Method for producing respiratory organoids

Abstract

A method for producing respiratory organoids comprises a step for culturing respiratory epithelial cells on an expansion culture medium that contains fibroblast growth factor (FGF), and a step for culturing the resulting culture on a differentiation culture medium that contains FGF. The method for producing respiratory organoids can produce respiratory organoids that not only can reproduce the life cycle of the pathogens of respiratory infections, but that can also be used to screen for prophylactic agents or therapeutic agents for respiratory infections.

Description

[Technical Field]

[0001] The present invention relates to a method for producing respiratory organoids. More specifically, the present invention relates to a method for producing respiratory organoids, respiratory organoids, a method for screening preventive or therapeutic agents for respiratory infections, a regenerative agent for damaged respiratory epithelial cells, and a culture medium for regenerating damaged respiratory epithelial cells. This application claims priority to U.S. Patent Application No. 63 / 029,567, provisionally filed in the United States on May 25, 2020, and U.S. Patent Application No. 63 / 146,720, provisionally filed in the United States on February 8, 2021, the contents of which are incorporated herein by reference. [Background technology]

[0002] Severe respiratory dysfunction is frequently observed in patients with novel coronavirus disease (COVID-19), not only during viral infection but also after the virus has been eliminated. While active efforts are being made both domestically and internationally to develop antiviral drugs for the treatment of COVID-19, the development of drugs capable of treating respiratory dysfunction after the virus has been eliminated has not progressed sufficiently. Although attempts have been made to treat the disease with anti-inflammatory drugs, respiratory dysfunction can remain as a residual effect even after the inflammation has subsided. Therefore, the development of new drugs capable of regenerating airway tissue damaged by bronchopneumonia and other conditions is essential.

[0003] In order to develop therapeutic drugs for COVID-19, it is essential to develop not only model animals but also excellent in vitro evaluation systems. To date, organoid technology, which can reproduce some organ functions in vitro, has been used to reproduce the life cycle of the novel coronavirus (SARS-CoV-2) and analyze organ responses to viral infection (see, for example, Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Takayama, K., In Vitro and Animal Models for SARS-CoV-2 research, Trends Pharmacol Sci., 41 (8), 513-517, 2020. Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention aims to provide respiratory organoid technology that not only can reproduce the life cycle of pathogens that cause respiratory infections, but also can screen preventive or therapeutic agents for respiratory infections. [Means for solving the problem]

[0006] The present invention includes the following aspects. [1] A method for producing respiratory organoids, comprising: (1-1) culturing respiratory epithelial cells in an expansion culture medium containing fibroblast growth factor (FGF); and (1-2) culturing the resulting culture in a differentiation medium containing FGF. [2] Before or after the step (1-2), the obtained culture is dissociated to obtain a gas-liquid The manufacturing method described in [1], further comprising a step of interface culture. [3] The method of [1] or [2], wherein the respiratory epithelial cells are one or more types of cells selected from the group consisting of bronchial epithelial cells, small airway epithelial cells, and alveolar epithelial cells. [4] The method according to any one of [1] to [3], wherein the respiratory epithelial cells are derived from frozen cells. [5] The method according to any one of [1] to [3], wherein the respiratory epithelial cells are cells obtained by inducing differentiation of somatic stem cells or pluripotent stem cells. [6] The production method according to any one of [1] to [5], wherein in the step (1-1), the respiratory epithelial cells are embedded in a gel and then cultured in the expansion culture medium. [7] The manufacturing method described in any of [1] to [6], wherein the expansion culture medium further contains one or more substances selected from the group consisting of a BMP signal inhibitor, a Wnt signal activator, and a p38 inhibitor. [8] The method of producing according to [7], wherein the BMP signal inhibitor is Noggin. [9] The method of producing according to [7] or [8], wherein the Wnt signal activator is R-spondin.

[10] The method of producing according to [9], wherein the R-spondin is R-spondin 1.

[11] The method according to any one of [7] to

[10] , wherein the p38 inhibitor is SB202190.

[12] The method according to any one of [1] to

[11] , wherein the differentiation medium further contains a TGF-β inhibitor.

[13] The method of producing according to

[12] , wherein the TGF-β inhibitor is A83-01.

[14] The method according to any one of [1] to

[13] , wherein the FGF is one or more substances selected from the group consisting of FGF2, FGF7, and FGF10.

[15] The method according to any one of [1] to

[14] , wherein the FGF is FGF2, FGF7, or FGF10.

[16] An artificial respiratory organoid comprising one or more cells from the following cell groups: basal stem cells, ciliated cells, goblet cells, club cells, pulmonary neuroendocrine cells, type I pneumocytes, and type II pneumocytes.

[17] The respiratory organoid described in

[16] , comprising: (i) any one or more cells from a group of cells consisting of basal stem cells, ciliated cells, goblet cells, club cells and pulmonary neuroendocrine cells; (ii) any one or more cells from a group of cells consisting of type I alveolar epithelial cells and type II alveolar epithelial cells; or (iii) the above (i) and (ii).

[18] The respiratory organoid according to

[16] or

[17] , wherein the apical surface is exposed.

[19] A respiratory organoid according to any one of

[16] to

[18] , produced by the production method according to any one of [1] to

[15] .

[20] A respiratory organoid according to any one of

[16] to

[19] , which exhibits an adult-type phenotype.

[21] Respiratory organoids described in

[20] that exhibit (i) angiotensin-converting enzyme 2 (ACE2)-positive and / or (ii) type II transmembrane serine protease (TMPRSS2)-positive phenotypes.

[22] A method for evaluating the infectivity and / or proliferation ability of a pathogen of a respiratory infection, comprising: (2-1) a step of infecting a respiratory organoid described in any one of

[16] to

[21] with the pathogen; and (2-2) a step of detecting the proliferation of the pathogen, wherein the pathogen is one or more pathogens selected from the group consisting of viruses, Hosozono, and Magozono.

[23] The method according to

[22] , wherein the step (2-2) is carried out by at least one step selected from the group consisting of (i) a step of detecting amplification of the pathogen's genome, (ii) a step of detecting a protein derived from the pathogen, (iii) a step of detecting cell pycnosis, and (iv) a step of detecting release of lactate dehydrogenase (LDH).

[24] The method according to

[22] or

[23] , wherein the pathogen is a virus.

[25] The method described in

[24] , wherein the virus is any one virus selected from the group consisting of influenza virus, coronavirus, and respiratory syncytial virus (RSV).

[26] The method described in

[25] , wherein the coronavirus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[27] A method for screening therapeutic agents for respiratory infections, comprising: (3-1) contacting a respiratory organoid described in any of

[16] to

[21] infected with a pathogen of the respiratory infection with a candidate substance; (3-2) detecting the proliferation of the pathogen in the respiratory organoid after contact with the candidate substance; and (3-3) selecting a candidate substance whose proliferation is inhibited compared to a negative control, wherein the pathogen is one or more pathogens selected from the group consisting of viruses, Hosozono, and Mazono.

[28] The method according to

[27] , wherein the step (3-2) is carried out by at least one step selected from the group consisting of (i) a step of detecting amplification of the pathogen's genome, (ii) a step of detecting a protein derived from the pathogen, (iii) a step of detecting cell pycnosis, and (iv) a step of detecting release of lactate dehydrogenase (LDH).

