflammatory mediators. These are associated with the stimulation of local parasympathetic nerve endings, leading to the development of cold symptoms. Kinins, prostaglandins, proinflammatory cytokines and chemokines may contribute to vasodilation, increased vascular permeability, influx of polymorphonuclear leukocytes, exocrine gland secretion and nerve endings stimulation, leading to nasal congestion, rhinorrhea, sneezing, coughing and sore throat.

[0088] Adenoviruses are nonenveloped icosahedral DNA viruses of the genus Mammalian Adenovirus in the family Adenoviridae. Respiratory infections caused by adenoviruses occur worldwide and have no apparent seasonality. Symptomatic infection may involve all parts of the respiratory tract and usually begins in the upper respiratory epithelium. Adenovirus infection causes necrosis of airway epithelial cells and may cause viremia through systemic viral dissemination in immunocompromised individuals. Bronchiolitis, interstitial pneumonia, and mononuclear cell infiltration are part of the inflammatory process in the lungs. In addition to lytic infection, adenoviruses may remain latent in epithelial cells and lymphocytes, which may be important for maintaining the virus in the population.

[0089] Until 2003, human coronavirus (HCoV) subtypes 229E and OC43 were the only coronaviruses identified in humans, but the identification of severe acute respiratory syndrome (SARS) coronavirus led to a renewed study of HCoVs. Coronaviruses are enveloped viruses with a distinctive virion morphology, displaying long, widely spaced, petal-shaped spikes on their surface, which give the virus its crown-like appearance and the origin of the name "corona." The viral envelope contains a long, helical nucleocapsid composed of a single, positively stranded chain. HCoVs are found worldwide and are considered the second most common cause of the common cold. HCoV-229E and -OC43 cause the common cold with varying frequency.

[0090] SARS-CoV-2 is a virus of the species severe acute respiratory syndrome-related coronavirus (SARSr-CoV), related to SARS-CoV-1. It is of zoonotic origin and shares close genetic similarity with bat coronaviruses. SARS-CoV-2 is a member of the subgenus Sarbecovirus (beta-CoV lineage B). Coronaviruses undergo frequent recombination. There are thousands of variants of SARS-CoV-2, which can be divided into larger clades.

[0091] Human bocavirus (HBoV) is a new member of the Parvoviridae family, provisionally classified, based on sequence homology and genome organization, into the genus Bocavirus, which already includes two other viruses: bovine parvovirus 1 and canine parvovirus. The HBoV virion consists of a small, nonenveloped, icosahedral particle with a single-stranded DNA genome of approximately 5300 nt, organized in a manner similar to other known bocaviruses, with three ORFs: two encoding the nonstructural proteins NS1 and NP-1, and the third enclosing the two capsid proteins, VP1 and VP2. HBoV circulates worldwide, and prevalence studies, primarily conducted by PCR in respiratory samples from children, have shown detection rates of 1.5–19%, depending on the region studied and the sensitivity of the assay. The site of HBoV replication has not been determined, but the virus is clearly not confined to the respiratory tract, as it has been detected in stool and serum, suggesting systemic infection.

[0092] Bacterial and protozoan pathogens are less common than viruses but may include Mycobacterium tuberculosis, Streptococcus pneumoniae, Mycoplasma pneumoniae, Haemophilus influenzae, Chlamydia pneumoniae, Chlamydia psittaci, Coxiella burnetii, Legionella pneumophila, Staphylococcus aureus, and Klebsiella pneumoniae. Cryptosporidiosis can cause outbreaks and sporadic cases of respiratory illness.

[0093] In one aspect, the spread and pathogenesis of respiratory pathogens can be studied using respiratory organoids, which can serve as highly physiologically relevant models to study the tropism and pathogenesis of respiratory pathogens.

[0094] 5.4 Compound Screening

[0095] In one aspect, the present disclosure provides a method for identifying a therapeutic agent effective in treating a respiratory disease. The method comprises providing a culture composition, infecting the culture composition with a respiratory pathogen, administering a therapeutic agent to the culture composition, and determining whether the therapeutic agent is effective in treating or preventing a respiratory disease caused by infection in a respiratory organoid. The therapeutic agent can be administered to the culture composition before or after the culture composition is infected with the respiratory pathogen.

[0096] Screening is performed using respiratory infectious pathogens, such as bacteria, viruses, and the like. In one aspect, a method is provided for in vitro screening of the effects of agents on cells of different cell types (e.g., epithelial, immune, and matrix present in organoids), including the initiation of viral infection and the treatment process. Optionally, before or after exposure to respiratory pathogens, the organoids cultured by the methods described herein are exposed to candidate agents. Agents of interest include pharmaceutical preparations, such as known therapeutic agents; small molecules, antibodies, peptides, and the like, and genetic agents, such as antisense, RNAi, expressible coding sequences, and the like, such as candidate secreted growth factors, cytokines, their receptors or inhibitors, or other expressible coding sequences of proteins of interest. In some embodiments, the effect of candidate therapeutic agents on viral infection-related immune responses or their downstream effects is determined, for example, wherein the agents may include, but are not limited to, chemotherapeutic agents, monoclonal antibodies, or other protein-based agents, radiation / radiosensitizers, cDNA, siRNA, shRNA, small molecules, and the like. Also provided is a method for using organoid cultures to screen agents that regulate tissue function.