[29] A method for screening therapeutic agents for respiratory infections, comprising: (4-1) contacting a respiratory organoid described in any of

[16] to

[21] , which has been damaged by infection with a pathogen of the respiratory infection, with a candidate substance; (4-2) evaluating the recovery of the respiratory organoid after contact with the candidate substance; and (4-3) selecting a candidate substance that enhances the recovery compared to a negative control, wherein the pathogen is one or more pathogens selected from the group consisting of viruses, Hosozono, and Mazono.

[30] A method for screening preventive agents for respiratory infections, comprising: (5-1) infecting a respiratory organoid described in any one of

[16] to

[21] that has been contacted with a candidate substance with a pathogen of the respiratory infection; (5-2) detecting the proliferation of the pathogen in the infected respiratory organoid; and (5-3) selecting a candidate substance whose proliferation is inhibited compared to a negative control, wherein the pathogen is one or more pathogens selected from the group consisting of viruses, Hosozono, and Mazono.

[31] The method according to

[30] , wherein the step (5-2) is carried out by at least one step selected from the group consisting of (i) a step of detecting amplification of the pathogen genome, (ii) a step of detecting a protein derived from the pathogen, (iii) a step of detecting cell pycnosis, and (iv) a step of detecting release of lactate dehydrogenase (LDH).

[32] The method according to

[30] or

[31] , wherein the pathogen is a virus.

[33] The method according to

[32] , wherein the virus is any one virus selected from the group consisting of influenza virus, coronavirus, and RSV.

[34] The method described in

[33] , wherein the coronavirus is SARS-CoV-2.

[35] A kit for use in the method described in any one of

[22] to

[34] , comprising the respiratory organoid described in any one of

[16] to

[21] .

[36] A regenerative agent for damaged respiratory epithelial cells, containing FGF10 as its active ingredient.

[37] A medium containing FGF10 for the regeneration of damaged respiratory epithelial cells.

[38] The medium for regenerating damaged respiratory epithelial cells according to

[37] , further comprising a ROCK inhibitor and a TGF-β inhibitor.

[0007] The present invention can also be said to include the following aspects. [P1] A method for producing respiratory organoids, comprising: (1-1) culturing respiratory epithelial cells in an expansion culture medium containing fibroblast growth factor (FGF); and (1-2) culturing the resulting culture in a differentiation medium containing FGF. [P2] The method according to [P1], wherein the respiratory epithelial cells are one or more types of cells selected from the group consisting of bronchial epithelial cells, small airway epithelial cells, and alveolar epithelial cells. [P3] The method according to [P1] or [P2], wherein the respiratory epithelial cells are derived from frozen cells. [P4] The method according to [P1] or [P2], wherein the respiratory epithelial cells are cells induced to differentiate from somatic stem cells or pluripotent stem cells. [P5] The method according to any one of [P1] to [P4], wherein the respiratory epithelial cells are embedded in Matrigel. [P6] A method according to any one of [P1] to [P5], wherein in step (1-1), the expansion culture medium further contains one or more substances selected from the group consisting of a BMP signal inhibitor and an R-spondin. [P7] The method according to any one of claims 1 to 6, wherein the FGF is one or more substances selected from the group consisting of FGF2, FGF7, and FGF10. [P8] The method according to any one of [P1] to [P7], wherein the FGF is FGF2, FGF7, or FGF10. [P9] The method according to any one of [P6] to [P8], wherein the BMP signal inhibitor is Noggin. [P10] The method according to any one of [P6] to [P9], wherein the R-spondin is R-spondin 1. [P11] An artificial respiratory organoid containing one or more cells from the following cell groups: basal cells (basal stem cells), ciliated cells, goblet cells, club cells, pulmonary neuroendocrine cells, type I pneumocytes, and type II pneumocytes. [P12] A respiratory organoid according to [P11], comprising (i) any one or more cells from a group of cells consisting of basal stem cells, ciliated cells, goblet cells, club cells, and pulmonary neuroendocrine cells, (ii) any one or more cells from a group of cells consisting of type I alveolar epithelial cells and type II alveolar epithelial cells, or (iii) the above (i) and (ii). [P13] A respiratory organoid according to [P11] or [P12], produced by a method according to any one of [P1] to [P10]. [P14] A respiratory organoid according to any one of [P11] to [P13], which exhibits an adult-type phenotype. [P15] The respiratory organoid described in [P14], wherein the phenotype is at least one phenotype selected from (i) high expression of angiotensin-converting enzyme 2 (ACE2) in respiratory epithelial cells of the respiratory organoid, and (ii) high expression of type II transmembrane serine protease (TMPRSS2) in respiratory epithelial cells of the respiratory organoid. [P16] A method for evaluating the infectivity and / or proliferation ability of a pathogen of a respiratory infection, comprising: (2-1) a step of infecting a respiratory organoid described in any of [P11] to [P15] with the pathogen; and (2-2) a step of detecting the proliferation of the pathogen, wherein the pathogen is one or more pathogens selected from viruses, bacteria, and fungi. [P17] The method according to [P16], wherein the step (2-2) is carried out by at least one step selected from (i) detecting amplification of the pathogen genome, (ii) detecting a protein derived from the pathogen, (iii) detecting cell pycnosis, and (iv) detecting release of lactate dehydrogenase (LDH). [P18] The method according to [P16] or [P17], wherein the pathogen is a virus. [P19] The method according to [P18], wherein the pathogen is any one of influenza virus, coronavirus, and respiratory syncytial virus (RSV). [P20] The method described in [P19], wherein the coronavirus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). [P21] A method for screening therapeutic agents for respiratory infections, comprising the steps of: (3-1) contacting a respiratory organoid described in any of [P11] to [P15] that has been infected with a pathogen of the respiratory infection with a candidate substance; (3-2) detecting the proliferation of the pathogen in the respiratory organoid after contact with the candidate substance; and (3-3) selecting a candidate substance whose proliferation is inhibited compared to a negative control, wherein the pathogen is one or more pathogens selected from viruses, bacteria, and fungi. [P22] The method according to [P21], wherein the step (3-2) is carried out by at least one step selected from (i) detecting amplification of the pathogen genome, (ii) detecting a protein derived from the pathogen, (iii) detecting cell pycnosis, and (iv) detecting release of lactate dehydrogenase (LDH). [P23] A method for screening preventive agents for respiratory infections, comprising: (4-1) infecting a respiratory organoid described in any one of [P11] to [P15] that has been contacted with a candidate substance with a pathogen of the respiratory infection; (4-2) detecting the proliferation of the pathogen in the infected respiratory organoid; and (4-3) selecting a candidate substance whose proliferation is inhibited compared to a negative control, wherein the pathogen is one or more pathogens selected from viruses, bacteria, and fungi. [P24] The method according to [P23], wherein the step (4-2) is carried out by at least one step selected from (i) detecting amplification of the pathogen genome, (ii) detecting a protein derived from the pathogen, (iii) detecting cell pycnosis, and (iv) detecting release of lactate dehydrogenase (LDH). [P25] The method according to any one of [P21] to [P24], wherein the pathogen is a virus. [P26] The method according to [P25], wherein the pathogen is any one of influenza virus, coronavirus, and RSV. [P27] The method described in [P25], wherein the coronavirus is SARS-CoV-2. [P28] A kit for use in the method described in any one of [P16] to [P27], comprising the respiratory organoid described in any one of [P11] to [P15]. [Effects of the Invention]

[0008] According to the present invention, a respiratory organoid technology can be provided that not only can reproduce the life cycle of pathogens of respiratory infections, but also can screen preventive or therapeutic agents for respiratory infections. [Brief explanation of the drawings]