[0097] 5.5 Pharmaceutical Compositions

[0098] Once the screening method of the present disclosure is used to identify that the compound can be used to affect the pathogen of respiratory organoids or infect respiratory organoids, a pharmaceutical composition comprising the identified compound can be prepared. According to the pharmaceutical composition of the present disclosure, it can be formulated for external application, such as the form of powder, granules, tablets, capsules, suspensions, emulsions, syrups or aerosols or sterile injectable solutions, the example of the carrier, excipient and diluent that can be included in the composition includes lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of preparation, preparation can be prepared by using diluent or excipient, such as commonly used filler, extender, adhesive, wetting agent, disintegrant and surfactant. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and solid preparations can be prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition, in addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration can correspond to suspensions, oral liquids, emulsions, syrups, etc., and in addition to water and liquid paraffin commonly used as simple diluents, various excipients such as wetting agents, sweeteners, aromatics, preservatives, etc. can also be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized agents, and suppositories. As non-aqueous solutions and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injection esters such as ethyl oleate, etc. can be used. As the basis of suppositories, witepsol, macrogol, Tween 61, cocoa butter, lauric acid, glycerin gelatin, etc. can be used.

[0099] The pharmaceutical composition of the present disclosure can be administered orally or parenterally, preferably parenterally. In the case of parenteral administration, it can be administered by intramuscular injection, intravenous injection, subcutaneous injection, intraperitoneal injection, topical administration, transdermal administration, and the like.

[0100] The appropriate dosage of the pharmaceutical composition of the present disclosure may vary depending on factors such as formulation method, administration method, patient's age, weight, sex, pathological condition, diet, administration time, administration route, excretion rate, and reaction sensitivity.

[0101] The pharmaceutical compositions of the present disclosure can be prepared into unit dosage forms using pharmaceutically acceptable carriers and / or excipients according to methods readily performed by those skilled in the art, or can be prepared by placing them in bulk containers. Here, the formulations can be in the form of solutions, suspensions, or emulsions in oil or aqueous media, or can be in the form of extracts, powders, granules, tablets, or capsules, and can additionally contain dispersants or stabilizers.

[0102] 5.6 Treatment

[0103] Respiratory diseases that can be treated using the methods of the present disclosure are physically apparent diseases or conditions associated with the respiratory system and are, for example, cystic fibrosis, respiratory distress syndrome, acute respiratory distress syndrome, tuberculosis, cough, bronchial asthma, cough based on increased airway hyperresponsiveness (bronchitis, influenza syndrome, asthma, obstructive lung disease, etc.), influenza syndrome, cough suppression, airway hyperresponsiveness, tuberculous disease, asthma (airway inflammatory cell infiltration, increased airway hyperresponsiveness, bronchoconstriction, mucus hypersecretion, etc.), chronic obstructive pulmonary disease, emphysema, pulmonary fibrosis, idiopathic pulmonary fibrosis, reversible airway obstruction, adult respiratory disease syndrome, pigeon breeder's disease, farmer's lung, bronchopulmonary dysplasia, airway disease, emphysema, allergic bronchopulmonary aspergillosis, allergic bronchitis bronchiectasis, occupational asthma, reactive airway disease syndrome, interstitial lung disease, parasitic lung disease, etc., but are not limited thereto.

[0104] 5.7 Reagent Kit

[0105] The present disclosure provides a kit for generating respiratory organoids for propagating microorganisms, comprising (a) a composition for generating respiratory organoids in a culture medium comprising a JAKL1 / JAK2 inhibitor or a TBK1 / IKKε inhibitor; and (b) respiratory microorganisms for infecting the respiratory organoids, wherein the microorganisms are propagated in the respiratory organoids.

[0106] The kit of the present disclosure may further include instructions. The instructions may include a production method, but are not limited thereto.

[0107] In certain embodiments, the test kit may include a container; instructions; and the like. The container can be used to package the materials, as well as to store and secure them. The container can take the form of, for example, a bottle, bucket, pouch, envelope, tube, ampoule, etc., which can be partially or entirely made of plastic, glass, paper, foil, etc. The container can be equipped with a completely or partially detachable stopper, which can initially be part of the container or attached to the container by mechanical, adhesive, or other means. The container can also be equipped with a stopper that allows needle access to the contents. The test kit can include an outer packaging that can include instructions for use of the components. 6. Examples

[0108] 6.1 Human Airway Organoids Susceptible to HRV-C-Positive Clinical Samples

[0109] A total of 9 HRV-C positive nasopharyngeal samples with variable viral loads were obtained. We inoculated these samples onto 2D human airway organoids, which are monolayers of airway organoids grown on transwell inserts. We harvested aliquots of culture medium from the top chamber on designated days after inoculation to measure viral load (copy number of viral 5'UTR) over time. Figure 1B As shown, 8 out of 9 samples showed an increase in viral load after inoculation, indicating productive HRV-C infection of airway organoids. Phylogenetic analysis of the 5'UTR of the 8 samples revealed that samples 1, 4, 5 and 7 were closely related to HRV-C15, while the other samples were related to HRV-C3, -C8, -C11 and -C45. Phylogenetic analysis based on the VP4 / VP2 genes, which are also commonly used for rhinovirus genotyping, showed similar results. However, sample 6 was genotyped as HRV-C45 and HRV-C11 based on the 5'UTR and VP4 / VP2 sequences, respectively. This inconsistency has been previously reported in HRV genotyping. 24 We then examined the growth and release of HRV-C virus in airway organoids. After inoculation of HRV-C3-positive (HRV-C3+) samples, we harvested culture medium from the top and bottom chambers for viral load detection. Figure 1C As shown, the viral load in the apical culture medium was significantly higher than that in the basolateral culture medium, indicating that progeny virions were preferentially released from the apical side. HRV-A was inoculated into airway organoids in parallel. We observed productive infection with HRV-A, with a similar pattern of viral release ( Figure 1C ).