[0009] [Figure 1] Figure 1 is a photograph showing the results of fluorescent immunostaining of acetylated α-tubulin and KRT5 in normal human bronchial epithelial cells (hereinafter sometimes referred to as "NHBE") in Experimental Example 1. Nuclei were stained with DAPI (4',6-diamidino-2-phenylindole). [Figure 2] Figure 2 is a graph showing the results of quantitative real-time PCR measurement of the expression levels of ACE2 and TMPRSS2 in a three-dimensional culture model of human bronchial organoids (hereinafter sometimes referred to as "hBOs") in Experimental Example 1. [Figure 3] FIG. 3 is a set of micrographs showing a phase-contrast image and a hematoxylin-eosin stained image of the hBO three-dimensional culture model taken in Experimental Example 1. [Figure 4] FIG. 4 is a graph showing the results of quantitative real-time PCR measurement of the expression levels of ACE2 and TMPRSS2 in the hBO three-dimensional culture model, NHBE, and the human lung cancer cell line A549 in Experimental Example 1. [Figure 5] FIG. 5 is a photograph showing the results of detecting the expression of ACE2 and TMPRSS2 in the hBO three-dimensional culture model by immunochemical staining in Experimental Example 1. [Figure 6]FIG. 6 is a set of photographs showing the results of detecting the expression of ACE2 and KRT5 in a hBO three-dimensional culture model by fluorescent immunostaining in Experimental Example 1. [Figure 7] FIG. 7 is a graph showing the measurement results of the expression levels of NGFR and PROM1, which are marker genes for basal stem cells, in the hBO three-dimensional culture model, NHBE, and A549 in Experimental Example 1. [Figure 8] FIG. 8 is a graph showing the measurement results of the expression levels of TUBA1A and MCIDAS, which are marker genes for ciliated cells, in the hBO three-dimensional culture model, NHBE, and A549 in Experimental Example 1. [Figure 9] FIG. 9 is a graph showing the measurement results of expression levels of MUC20 and MUC5B, which are marker genes for goblet cells, in the hBO three-dimensional culture model, NHBE, and A549 in Experimental Example 1. [Figure 10] FIG. 10 is a graph showing the measurement results of the expression levels of SCGB1A1 and KLF5, which are marker genes for club cells, in the hBO three-dimensional culture model, NHBE, and A549 in Experimental Example 1. [Figure 11] Figure 11 is a micrograph showing the results of immunochemical staining of the hBO three-dimensional culture model in Experimental Example 1 to detect the expression of KRT5 (a marker for basal stem cells), acetylated α-tubulin (a marker for ciliated cells), MUC5AC (a marker for goblet cells), and CC10 (a marker for club cells). [Figure 12] FIG. 12 is a transmission electron microscope photograph of an ultrathin section of the hBO three-dimensional culture model taken in Experimental Example 1. [Figure 13] FIG. 13 is a transmission electron microscope photograph of an ultrathin section of the hBO three-dimensional culture model taken in Experimental Example 1. [Figure 14] FIG. 14 is a graph showing the results of measuring the expression levels of bronchial markers KRT5, MUC20, MCIDAS, NGFR, MUC5B, and SCGB1A1 in an hBO three-dimensional culture model by quantitative real-time PCR in Experimental Example 2. [Figure 15] Figure 15 is a graph showing the results of quantitative real-time PCR measurement of the expression levels of TMPRSS2, MCIDAS, MUC20, MUC5B, and SCGB1A1 in NHBE, expanded hBO three-dimensional culture models, and differentiated hBO three-dimensional culture models in Experimental Example 3. [Figure 16] Figure 16 is a heat map of bronchial markers created based on the results of RNA-seq analysis of the hBO three-dimensional culture model in Experimental Example 4. [Figure 17] FIG. 17 is a schematic diagram showing the experimental schedule for Experimental Example 5. [Figure 18] Figure 18 is a photograph showing the results of immunohistochemical staining of a SARS-CoV-2-infected hBO three-dimensional culture model in Experimental Example 5, in which the S protein (SP) of SARS-CoV-2 was detected. [Figure 19] Figure 19 is a photograph showing the results of detecting SP and KRT5 by fluorescent immunostaining of a hBO three-dimensional culture model infected with SARS-CoV-2 in Experimental Example 5. [Figure 20] Figure 20 is a photograph showing the results of detecting SP and CC10 by fluorescent immunostaining of a hBO three-dimensional culture model infected with SARS-CoV-2 in Experimental Example 5. [Figure 21] Figure 21 is a graph showing the results of Experimental Example 5, in which a hBO three-dimensional culture model was infected with SARS-CoV-2 in the presence or absence of Camostat, and the virus titer was measured by TCID50 assay. [Figure 22] Figure 22 is a graph showing the results of Experimental Example 5, in which a hBO three-dimensional culture model was infected with SARS-CoV-2 in the presence or absence of camostat, and a lactate dehydrogenase (LDH) assay was performed 1, 2, 3, 4, and 5 days after viral infection. [Figure 23]Figure 23 is a graph showing the results of parametric gene set enrichment analysis (PGSEA) of the GO biological gene set based on the results of RNA-seq in the non-virus-infected hBO 3D culture model (control), the virus-infected hBO 3D culture model (SARS-CoV-2), and the virus-infected hBO 3D culture model in the presence of Camostat (SARS-CoV+Camostat) in Experimental Example 5. [Figure 24] Figure 24 is a graph showing the results of quantitative real-time PCR measurement of the expression levels of interferon (IFN)-α, IFN-β, ISG56, and ISG15 genes in an hBO three-dimensional culture model not infected with a virus (control), an hBO three-dimensional culture model infected with a virus (SARS-CoV-2), and an hBO three-dimensional culture model infected with a virus in the presence of Camostat (SARS-CoV+Camostat) in Experimental Example 5. [Figure 25] Figure 25 shows a heat map of genes related to type I IFN signaling, created based on the results of RNA-seq in the non-virus-infected hBO 3D culture model (control) and the virus-infected hBO 3D culture model (SARS-CoV-2) in Experimental Example 5. [Figure 26] FIG. 26 is a schematic diagram showing the experimental schedule for Experimental Example 6. [Figure 27] Figure 27 is a photograph showing the results of fluorescent immunostaining of a human respiratory organoid-derived air-liquid interface cell culture model (hereinafter sometimes referred to as "hBO-ALI") that was not infected with a virus in Experimental Example 6, detecting acetylated α-tubulin and KRT5. [Figure 28] FIG. 28 is a photograph showing the results of detecting acetylated α-tubulin and ACE2 by fluorescent immunostaining of non-virus-infected hBO-ALI in Experimental Example 6. [Figure 29]Figure 29 is a graph showing the results of quantitative real-time PCR measurement of the expression levels of the ACE2, TMPRSS2, FURIN, NGFR, MCIDAS, MUC5B, and SCGB1A genes in the hBO three-dimensional culture model, hBO-ALI, and bronchial basal stem cells (basal stem cells) in Experimental Example 6. [Figure 30] Figure 30 is a graph showing the results of measuring infectious virus in the culture supernatant of the hBO three-dimensional culture model and hBO-ALI infected with SARS-CoV-2 using a TCID50 assay in Experimental Example 6. [Figure 31] Figure 31 is a photograph showing the results of fluorescent immunostaining of hBO-ALI two days after infection with SARS-CoV-2 in Experimental Example 6, in which SARS-CoV-2 SP, acetylated α-tubulin, and KRT5 were detected. [Figure 32] Figure 32 is a photograph showing the results of Experimental Example 7, in which hBO-ALI were infected with SARS-CoV-2 and acetylated α-tubulin, SARS-CoV-2 SP, and KRT5 were detected by fluorescent immunostaining 7 days after infection. [Figure 33] Figure 33 is a photograph showing the results of Experimental Example 7, in which hBO-ALI was infected with SARS-CoV-2 and acetylated α-tubulin and KRT5 were detected by fluorescent immunostaining 15 days after infection. [Figure 34] Figure 34 is a graph showing the results of Experimental Example 7, in which hBO-ALI were infected with SARS-CoV-2 and cultured for two days in a differentiation medium containing or not containing FGF2, FGF7, and FGF10, and then the infectious virus contained in the culture supernatant was measured by TCID50 assay. [Figure 35] FIG. 35 is a photograph showing the results of detecting acetylated α-tubulin and KRT5 by fluorescent immunostaining 15 days after virus infection in Experimental Example 7. [Figure 36]Figure 36 is a graph showing the results of Experimental Example 8, in which hBO-ALIs prepared from NHBEs derived from different donors were infected with SARS-CoV-2, and the infectious virus contained in the culture supernatant was measured by TCID50 assay two days after infection. DETAILED DESCRIPTION OF THE INVENTION

[0010] [Method for producing respiratory organoids] In one embodiment, the present invention provides a method for producing respiratory organoids, comprising the steps of: (1-1) culturing respiratory epithelial cells in an expansion culture medium containing fibroblast growth factor (FGF); and (1-2) culturing the resulting culture in a differentiation medium containing FGF.

[0011] As described below in the Examples, the manufacturing method of this embodiment not only allows for the reproduction of the life cycle of pathogens of respiratory infections, but also allows for the production of respiratory organoids (hereinafter sometimes referred to as "BOs"; human BOs may also be referred to as "hBOs") that can be used to screen for preventive or therapeutic agents for respiratory infections.

[0012] The respiratory epithelial cells cultured in step (1-1) can be one or more types of cells selected from the group consisting of bronchial epithelial cells, small airway epithelial cells, and alveolar epithelial cells. The respiratory epithelial cells may be cells collected from the living body of a human or non-human animal, or may be cryopreserved cells. Alternatively, the respiratory epithelial cells may be cells obtained by inducing differentiation of somatic stem cells or pluripotent stem cells derived from a human or non-human animal. Examples of pluripotent stem cells include embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). As used herein, non-human animals are not particularly limited and include mice, rats, rabbits, pigs, sheep, goats, cows, monkeys, etc.

[0013] First, in step (1-1), respiratory epithelial cells are cultured in an expansion culture medium containing fibroblast growth factor (FGF). The culture method may be plate culture, three-dimensional culture, or air-liquid interface culture. Organoids may also be cultured without extracellular matrix components. This method makes it possible to produce organoids with their apical ends exposed (apical-out 3D organoids). This results in the formation of respiratory organoids. In step (1-1), respiratory epithelial cells are preferably embedded in a gel and then cultured in an expansion culture medium. This facilitates the formation of respiratory organoids.

[0014] Examples of gels include gels made from gelled extracellular matrix components and gels made from components other than extracellular matrix components. Examples of extracellular matrix components include components contained in basement membranes and glycoproteins present in intercellular spaces. Examples of components contained in basement membranes include type IV collagen, laminin, heparan sulfate proteoglycans, and entactin. Examples of glycoproteins present in intercellular spaces include collagen, laminin, entactin, fibronectin, fibrinogen, heparin sulfate, and the like. Examples of gels made from components other than extracellular matrix components include gelled carboxymethylcellulose and calcium alginate gel. Among these, Matrigel (Corning) is preferably used as a gel.

[0015] For example, cells may be embedded in Matrigel to form a dome structure on the surface of a cell culture dish, and an expansion culture medium may be added thereto for culture.

[0016] In addition to FGF, the expansion culture medium preferably further contains one or more substances selected from the group consisting of a BMP signal inhibitor, a Wnt signal activator, and a p38 inhibitor.

[0017] The FGF contained in the expansion culture medium is preferably one or more substances selected from the group consisting of FGF2, FGF7, and FGF10, and more preferably FGF2, FGF7, and FGF10. NCBI accession numbers for human FGF2 protein are NP_001348594.1, NP_001997.5, etc. NCBI accession numbers for human FGF7 protein are NP_002000.1, etc. NCBI accession numbers for human FGF10 protein are NP_004456.1, etc. The concentration of FGF contained in the expansion culture medium may be approximately 1 ng / mL to 1,000 ng / mL.

[0018] Examples of BMP signal inhibitors include Noggin, Chordin, LDN-193189 (CAS No.: 1062368-62-0), and DMH-1 (CAS No.: 1206711-16-1), with Noggin being preferred. The concentration of the BMP signal inhibitor contained in the expansion culture medium may be approximately 1 ng / mL to 1,000 ng / mL.

[0019] Examples of Wnt signal activators include R-spondin and CHIR99021 (CAS number: 252917-06-9), with R-spondin being preferred. Examples of R-spondin include R-spondin 1, R-spondin 2, and R-spondin 3, with R-spondin 1 being preferred. The concentration of the Wnt signal activator contained in the expansion culture medium may be approximately 1 ng / mL to 1,000 ng / mL.

[0020] Examples of p38 inhibitors include SB202190 (CAS No.: 152121-30-7), doramapimod (CAS No.: 285983-48-4), SB203580 (CAS No.: 152121-47-6), and FR167653 (CAS No.: 158876-66-5), with SB202190 being preferred. The concentration of the p38 inhibitor contained in the expansion culture medium may be approximately 10 μM to 1,000 μM.

[0021] The respiratory organoids obtained by culturing in expansion culture medium can be passaged. Passage can be carried out by dissociating the respiratory organoids once and culturing them again in expansion culture medium. Dissociation of the respiratory organoids can be carried out by mechanical shearing treatment and / or enzyme treatment. Even when passaged, it is preferable to embed the dissociated respiratory organoids in gel and then culture them in expansion culture medium.

[0022] Subsequently, in step (1-2), the culture (respiratory organoid) obtained in step (1-1) is cultured in a differentiation medium containing FGF.As will be described later in the examples, by culturing respiratory organoids in a differentiation medium, the expression level of bronchial markers in respiratory organoids can be increased and matured.Bronchial markers include TMPRSS2, MCIDAS, MUC20, MUC5B, SCGB1A1, etc.

[0023] The differentiation medium preferably further contains a TGF-β inhibitor in addition to FGF.

[0024] The FGF contained in the differentiation medium is preferably one or more substances selected from the group consisting of FGF2, FGF7, and FGF10, and more preferably FGF2, FGF7, and FGF10. The concentration of FGF contained in the differentiation culture medium may be about 1 ng / mL to 1,000 ng / mL.

[0025] Examples of TGF-β inhibitors include A83-01 (CAS No.: 909910-43-6), SB525334 (CAS No.: 356559-20-1), SB431542 (CAS No.: 301836-41-9), and LY2109761 (CAS No.: 700874-71-1), with A83-01 being preferred. The concentration of the TGF-β inhibitor contained in the expansion culture medium may be approximately 0.1 μM to 100 μM.