[0110] Understanding viral biology always begins with the isolation and culture of reproducible virus in sufficient quantities, followed by a series of in vitro and in vivo characterizations of viral biology and virus-host interactions. Given the susceptibility of airway organoids to HRV-C and the ongoing active viral replication, we reasoned that airway organoids might be able to sustain serial viral passages, thereby enabling reproducible HRV-C isolation and culture. We were prompted to use medium from a first round of infection with an HRV-C+ clinical sample to propagate progeny virions. However, when HRV-C+ medium was used to inoculate a new batch of airway organoids, we did not observe any signs of viral growth. In other words, despite being susceptible to HRV-C, airway organoids were unable to sustain serial viral passages.

[0111] 6.2 Immunosuppression enables serial passage of HRV-C in airway organoids

[0112] Previous studies in experimental mice and human epithelial organoids have documented that mucosal epithelial cells mount a robust antiviral response upon viral infection. 25-29 We hypothesized that the antiviral response triggered by HRV-C infection in airway organoids might inhibit serial viral passages. CYT387, a drug for the treatment of myelofibrosis, inhibits interferon and interferon-stimulated gene expression through the JAK1 / JAK2 (kinases that mediate IFN signaling) and TBK1 / IKKε (kinases that mediate RLR and TLR signaling) pathways. 28 Clinical studies have reported that JAK1 / JAK2 inhibition puts patients at higher risk of rhinovirus infection 30 Therefore, we hypothesized that treatment with CYT387 or other molecules could suppress virus-induced antiviral responses in airway organoids, promote viral proliferation, and enable serial HRV-C passaging.

[0113] We examined HRV-C replication in airway organoids in the presence or absence of CYT387 ( Figure 2A Briefly, we inoculated HRV-C3+ culture medium (collected from organoids infected with HRV-C3+ clinical samples) onto airway organoids pretreated with 1 μl of CYT387 or DMSO. The infected organoids were further incubated with CYT387 (hereafter referred to as CYT) or DMSO for 96 h, after which we harvested the culture medium (P1) to detect viral growth. P1 culture medium from CYT- and mock-treated organoids was passaged to P2 in the presence of CYT and DMSO, respectively. Figure 2A ).like Figure 2B As shown, CYT treatment resulted in an increase in viral load in P1 culture medium at 96 hours post infection (hpi). In P2, CYT-treated organoids still maintained robust viral replication, while no viral growth was observed in DMSO-treated organoids. In the subsequent P3 passage, we had to inoculate the CYT-treated P2 culture medium onto airway organoids, where we continued CYT or DMSO treatment to verify the enhancing effect of CYT. After CYT or DMSO treatment, the CYT-treated culture medium inoculation was repeated in the subsequent P4 and P5 passages. In the presence of CYT, HRV-C3 was passaged five times in the airway organoids, and its viral load was higher than that of the DMSO-treated organoids ( Figure 2B Furthermore, HRV-C virions continuously propagated in CYT-treated organoids established productive infection in airway organoids in the absence of CYT, indicating that progeny virions passaged in airway organoids in the presence of CYT are highly infectious. Overall, these results demonstrate that airway organoids are capable of continuous HRV-C propagation in the presence of CYT and possibly other immunosuppressive molecules.

[0114] 6.3 Nasal mucosal organoids that maintain continuous HRV-C proliferation

[0115] We then performed viral passage in nasal mucosal organoids, a model with a cellular composition comparable to airway organoids but more similar to the upper airway epithelium. 17 We inoculated nasal mucosal organoids with HRV-C3+ culture medium collected from organoids infected with HRV-C3+ clinical samples in the presence of CYT or DMSO and collected the culture medium at 96 h.pi. Due to the high viral load in DMSO-treated nasal mucosal organoids in preliminary experiments, we changed the experimental design. Briefly, virus-containing culture medium from CYT- and DMSO-treated organoids was used for the next round of infection with CYT and DMSO, respectively ( Figure 3A ). Notably, CYT treatment significantly promoted viral growth. Despite this, in the absence of CYT, nasal mucosal organoids from two different donors sustained four serial passages of HRV-C3. We also tested HRV-C11+ and HRV-C15+ culture media in nasal mucosal organoids derived from another donor (culture media collected from organoids infected with HRV-C11+ and HRV-C15+ clinical samples, respectively). Both subtypes of HRV-C were serially passaged three times with high viral loads and, if necessary, could be further passaged more times. Overall, the nasal mucosal organoids themselves maintained the continuous proliferation of HRV-C in the absence of any intervention. To verify infectivity, we demonstrated that HRV-C passaged multiple times in nasal mucosal organoids established productive infection in nasal mucosal organoids ( Figure 3A HRV-C virions released from nasal mucosal organoids were concentrated and applied to transmission electron microscopy (TEM) for examination. Virion particles and empty capsid particles of approximately 30 nm in size were easily distinguished ( Figure 3C We performed whole-genome sequencing of viruses from the initial clinical sample and after one and six serial passages following CYT treatment; no adaptive mutations were identified. Overall, airway and nasal mucosal organoids enabled reproducible virus isolation and serial passage of the poorly culturable HRV-C.

[0116] 6.4 Receptor Blockade, Antiviral Inhibition, and Viral Titration in Nasal Mucosal Organoids

[0117] HRV infection is initiated by viral binding to its cognate receptor and receptor-mediated endocytosis. CDHR3, the cellular receptor for HRV-C6, a member of the cadherin family of transmembrane proteins, is highly expressed in the human airway epithelium, particularly in ciliated cells in the natural airway epithelium. 31First, we examined the expression and distribution of CDHR3 in airway and nasal mucosal organoids and compared them with their undifferentiated counterparts used to maintain and expand organoid cultures. Flow cytometric analysis showed that the percentage of CDHR3+ cells increased from less than 20% in undifferentiated organoids to more than 60% and 80% in differentiated airway and nasal mucosal organoids, respectively ( Figure 4A Confocal imaging revealed high expression of CDHR3 in differentiated nasal and airway organoids ( Figure 4A ); Most ACCTUB+-positive ciliated cells express CDHR3 on the apical surface, and a small fraction of CDHR3+ cells are non-ciliated cells.

[0118] We then performed antibody blocking experiments in nasal mucosal organoids to assess the role of CDHR3 in HRV-C replication. We examined HRV-C replication in nasal mucosal organoids in the presence of two α-CDHR3 antibodies or IgG isotypes. Blocking CDHR3 with specific antibodies significantly reduced viral growth, confirming that CDHR3 is required for HRV-C entry into nasal mucosal organoids ( Figure 4B To demonstrate the utility of airway organoids for identifying antiviral agents against HRV-C, we tested the HRV inhibitor, lupinquér, which targets the HRV 3C protease, as a proof-of-concept. Itraconazole was also tested. 32 , a repurposed antifungal drug with documented efficacy against rhinoviruses. We examined the effects of these two drugs on HRV-C and HRV-A in airway organoids and observed that both drugs had a more pronounced inhibitory effect on HRV-C than on HRV-A ( Figure 4B right).

[0119] Currently, few in vitro assays are available for quantification of HRV-C infectious particles, since, apart from the one described in Bochkov et al. 33 No standard cell line is susceptible to this virus except for the adapted strain C15a that develops cytopathic effects and forms plaques in a stable cell line expressing CDHR3. Quantification of HRV-C mainly relies on RT-qPCR assays to determine viral gene copy number, as we demonstrated above. Given the susceptibility of airway and nasal mucosal organoids to HRV-C, we attempted to establish an organoid-based immunofluorescence assay (IFA) to quantify HRV-C infectious virions. 11 Aliquots of HRV-C collected from infected organoids with 100 viral gene copies / ml were serially diluted and used in triplicate to inoculate nasal mucosal organoids. Organoid monolayers were fixed at 24 hours p.i. and immunostained with an α-VP3 antibody to mark HRV-C-infected cells, followed by analysis using a high-content imaging analysis system. Figure 4C The right panel shows representative images of organoids infected with serial dilutions of virus. A total of three randomly selected areas were selected from each organoid monolayer from which VP3 was counted. + The number of cells. VP3 + The average number of cells Figure 4C As shown in the figure on the left, we based our analysis on the highest dilution factor of 10. 3 Viral titers were calculated from image analysis of seeded organoids; the number of infectious particles in the sample was approximately 3.7 × 10 6 IFU / ml.

[0120] 6.5 Discussion

[0121] We have established the first human airway epithelial organoid culture system derived from primary lung tissue and nasal epithelial cells, which allows reconstruction and expansion of the entire human airway epithelium in culture plates with excellent efficiency and stability. 13-17 Given that nasal and airway organoids accurately mimic the natural airway epithelium, we hypothesized that these airway organoids could potentially sustain productive infection with HRV-C, a common respiratory virus that is challenging to culture routinely. Furthermore, the high stability and ease of access to these airway organoids provided by the robust culture system could provide a reproducible culture system for HRV-C, eliminating a long-standing barrier to understanding this common human respiratory virus. Indeed, upon inoculation with HRV-C + Following clinical samples, we observed active viral growth, demonstrating that airway organoids can recapitulate the susceptibility of human airway epithelium to HRV-C ( Figure 1B However, continuous HRV-C proliferation in airway organoids was unsuccessful unless the immunosuppressive molecule CYT387 was applied to attenuate the antiviral response triggered in infected organoids ( Figure 2B and 2C ).

[0122] After first demonstrating HRV-C infection in surgically resected sinonasal mucosa and nasal polyps, Bochkov et al. identified the cellular receptor CDHR3 for HRV-C6. This landmark discovery was followed by the establishment of a stable Hela-E8 cell line carrying variants with higher cell surface CDHR3 expression. By serial passage in Hela-E8CDHR3 cells, an HRV-C15a strain with enhanced fitness due to adaptive mutations was obtained. 33. However, these two adaptive mutations only partially confer the enhanced phenotype on other recombinant HRV-C subtypes. So far, this C15a strain appears to be the only one that can be propagated to yields sufficient for experimental use. In addition, the adaptive mutations are cell type specific; HRV-C15a viruses propagated in Hela-E8CDHR3 cells appear to replicate less actively than wild-type C15 viruses in primary human bronchial epithelial cells. 33 , which may hinder the application of adaptive virus strains for understanding human HRV-C respiratory infections. In this study, we randomly selected three HRV-C+ clinical samples with different subtypes, all of which were serially passaged in nasal mucosal organoids with excellent efficiency and remarkable viral genome stability ( Figure 3B 、 3C ). Therefore, we have established a robust culture system to reproducibly propagate the poorly culturable HRV-C.