[0026] As described later in the Examples, respiratory organoids produced by the production method including steps (1-1) and (1-2) contain at least basal stem cells, ciliated cells, goblet cells, and club cells, just like living organisms. Respiratory organoids are spherical with a diameter of approximately 100 to 200 μm, and the outer edge of the organoid contains basal stem cells, while the lumen of the organoid contains ciliated cells.

[0027] Furthermore, as described later in the Examples, the inventors have demonstrated that the infection and replication efficiency of the novel coronavirus (SARS-CoV-2) is low in basal stem cells of respiratory organoids, but high in ciliated cells. Therefore, when respiratory organoids containing basal stem cells on the periphery and ciliated cells in the lumen are infected with SARS-CoV-2, SARS-CoV-2 infects the basal stem cells, resulting in low infection and replication efficiency.

[0028] In the method for producing respiratory organoids of this embodiment, it is preferable to further include a step of dissociating the obtained culture (respiratory organoids) and culturing them at an air-liquid interface before or after the above step (1-2).

[0029] By dissociating respiratory organoids and culturing them at the air-liquid interface, a respiratory organoid-derived air-liquid interface cell culture model (BO-ALI) can be obtained. As described in the Examples section, the apical surface where ciliated cells reside is exposed in the BO-ALI. As a result, SARS-CoV-2 infection and replication efficiency can be significantly improved compared to the BO 3D culture model.

[0030] [Respiratory organoids] In one embodiment, the present invention provides an artificial respiratory organoid, comprising any one or more cells from the cell group consisting of basal stem cells, ciliated cells, goblet cells, club cells, pulmonary neuroendocrine cells, type I pneumocytes and type II pneumocytes.The respiratory organoid of this embodiment can be produced by the above-mentioned production method.

[0031] The respiratory organoid preferably has an exposed apical surface. The respiratory organoid having an exposed apical surface where ciliated cells are present can be obtained by dissociating the respiratory organoid and culturing it on a plate, preferably at an air-liquid interface.

[0032] The respiratory organoids of this embodiment preferably exhibit (i) angiotensin-converting enzyme 2 (ACE2)-positive and / or (ii) type II transmembrane serine protease (TMPRSS2)-positive phenotypes.

[0033] Here, being ACE2 positive means that the expression level of the ACE2 gene or ACE2 protein is significantly increased compared to the respiratory epithelial cells used to produce the respiratory organoids.

[0034] Furthermore, being TMPRSS2 positive means that the expression level of the TMPRSS2 gene or TMPRSS2 protein is significantly increased compared to the respiratory epithelial cells used to produce the respiratory organoids.

[0035] ACE is a receptor for SARS-CoV-2, and TMPRSS2 is a protease that cleaves and activates the S protein of SARS-CoV-2. Therefore, respiratory organoids expressing either ACE2 or TMPRSS2, or preferably both, are useful as an in vitro model that can recapitulate the SARS-CoV-2 life cycle and efficiently infect and replicate SARS-CoV-2.

[0036] [Method for screening preventive or therapeutic agents for respiratory infections] In one embodiment, the present invention provides a method for screening preventive or therapeutic agents for respiratory infections, comprising the steps of: (3-1) contacting any of the above-mentioned respiratory organoids infected with a pathogen of the respiratory infection with a candidate substance; (3-2) detecting proliferation of the pathogen or damage caused by infection with the pathogen in the respiratory organoids after contact with the candidate substance; and (3-3) selecting a candidate substance that inhibits the proliferation or damage compared to a negative control, wherein the pathogen is one or more pathogens selected from the group consisting of viruses, Hosozono, and Mazono.

[0037] First, in step (3-1), respiratory organoids infected with a pathogen of a respiratory infection are contacted with a candidate substance.

[0038] Here, examples of respiratory organoids include those described above, and specifically, BO or BO-ALI are preferably used.

[0039] The pathogen may be one or more pathogens selected from the group consisting of viruses, Hosozono, and Mazono. Depending on the purpose, a single pathogen may be used for infection, or two or more pathogens may be used in combination. The pathogen may be a virus. Examples of viruses include influenza virus, coronavirus, respiratory syncytial virus (RSV), etc. Examples of coronaviruses include severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0040] Candidate substances are not particularly limited, and examples thereof include natural compound libraries, synthetic compound libraries, existing drug libraries, metabolite libraries, and the like.

[0041] Next, in step (3-2), pathogen proliferation or damage caused by infection with the pathogen is detected in the respiratory organoids after contact with the candidate substance.

[0042] The method for detecting the proliferation of pathogens is not particularly limited, and examples thereof include a method for detecting the amplification of the genome of the pathogen, a method for detecting proteins derived from the pathogen, etc. Furthermore, the method for detecting the damage to respiratory organoids caused by infection with a pathogen is not particularly limited, and examples thereof include a method for detecting the pycnosis of cells constituting respiratory organoids, a method for detecting the release of lactate dehydrogenase (LDH) from respiratory organoids, etc. These methods may be carried out either alone or in combination of two or more.

[0043] A candidate substance that inhibits the proliferation or damage compared to a negative control can be said to be a preventive or therapeutic agent for respiratory tract infections. Here, the negative control can be respiratory organoids that have not been contacted with the candidate substance.

[0044] In step (3-1), the pathogen, respiratory organoids, and candidate substance may be contacted in any order. For example, the pathogen may be contacted with respiratory organoids, and then the candidate substance may be contacted after infection. In this case, the candidate substance that suppresses the growth of the pathogen, or the candidate substance that suppresses the damage caused by the infection of the pathogen (recovers the damage caused by the infection of the pathogen) can be said to be a therapeutic agent for respiratory infection.

[0045] Alternatively, in step (3-1), the respiratory organoids may be contacted with a candidate substance and then contacted with a pathogen. In this case, a candidate substance that suppresses the growth of the pathogen or that suppresses the damage caused by infection with the pathogen (prevents the damage caused by infection with the pathogen) can be said to be a preventive agent for respiratory infections.

[0046] As described later in the Examples section, the inventors demonstrated that after SARS-CoV-2 infection of BO-ALI, ciliated cells die, but basal stem cells remain. Because basal stem cells can differentiate into other epithelial cells that make up the airway, elucidating the mechanisms of basal stem cell replication and differentiation and freely controlling basal stem cells may enable airway tissue regeneration targeting basal stem cells. If such efforts lead to the development of drugs with different modes of action than antiviral or anti-inflammatory drugs, it is expected that the number of treatment options for COVID-19 will increase.

[0047] [Regenerator of damaged respiratory epithelial cells] In one embodiment, the present invention provides a regenerating agent for damaged respiratory epithelial cells, which contains FGF10 as an active ingredient. As described later in the Examples, the inventors have demonstrated that FGF10 is essential for the regeneration of respiratory epithelial cells damaged by infection with pathogens of respiratory infections.

[0048] Therefore, the regenerating agent of this embodiment can regenerate respiratory epithelial cells that have been damaged by infection with pathogens of respiratory infections.

[0049] The pathogen is the same as that described above, and includes one or more pathogens selected from the group consisting of viruses, Hosozono, and Magozono. The pathogen may be a virus. Examples of the virus include influenza virus, coronavirus, RSV, etc. Examples of the coronavirus include SARS-CoV-2.

[0050] The regenerating agent of this embodiment can be said to be a therapeutic agent for respiratory infections by regenerating respiratory epithelial cells damaged by infection with pathogens of respiratory infections.