[0123] We observed that the HRV-C receptor CDHR3 was abundantly expressed on the apical surface of airway and nasal mucosal organoids, particularly on the apical surface of ciliated cells ( Figure 4A ), which is consistent with the results of single-cell RNA sequencing studies of native human airway epithelial cells 31 Blocking CDHR3 with a specific antibody significantly inhibited HRV-C viral growth ( Figure 4B We also demonstrated the efficacy of two antiviral drugs targeting HRV as proof of concept ( Figure 4B In addition, we developed an organoid-based IFA to quantify HRV-C infectious particles ( Figure 4C Overall, our study demonstrates the unique advantages of respiratory organoids for studying HRV-C, from the reproducible propagation of poorly culturable viruses to the detailed dissection of virus-host interactions and the development of antiviral agents. Given that receptor blockade with α-CDHR3 inhibits viral growth, organoid-based neutralization assays can be developed to evaluate the neutralizing activity of vaccinated sera and engineered antibodies against HRV-C. Importantly, our study establishes a new paradigm for the propagation and study of other non-culturable human and animal viruses.

[0124] 6.6 Materials and Methods

[0125] 6.6.1. Establishment, Maintenance, and Differentiation of Respiratory Organoids

[0126] This study was approved by the Ethics Review Committee of the University of Hong Kong / Hospital Authority Hong Kong West Cluster (UW13-364 and UW21-695). Informed consent was obtained from patients and volunteers to collect human lung tissue and nasal cells. Multiple organoid lines were established from surgically resected human lung tissue according to our previously published protocols.13,15,18,20 To obtain lung organoids, we used normal lung tissue adjacent to diseased tissue that was randomly provided to us. These lung tissues typically contain bronchioles of varying sizes, surrounded by alveolar sacs. After one to two passages, the fibroblasts and other non-epithelial elements in the initial culture gradually decreased. Subsequently, cultures of pure epithelial organoids expanded stably in expansion medium for more than one year. Nasal mucosal organoids were derived from nasal epithelial cells that were non-invasively collected from healthy donors with perfect efficiency and serially passaged in expansion medium for up to 6 months 17 Undifferentiated lung and nasal mucosa organoids were passaged every 2 to 3 weeks at a ratio of 1:3 to 1:10, depending on whether mechanical shearing or enzymatic digestion was used to split the organoids. Proximal differentiation protocols for generating mature airway and nasal mucosa organoids were previously described. 17,20 Unless otherwise stated, mature airway and nasal mucosal organoid monolayers grown on transwell inserts were used throughout the studies.

[0127] 6.6.2 Virus isolation, infection and detection

[0128] A total of 9 HRV-C positive archived nasopharyngeal samples from clinical patients were used in this study. Mature human airway and nasal mucosal organoids grown on 24-transwell inserts were washed twice with basal medium (Advanced DMEM / F-12 (Gibco) supplemented with 1% HEPES, 1% GlutaMAX and 1% penicillin / streptomycin) and then inoculated with the indicated viral gene copies, followed by incubation at 37°C for 4 hours in PD medium (PneumaCult-ALI medium (STEMCELL Technologies) supplemented with 10μ0 times, then 10 times MY27632). After incubation, the airway or nasal mucosal organoids were washed three times with basal medium to remove residual inoculum. The apical and basolateral chambers were supplemented with 300μl and 500μl PD medium, respectively. For CYT387 treatment, the airway and nasal mucosal organoids were inoculated with 1, and the airway and nasal mucosal organoids were inoculated with 1. For organoids, cells were pre-incubated overnight in PD medium supplemented with α-cy87 or DMSO. After viral inoculation, PD medium supplemented with 1 ml of CYT387 or DMSO was dispensed into the apical and basolateral chambers to maintain the organoids. To evaluate the effect of temperature on viral growth, we infected nasal mucosal organoids with HRV-C3 and HRV-A1 at 100 viral gene copies / cell and maintained the organoids at 33°C or 37°C. To demonstrate the effect of CDHR3 on HRV-C viral replication, we pre-treated organoids with 20 μg of two antibodies against CDHR3 (Abcam, ab121337; Sigma-Aldrich, HPA011218) and autologous IgG (Abcam, ab172730) for 2 hours, then incubated with HRV-C3 at 1000 viral gene copies / cell and further incubated in the presence of α-CDHR3 and IgG, respectively. At the indicated hpi, culture medium was harvested from infected organoids to assay for viral replication. To investigate the antiviral effects of inhibitors against HRV-C replication, organoids were inoculated with HRV-A1 or HRV-C3 at 1000 viral gene copies / cell and incubated in PD medium in the presence of 1 μg of lupinquvir, 3 μM itraconazole, or DMSO. Culture medium was collected at the indicated hpi for viral load assays.

[0129] To examine viral replication, we harvested the cell-free culture medium from the apical chamber at the indicated hours after infection and then extracted RNA using the RNAeasy mini kit (Qiagen). The viral load was detected by RT-qPCR assay targeting the human rhinovirus 5′UTR gene using the QuantiNova Probe RT-PCR Kit (Qiagen) and the primers listed in Supplementary Table 1.

[0130] Supplementary Table 1.

[0131]

[0132] Thermal cycling conditions included reverse transcription at 45°C for 10 min, initial denaturation at 95°C for 5 min, followed by 40 cycles at 95°C for 5 s and 55°C for 30 s. All live virus experiments were performed in a biosafety level 2 laboratory.

[0133] 6.6.3 Flow cytometry analysis

[0134] Airway and nasal mucosal organoids were infected with HRV-C at 10,000 viral gene copies / cell or mock infected. At 24 and 48 h p.i., organoids were dissociated into single cells with 10 mM EDTA (Invitrogen) at 37°C for 30–60 min and fixed with 4% PFA for 30 min at room temperature. After permeabilization with 0.1% Triton X-100 for 5 min at 4°C, cells were incubated with primary antibodies, α-rhinovirus VP3 (Invitrogen, MA5-18249), α-ACCTUB (Abcam, ab179504), and α-CDHR3 (Abcam, ab121337), for 1 h, followed by incubation with secondary antibodies. Immunostained cells were resuspended in 2% FBS / PBS and analyzed using an Agilent NovoCyte Quanteon analyzer. FlowJo software was used for data processing.

[0135] 6.6.4 Scanning and Transmission Electron Microscopy

[0136] Viral and mock-infected airway organoids were fixed with 2.5% glutaraldehyde (GTA). HRV-C3 virions in the culture medium were concentrated by ultracentrifugation and fixed with 2.5% GTA. Sample processing was performed by the Electron Microscopy Unit of the University of Hong Kong. Images were acquired using a LEO 1530 FEG scanning electron microscope and a Philips CM100 transmission electron microscope.

[0137] 6.6.5 Immunofluorescence staining and confocal imaging

[0138] To identify virus-infected cells, we stained organoids and performed confocal imaging as described elsewhere. 21,22,26,29. Briefly, airway and nasal mucosal organoids were infected with HRV-C at 1000 viral gene copies / cell or mock infected and incubated for 24 hours. After fixation with 4% paraformaldehyde (PFA) for 1 hour at room temperature, the membrane inserts seeded with organoids were cut and removed from the Transwell and used for immunostaining. The fixed organoid monolayers were permeabilized in 0.1% Triton-X100 for 10 minutes at room temperature and blocked with 3% bovine serum albumin (BSA) for 1 hour. Subsequently, the organoids were incubated with primary antibodies, including α-rhinovirus VP3 (Invitrogen, MA5-18249), α-CDHR3 (Abcam, ab121337), and α-ACCTUB (Sigma-Aldrich, T7941; Abcam, ab179504), overnight at 4°C, and then incubated with secondary antibodies. The nuclei and actin filaments were counterstained with DAPI (ThermoFisher) and phalloidin-647 (Sigma-Aldrich), respectively. TM Organoids were mounted whole on glass slides using GSigma-Aldrich lass Antifade Mountant (Invitrogen), and confocal images were acquired using a Carl Zeiss LSM 980 confocal microscope.

[0139] 6.6.6 Organoid-based Immunofluorescence Assay (IFA)

[0140] The culture medium harvested from HRV-C3 infected nasal mucosal organoids was used to seed nasal mucosal organoids after 10-fold serial dilution. At 24 h.pi, the organoid monolayer was fixed and permeabilized with 0.1% Triton X-100 for 10 minutes and blocked with 3% BSA for 1 hour, then incubated with α-rhinovirus VP3 (Invitrogen, MA5-18249) and secondary antibodies. The cell nuclei were counterstained with DAPI (Thermo Fisher). ProLong was then used to stain the cells. TM Organoids were mounted whole on glass slides using Glass Antifade Mountant (Invitrogen). High-content confocal images were acquired using the IN Cell Analyzer 6500HS (GE Healthcare), a laser-based line-scanning high-content imaging system. Images were processed using IN Carta and Image J software.

[0141] 6.6.7 Statistical analysis

[0142] Statistical analysis was performed using GraphPad Prism 9.0. Two-tailed Student's t-test was used to determine statistical significance. The number of replicates is indicated in the figure legends. *P < 0.05, **P < 0.01, ***P < 0.001.

[0143] 7. Exemplary products, systems, and methods are set forth in the following items:

[0144] 1. A composition for culturing respiratory microorganisms, the composition comprising: (i) a respiratory organoid selected from the group consisting of an airway organoid and a nasal mucosal organoid; and (ii) respiratory microorganisms, wherein the respiratory microorganisms maintain continuous proliferation in the respiratory organoid.

[0145] 2. The composition of claim 1 , wherein the airway organoid is an airway organoid derived from lung tissue.

[0146] 3. The composition of any of the preceding items, wherein the respiratory organoid is a nasal mucosal organoid derived from nasal epithelial cells.

[0147] 4. The composition of any of the preceding items, wherein the respiratory microorganism is maintained for at least 3-6 passages and has genomic stability.

[0148] 5. The composition of any of the preceding items, further comprising an immunosuppressive compound.

[0149] 6. The composition of any of the preceding items, wherein the immunosuppressive compound interferes with the antimicrobial response in the respiratory organoid.

[0150] 7. The composition of any of the preceding items, wherein the immunosuppressive compound is a JAK1 / JAK2 inhibitor or a TBK1 / IKKε inhibitor.

[0151] 8. The composition of any of the preceding items, wherein the immunosuppressive compound is molotinib (CYT387).

[0152] 9. The composition of any of the preceding items, wherein the respiratory microorganism is a respiratory virus or a respiratory bacterium.