[0051] In one embodiment, the present invention provides a method for treating a respiratory infection, comprising administering an effective amount of FGF10 to a patient in need of treatment. FGF10 is preferably formulated into a dosage form such as an injection, nasal spray, or air spray.

[0052] In one embodiment, the present invention provides FGF10 for use in the treatment of respiratory infections.

[0053] In one embodiment, the present invention provides use of FGF10 for the manufacture of a therapeutic agent for a respiratory infection.

[0054] [Culture medium for regenerating damaged respiratory epithelial cells] In one embodiment, the present invention provides a culture medium for regenerating damaged respiratory epithelial cells, comprising FGF10. As described later in the Examples, the inventors have demonstrated that a culture medium containing FGF10 can regenerate respiratory epithelial cells damaged by infection with a pathogen causing a respiratory infection.

[0055] The medium of this embodiment preferably further contains a Rho-kinase (ROCK) inhibitor and a TGF-β inhibitor.

[0056] Examples of ROCK inhibitors include Y-27632 (CAS number: 129830-38-2), HA1077 (CAS number: 103745-39-7), H-1152 (CAS number: 871543-07-6), etc. Examples of TGF-β inhibitors are the same as those described above. [Example]

[0057] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0058] [material and method] (Cultivation of human respiratory organoid (hBO) three-dimensional culture model and human respiratory organoid-derived air-liquid interface cell culture model (hBO-ALI)) To prepare the hBO 3D culture model, normal human bronchial epithelial cells (NHBE, Lonza) were first suspended in 10 mg / mL chilled Matrigel (growth factor-reduced, GFR). Next, 50 μL droplets of the cell suspension were placed on a 24-well plate (Nunc) preheated to 37°C and incubated for 10 minutes to solidify. Next, 500 μL of expansion culture medium was added to each well. The composition of the expansion culture medium is shown in Table 1 below. The medium was changed every two days.

[0059] The formed hBO 3D culture models were passaged as follows: The hBO 3D culture models were suspended in 1 mL of 0.5 mM EDTA / PBS (Nacalai Tesque) and mechanically sheared using a P1000 pipette tip. 2 mL of TrypLE Select (Thermo Fisher Scientific) was then added to the suspension. After 5 minutes of incubation at room temperature, the hBO 3D culture models were again mechanically sheared using a P1000 pipette tip. 7 mL of expansion culture medium was then added, the tube was transferred to, and centrifuged at 400 rpm. The organoid fragments were then resuspended in chilled expansion culture medium and seeded as described above. The hBO 3D culture models were passaged every 10 days.

[0060] To mature the hBO 3D culture model, the expanded hBO 3D culture model was cultured in differentiation medium for 5 days. The composition of the differentiation medium is shown in Table 1 below. In Table 1, "+" indicates the presence of the agent, and "-" indicates the absence of the agent. The hBO 3D culture model could be cryopreserved using STEM-CELLBANKER GMP grade (Takara Bio).

[0061] To prepare hBO-ALIs, we dissociated growing hBO 3D culture models cultured in 24-well plates and seeded them into Transwell inserts (Corning) mounted in the 24-well plates. To promote maturation, we cultured the hBO-ALIs in differentiation medium for 5 days.

[0062] [Table 1]

[0063] (A549 culture) Human lung cancer cell line A549 was cultured in Ham's F12 medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS), 1x GlutaMAX (Thermo Fisher Scientific), and penicillin-streptomycin. A549 was passaged every 4 days.

[0064] (Preparation of SARS-CoV-2) SARS-CoV-2 (SARS-CoV-2 / Hu / DP / Kng / 19-020 and SARS-CoV-2 / Hu / DP / Kng / 19-027 strains) were obtained from the Kanagawa Prefectural Institute of Public Health. SARS-CoV-2 was isolated from COVID-19 patients in Japan (GenBank accession numbers: LC528232.1 and LC528233.1, respectively). Each virus was plaque purified and propagated in Vero cells. SARS-CoV-2 was stored at -80°C. All experiments, including virus infection experiments, were performed in strict accordance with regulations in biosafety level 3 facilities at Kyoto University and Osaka University.

[0065] (SARS-CoV-2 infection and drug treatment) 0.7 × 10 organoids were cultured in a 24-well plate (approximately 100 organoids) for the hBO three-dimensional culture model and in a Transwell insert (prepared from approximately 100 organoids) for the hBO-ALI cultured in a 24-well plate (approximately 100 organoids). 5 or 1.3 x 10 5 They were infected with TCID50 of SARS-CoV-2.

[0066] In the infection experiment of the hBO 3D culture model, half of the differentiation medium containing SARS-CoV-2 was replaced with fresh medium every day. In the drug treatment experiment, the infected hBO 3D culture model was cultured for 5 days in differentiation medium containing Camostat (catalog number SML0057, Sigma-Aldrich). Camostat is one of the compounds currently undergoing clinical trials as a treatment for COVID-19.

[0067] For the infection experiment, hBO-ALI cells were cultured in differentiation medium containing SARS-CoV-2 for 90 minutes, after which the differentiation medium containing SARS-CoV-2 was replaced with fresh differentiation medium.

[0068] (SARS-CoV-2 virus titer measurement) Viral titers were measured by median tissue culture infectious dose (TCID50) assay in a biosafety level 3 laboratory at Kyoto University.

[0069] TMPRSS2 / Vero cells (catalog number "JCRB1818", JCRB Cell Bank) were seeded in a 96-well plate (Thermo Fisher Scientific) in Minimum Essential Media (MEM, Sigma-Aldrich) supplemented with 5% FBS and 1% penicillin / streptomycin.

[0070] The sample was 10 -1 ~10 -8 The diluted samples were added to TMPRSS2 / Vero cells and incubated at 37°C for 96 hours (n=3). The cytopathic effect was assessed by microscopic observation, and TCID50 / mL was calculated using the Reed-Münch method.

[0071] Total RNA was extracted from the hBO 3D culture model, hBO-ALI, and bronchial basal stem cells using ISOGENE II (Nippon Gene). Subsequently, cDNA was synthesized from 500 ng of total RNA using the Superscript VILO cDNA synthesis kit (Thermo Fisher Scientific).

[0072] Real-time RT-PCR was performed using SYBR Green PCR Master Mix (Thermo Fisher Scientific) and a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific).

[0073] Relative quantification of target mRNA expression levels is 2 -ΔΔCT The values ​​were normalized to the value of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase. The base sequences of the primers used are shown in Table 2 below.

[0074] [Table 2]

[0075] (Transmission electron microscope observation of ultrathin sections) The hBO 3D culture model was fixed in phosphate-buffered 2% glutaraldehyde, followed by postfixation in 2% osmium tetroxide at 4°C for 2 hours. After fixation, the specimens were dehydrated in an ethanol series and embedded in epoxy resin. Ultrathin sections were then cut, stained with uranyl acetate and lead stain, and observed at 100 kV using a Hitachi H-7600 electron microscope.

[0076] (Histopathology and Immunofluorescence) The fixed hBO 3D culture model samples were processed and embedded in paraffin. Subsequently, sections were cut at 2 μm thickness, deparaffinized, rehydrated, and stained with hematoxylin and eosin (HE). The sections were observed using a microscope (BX53, Olympus) and a camera (DP73, Olympus).