[0153] 10. The composition of any of the preceding items, wherein the respiratory virus is HRV-C, HRV-C3, HRV-C8, HRV-C11, HRV-C15, HRV-C45, influenza virus, adenovirus, human bocavirus, human coronavirus including SARS-CoV1 and SARS-CoV2, human metapneumovirus, human parainfluenza virus, human respiratory syncytial virus, or human rhinovirus.

[0154] 11. The composition of any of the preceding items, wherein the respiratory bacteria is Mycobacterium tuberculosis, Streptococcus pneumoniae, Mycoplasma pneumoniae, Haemophilus influenzae, Chlamydia pneumoniae, Chlamydia psittaci, Coxiella burnetii, Legionella pneumophila, Staphylococcus aureus or Klebsiella pneumoniae.

[0155] 12. The composition of any of the preceding items, wherein the airway organoid is a human airway organoid comprising human airway epithelial cells.

[0156] 13. The composition of any of the preceding items, wherein the airway organoid comprises human lung epithelial cells.

[0157] 14. The composition of any of the preceding items, wherein the respiratory organoid comprises human nasal epithelial cells.

[0158] 15. The composition of any of the preceding items, wherein the airway organoids are derived from human epithelial stem cells.

[0159] 16. The composition of any of the preceding items, wherein the airway organoid survives in culture for a period of at least 30 days.

[0160] 17. A method for culturing respiratory microorganisms, the method comprising: (i) preparing a respiratory organoid, wherein the respiratory organoid is selected from the group consisting of an airway organoid and a nasal mucosal organoid; and (ii) infecting the respiratory organoid with a microorganism, wherein the microorganism maintains proliferation in the respiratory organoid.

[0161] 18. The method of claim 17, further comprising the steps of isolating and culturing the respiratory microorganisms.

[0162] 19. The method of any of the preceding items, wherein the airway organoid is an airway organoid derived from lung tissue.

[0163] 20. The method of any of the preceding items, wherein the respiratory organoid is a nasal mucosal organoid derived from nasal epithelial cells.

[0164] 21. The method of any of the preceding items, wherein the airway organoid is prepared by (a) providing a fragment of airway tissue or nasal tissue; and (b) culturing the fragment of airway or nasal tissue in culture medium for a period of time sufficient to form the airway organoid.

[0165] 22. The method of any of the preceding items, wherein the culture medium further comprises an immunosuppressive compound.

[0166] 23. The method of any of the preceding items, wherein the immunosuppressive compound interferes with the antimicrobial response in the respiratory organoid.

[0167] 24. The method of any of the preceding items, wherein the immunosuppressive compound is a JAK1 / JAK2 inhibitor or a TBK1 / IKKε inhibitor.

[0168] 25. The method of any of the preceding items, wherein the immunosuppressive compound is molotinib (CYT387), amlexanob, BX795, MRT67307, ruxolitinib, or a combination thereof.

[0169] 26. The method of any of the preceding items, wherein the respiratory microorganism is maintained for at least 3-6 passages and has genomic stability.

[0170] 27. The method of any of the preceding items, wherein the respiratory microorganism is a respiratory virus or a respiratory bacterium.

[0171] 28. The method of any of the preceding items, wherein the respiratory virus is HRV-C, HRV-C3, HRV-C8, HRV-C11, HRV-C15, HRV-C45, influenza virus; adenovirus; human bocavirus; human coronaviruses including SARS-CoV1 and SARS-CoV2; human metapneumovirus; human parainfluenza virus; human respiratory syncytial virus; and / or human rhinovirus, or a combination thereof.

[0172] 29. The method of any of the preceding items, wherein the respiratory bacteria is Mycobacterium tuberculosis, Streptococcus pneumoniae, Mycoplasma pneumoniae, Haemophilus influenzae, Chlamydia pneumoniae; Chlamydia psittaci; Coxiella burnetii; Legionella pneumophila, Staphylococcus aureus; and / or Klebsiella pneumoniae.

[0173] 30. The method of any of the preceding items, wherein the airway organoid is a human airway organoid comprising human airway epithelial cells.

[0174] 31. The method of any of the preceding items, wherein the airway organoid survives in culture for a period of at least 30 days.

[0175] 32. A system for culturing respiratory microorganisms, the system comprising: (i) culturing lung tissue fragments in a culture medium comprising a JAK1 / JAK2 inhibitor or a TBK1 / IKKε inhibitor for a period of time sufficient for respiratory organoids to form; and (ii) maintaining continuously proliferating respiratory microorganisms in the respiratory organoids.

[0176] The foregoing description of the specific embodiments will fully reveal the overall nature of the present disclosure so that others can easily modify and / or adjust these specific embodiments for various applications without departing from the general concept of the present disclosure by applying the knowledge within the technical scope of the relevant art (including the contents of the documents cited and incorporated herein by reference) without excessive experimentation. Therefore, based on the teachings and guidance presented herein, such adjustments and modifications are intended to be within the meaning and scope of the equivalents of the disclosed embodiments. It should be understood that the words or terms herein are used for descriptive and non-limiting purposes, so that the terms or terms of this specification will be interpreted by those skilled in the art in view of the teachings and guidance presented herein, in conjunction with the knowledge of those skilled in the relevant art.

[0177] Although various embodiments of the present disclosure have been described above, it should be understood that they have been presented by way of example and not limitation. Various changes in form and detail may be made therein without departing from the spirit and scope of the present disclosure, as will be apparent to those skilled in the relevant art. Therefore, the present disclosure should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

[0178] All references cited herein are incorporated by reference in their entirety and for all purposes to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0179] References

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Claims

1. A composition for culturing respiratory microorganisms, the composition comprising: (i) a respiratory organoid selected from the group consisting of an airway organoid and a nasal mucosal organoid; and (ii) respiratory microorganisms, wherein the respiratory microorganisms maintain continuous proliferation in the respiratory organoid.

2. The composition of claim 1, wherein the airway organoid is an airway organoid derived from lung tissue.

3. The composition of claim 1, wherein the respiratory organoid is a nasal mucosal organoid derived from nasal epithelial cells.

4. The composition of claim 1, wherein the respiratory microorganism is maintained for at least 3-6 passages and has genomic stability.

5. The composition of claim 1, further comprising an immunosuppressive compound.

6. The composition of claim 5, wherein the immunosuppressive compound interferes with the antimicrobial response in the respiratory organoid.

7. The composition of claim 6, wherein the immunosuppressive compound is a JAK1 / JAK2 inhibitor or a TBK1 / IKKε inhibitor.

8. The composition of claim 6, wherein the immunosuppressive compound is molotinib (CYT387), amlexanob, BX795, MRT67307, ruxolitinib, or a combination thereof.

9. The composition of claim 1, wherein the respiratory microorganism is a respiratory virus or a respiratory bacteria.

10. The composition of claim 9, wherein the respiratory virus is HRV-C, HRV-C3, HRV-C8, HRV-C11, HRV-C15, HRV-C45, influenza virus, adenovirus, human bocavirus, human coronavirus including SARS-CoV1 and SARS-CoV2, human metapneumovirus, human parainfluenza virus, human respiratory syncytial virus, or human rhinovirus.

11. The composition of claim 9, wherein the respiratory bacteria is Mycobacterium tuberculosis, Streptococcus pneumoniae, Mycoplasma pneumoniae, Haemophilus influenzae, Chlamydia pneumoniae, Chlamydia psittaci, Coxiella burnetii, Legionella pneumophila, Staphylococcus aureus, or Klebsiella pneumoniae.

12. The composition of claim 1, wherein the airway organoid is a human airway organoid comprising human airway epithelial cells.

13. The composition of claim 1, wherein the airway organoid comprises human lung epithelial cells.

14. The composition of claim 1, wherein the airway organoid comprises human nasal epithelial cells.

15. The composition of claim 1, wherein the airway organoids are derived from human epithelial stem cells.

16. The composition of claim 1, wherein the airway organoid survives in culture for a period of at least 30 days.

17. A method for culturing respiratory microorganisms, the method comprising: (i) preparing a respiratory organoid, wherein the respiratory organoid is selected from the group consisting of an airway organoid and a nasal mucosa organoid; and (ii) infecting the airway organoid with a microorganism, wherein the microorganism maintains proliferation in the airway organoid.

18. The method of claim 17, further comprising the steps of isolating and culturing the respiratory microorganisms.

19. The method of claim 17, wherein the airway organoid is an airway organoid derived from lung tissue.

20. The method of claim 17, wherein the airway organoid is a nasal mucosal organoid derived from nasal epithelial cells.

21. The method of claim 17, wherein the airway organoid is prepared by (a) providing a fragment of airway tissue or nasal tissue; and (b) culturing the fragment of airway or nasal tissue in culture for a period of time sufficient to form the airway organoid.

22. The method of claim 21, wherein the culture medium further comprises an immunosuppressive compound.

23. The method of claim 22, wherein the immunosuppressive compound interferes with the antimicrobial response in the respiratory organoid.

24. The method of claim 22, wherein the immunosuppressive compound is a JAK1 / JAK2 inhibitor or a TBK1 / IKKε inhibitor.

25. The method of claim 22, wherein the immunosuppressive compound is molotinib (CYT387).

26. The method of claim 17, wherein the respiratory microorganism is maintained for at least 3-6 passages and has genomic stability.

27. The method of claim 17, wherein the respiratory microorganism is a respiratory virus or a respiratory bacteria.

28. The method of claim 27, wherein the respiratory virus is HRV-C, HRV-C3, HRV-C8, HRV-C11, HRV-C15, HRV-C45, influenza virus; adenovirus; human bocavirus; human coronaviruses including SARS-CoV1 and SARS-CoV2; human metapneumovirus; human parainfluenza virus; human respiratory syncytial virus; and / or human rhinovirus, or a combination thereof.

29. The method of claim 27, wherein the respiratory bacteria is Mycobacterium tuberculosis, Streptococcus pneumoniae, Mycoplasma pneumoniae, Haemophilus influenzae, Chlamydia pneumoniae; Chlamydia psittaci; Coxiella burnetii; Legionella pneumophila, Staphylococcus aureus; and / or Klebsiella pneumoniae.

30. The method of claim 17, wherein the airway organoid is a human airway organoid comprising human airway epithelial cells.

31. The method of claim 17, wherein the airway organoid survives in culture for a period of at least 30 days.

32. A system for culturing respiratory microorganisms, the system comprising: (i) culturing lung tissue fragments in a culture medium comprising a JAK1 / JAK2 inhibitor or a TBK1 / IKKε inhibitor for a period of time sufficient for respiratory organoids to form; and (ii) maintaining continuously proliferating respiratory microorganisms in the respiratory organoids.

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