[0077] Immunostaining was performed as follows. Formalin-fixed, paraffin-embedded hBO three-dimensional culture model samples were treated in citrate buffer (pH 6.0) at 125°C for 30 seconds using a pressure cooker (Dako Japan) for antigen retrieval. Sections were reacted with each antibody, followed by Histofine Simple Stain MAX-PO (Nichirei Biosciences). The antibodies used are listed in Table 3 below. Sections were stained with Peroxidase Stain DAB Kit (Nacalai Tesque) and then counterstained with Mayer's hematoxylin solution.

[0078] [Table 3]

[0079] Double immunofluorescence staining of infected hBO 3D culture models was performed as follows. Sections were deparaffinized and treated with 0.5% trypsin for 30 minutes for antigen retrieval. To suppress nonspecific reactions, sections were blocked with 5% skim milk and albumin (derived from fetal bovine serum Cohn Fraction V, pH 7.0, Fujifilm Wako Pure Chemical Industries) in PBS at room temperature for 30 minutes. The sections were then incubated with primary antibodies (Table 3) overnight at 4°C, washed, and incubated with secondary antibodies at room temperature for 1 hour.

[0080] Double immunofluorescence staining of uninfected and infected hBO-ALI was performed as follows. Cells were fixed in PBS containing 4% paraformaldehyde at 4°C. Then, cells were blocked for 45 minutes at room temperature in PBS containing 2% bovine serum albumin and 0.2% Triton X-100. The cells were then incubated with primary antibodies (Table 3) overnight at 4°C, washed, and incubated with secondary antibodies at room temperature for 1 hour.

[0081] (RNA-seq) Total RNA was prepared using the RNeasy Mini Kit (Qiagen). RNA integrity was then confirmed using the 2100 Bioanalyzer (Agilent Technologies). Library preparation was then performed using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB) or the TruSeq stranded mRNA sample prep kit (Illumina) according to the manufacturer's instructions.

[0082] NextSeq500 (Illumina) or NovaSeq6000 (Illumina) were used to sequence the sequences in single-end mode, with 152 or 101 bases, respectively. Fastq files were then generated using bcl2fastq2. Adapter sequences were then trimmed from the raw reads using cutadapt version 2.7. The trimmed reads were mapped to the human reference genome sequence (hg19) using HISAT2 version 2.1.0.

[0083] Raw counts were calculated using featureCounts ver 2.0.0 and used for heatmap visualization using integrated differential expression and pathway analysis (iDEP, http: / / ge-lab.org / idep / ). The raw data from this experiment were submitted to Gene Expression Omnibus (GEO) under the accession number GSE150819.

[0084] (LDH assay) Following SARS-CoV-2 infection, lactate dehydrogenase (LDH) present in 250 μL of culture supernatant was monitored using the LDH-Glo cytotoxicity assay (Promega) according to the manufacturer's instructions. Absorbance was measured at 490 nm using a Bio-Rad microplate reader (Bio-Rad). LDH release in uninfected cells was used as a control.

[0085] (statistical analysis) Statistical analysis was performed using an unpaired, two-tailed Student's t-test. Statistical significance was assessed by one-way analysis of variance (ANOVA) followed by Tukey's or Dunnett's post-hoc test. Representative results from three independent experiments were used.

[0086] [Experimental Example 1] (Generation of human respiratory organoids from cryopreserved adult-derived bronchial epithelial cells) The inventors investigated the conditions under which a three-dimensional culture model of human respiratory organoids (hBOs) can be created from cryopreserved adult-derived bronchial epithelial cells (NHBEs).

[0087] Figure 1 shows the results of immunofluorescence staining of acetylated α-tubulin and KRT5 in NHBEs. Nuclei were stained with DAPI (4',6-diamidino-2-phenylindole). The results revealed that most NHBEs were KRT5-positive but acetylated α-tubulin-negative, indicating that most NHBEs are basal stem cells.

[0088] Furthermore, the results of this study revealed that a three-dimensional hBO culture model could be produced by embedding NHBE in Matrigel and culturing them in advanced DMEM / F12 medium (expansion culture medium, composition shown in Table 1 above) containing FGF2, FGF7, FGF10, Noggin, R-spondin 1, Y-27632, and SB202190.

[0089] Furthermore, it was found that the hBO three-dimensional culture model can be matured by culturing it in advanced DMEM / F12 medium (differentiation medium, composition shown in Table 1 above) containing FGF2, FGF7, FGF10, Y-27632, and A83-01.

[0090] The expanded hBO 3D culture models were then cultured for 5 days in a medium containing FGF2, EGF, HGF, R-spondin 1, or Noggin. The expression levels of ACE2 and TMPRSS2 were then measured by quantitative real-time PCR. Figure 2 shows the results of quantitative real-time PCR (n = 3). Statistical significance was assessed by one-way analysis of variance (ANOVA) followed by Dunnett's post-hoc test. The values ​​shown in Figure 2 are relative values, with the value measured in the medium containing FGF2 set at 1. In Figure 2, "*" indicates a significant difference at p < 0.05, and "**" indicates a significant difference at p < 0.01. The results revealed that, among the growth factors contained in the medium, FGF2 is important for enhancing the expression levels of ACE2 and TMPRSS2.

[0091] Figure 3 shows micrographs of the three-dimensional culture model of hBOs, both phase-contrast and hematoxylin-eosin stained. Approximately 100 hBOs were present in 50 μL of Matrigel, with each hBO measuring approximately 100–200 μm in diameter.

[0092] The expression levels of ACE2 and TMPRSS2 in the hBO 3D culture model, NHBE, and A549 were measured by quantitative real-time PCR. Figure 4 shows the results of quantitative real-time PCR (n = 3). Statistical significance was assessed by one-way analysis of variance (ANOVA) followed by Dunnett's post-hoc test. The values ​​shown in Figure 4 are expressed relative to the values ​​measured in the hBO 3D culture model, with the value measured in the hBO 3D culture model set at 1. In Figure 4, groups not sharing the same letter indicate significant differences at p < 0.05. The results revealed that the expression levels of ACE2 and TMPRSS2 in the hBO 3D culture model were significantly higher than those in NHBE and A549.

[0093] Next, the expression of ACE2 and TMPRSS2 in the hBO 3D culture model was detected by immunochemical staining. Figure 5 is a photograph showing the results of immunochemical staining. The scale bar is 20 μm. The arrow in Figure 5 indicates the location where TMPRSS2 expression was detected. Furthermore, the expression of ACE2 and KRT5 in the hBO was detected by fluorescent immunostaining. KRT5 is a marker for basal stem cells. Figure 6 is a photograph showing the results of fluorescent immunostaining. The scale bar is 20 μm. Nuclei were counterstained with DAPI. The results revealed that ACE2 was expressed in part of the outer edge of the hBO, and TMPRSS2 was expressed in part of the outer edge and part of the lumen of the hBO.

[0094] The bronchi contain basal stem cells, ciliated cells, goblet cells, and club cells, so we measured the expression levels of marker genes for each of these cells in the hBO 3D culture model using quantitative real-time PCR.

[0095] Figure 7 is a graph showing the results of measuring the expression levels of basal stem cell marker genes NGFR and PROM1 in the hBO 3D culture model, NHBE, and A549 (n=3). Figure 8 is a graph showing the results of measuring the expression levels of ciliated cell marker genes TUBA1A and MCIDAS in the hBO 3D culture model, NHBE, and A549 (n=3). Figure 9 is a graph showing the results of measuring the expression levels of goblet cell marker genes MUC20 and MUC5B in the hBO 3D culture model, NHBE, and A549 (n=3). Figure 10 is a graph showing the results of measuring the expression levels of club cell marker genes SCGB1A1 and KLF5 in the hBO 3D culture model, NHBE, and A549 (n=3).

[0096] The values ​​shown in Figures 7-10 are relative values, with the measured values ​​in the hBO 3D culture model taken as 1. Statistical significance was assessed by one-way analysis of variance (ANOVA) followed by Dunnett's post-hoc test. The values ​​shown in Figures 7-10 are relative values, with the measured values ​​in the hBO 3D culture model taken as 1. In Figures 7-10, groups that do not share the same letter indicate significant differences with each other at p<0.05.

[0097] As a result, it was revealed that the expression levels of marker genes for basal stem cells, ciliated cells, goblet cells, and club cells in the hBO three-dimensional culture model were significantly higher than those in the NHBE.

[0098] Figure 11 shows micrographs showing the results of immunochemical staining of the hBO 3D culture model to detect the expression of KRT5 (a marker for basal stem cells), acetylated α-tubulin (a marker for ciliated cells), MUC5AC (a marker for goblet cells), and CC10 (a marker for club cells). The immunochemical staining results also confirmed that the hBO 3D culture model expresses KRT5, acetylated α-tubulin, MUC5AC, and CC10. Furthermore, it was revealed that the outer periphery of the hBO was positive for KRT5, and the lumen was positive for acetylated α-tubulin.

[0099] These results indicate that basal stem cells express both ACE2 and TMPRSS2, while ciliated cells express only TMPRSS2.

[0100] 12 and 13 are transmission electron micrographs of ultrathin sections of the hBO 3D culture model, revealing ciliated cells, goblet cells, basal stem cells, 9+2 structures, cilia, and microvilli.

[0101] These results demonstrate that functional hBOs capable of expansion could be generated from cryopreserved adult-derived NHBEs.

[0102] [Experimental Example 2] (FGF2 treatment increased the expression levels of bronchial markers) The expanded hBO 3D culture models were cultured for 5 days in medium containing FGF2, EGF, HGF, R-spondin 1, or Noggin. Subsequently, the expression levels of bronchial markers KRT5, MUC20, MCIDAS, NGFR, MUC5B, and SCGB1A1 were measured by quantitative real-time PCR. Figure 14 shows the results of quantitative real-time PCR (n = 3). Statistical significance was assessed by one-way analysis of variance (ANOVA) followed by Dunnett's post-hoc test. The values ​​shown in Figure 14 are expressed relative to the value measured in the medium containing FGF2, where 1 is taken as the mean. In Figure 14, "*" indicates a significant difference at p < 0.05, "**" indicates a significant difference at p < 0.01, "***" indicates a significant difference at p < 0.001, and "****" indicates a significant difference at p < 0.0001. As a result, it was revealed that among the growth factors contained in the culture medium, FGF2 was important for enhancing the expression levels of bronchial markers.

[0103] [Experimental Example 3] (Comparison of bronchial marker expression levels in NHBE, expanded hBO 3D culture model, and differentiated hBO 3D culture model) The expression levels of bronchial markers, including TMPRSS2, MCIDAS, MUC20, MUC5B, and SCGB1A1, were measured in NHBE, expanded hBO 3D culture models, and differentiated hBO 3D culture models by quantitative real-time PCR.

[0104] Figure 15 is a graph showing the results of quantitative real-time PCR (n = 3). Statistical significance was assessed by one-way analysis of variance (ANOVA) followed by Dunnett's post-hoc test. The values ​​shown in Figure 15 are expressed relative to the measured values ​​in NHBE, with the measured values ​​in NHBE set at 1. The results revealed that the expression levels of bronchial markers increased in the following order: NHBE, expanded hBO 3D culture model, and differentiated hBO 3D culture model.

[0105] [Experimental Example 4] (RNA-seq analysis of hBO 3D culture model) We performed RNA-seq analysis of the hBO 3D culture model (n=3). For comparison, we also performed RNA-seq analysis on NHBE and A549. Heat map analysis and scatter plots of principal component analysis (PCA) gene expression profiles all showed that the hBO 3D culture model was closer to NHBE than to A549.

[0106] Figure 16 is a heat map created for bronchial epithelial cell markers. Figure 16 reveals that the hBO three-dimensional culture model expresses bronchial epithelial cell markers more strongly than NHBE or A549.

[0107] These results indicate that the hBO three-dimensional culture model has higher bronchial function than NHBE or A549.

[0108] [Experimental Example 5] SARS-CoV-2 infection and replication in a human bovine serum (hBO) three-dimensional culture model hBOs were infected with SARS-CoV-2 and cultured in differentiation medium for 5 days. Figure 17 is a schematic diagram showing the experimental schedule.

[0109] The virus contained in the medium infected the hBO from the basal membrane side. Figure 18 shows the results of immunohistochemical staining of a SARS-CoV-2-infected hBO 3D culture model to detect the SARS-CoV-2 S protein (SP). The scale bar is 20 μm. In Figure 18, "control" represents the results of the hBO 3D culture model not infected with SARS-CoV-2, while "SARS-CoV-2" represents the results of the hBO 3D culture model infected with SARS-CoV-2. As a result, SP-positive cells were observed in part of the outer edge of the hBO.

[0110] Figure 19 shows the results of immunofluorescence staining of a SARS-CoV-2-infected hBO 3D culture model. The scale bar is 20 μm. SP and KRT5 were detected. Nuclei were counterstained with DAPI. Figure 20 shows the results of immunofluorescence staining of a SARS-CoV-2-infected hBO. The scale bar is 20 μm. SP and CC10 were detected. Nuclei were counterstained with DAPI. The results revealed that SP colocalized with KRT5 but not with CC10-positive club cells. This indicates that SARS-CoV-2 has difficulty replicating in the hBO 3D culture model.

[0111] Figure 21 shows the results of infecting hBO 3D culture models with SARS-CoV-2 in the presence or absence of camostat and measuring viral titers by TCID50 assay. Small amounts of infectious virus were detected in the infected hBO 3D culture models. Furthermore, camostat treatment reduced the production of this virus.

[0112] Figure 22 shows the results of 1.3 x 10 cells cultured in a hBO three-dimensional culture model in the presence or absence of 10 μM Camostat. 5 This graph shows the results of LDH assays performed on days 1, 2, 3, 4, and 5 after infection with TCID50 / mL of SARS-CoV-2 and culture in differentiation medium for 5 days. Statistical significance was assessed by one-way analysis of variance (ANOVA) followed by Dunnett's post-hoc test, comparing the results to those obtained when SARS-CoV-2 was infected in the absence of 10 μM Camostat. No accumulation of lactate dehydrogenase (LDH) was observed in the culture medium of the infected hBO 3D culture model. This result suggests that cytotoxicity is not the cause of viral infection.

[0113] Figure 23 shows the results of parametric gene set enrichment analysis (PGSEA) of the GO biological gene set based on the RNA-seq results for the non-infected hBO 3D culture model (control), the infected hBO 3D culture model (SARS-CoV-2), and the infected hBO 3D culture model in the presence of 10 μM Camostat (SARS-CoV+Camostat).

[0114] Figure 24 is a graph showing the results of quantitative real-time PCR measurement of the expression levels of interferon (IFN)-α, IFN-β, ISG56, and ISG15 genes in a non-virus-infected hBO 3D culture model (control), a virus-infected hBO 3D culture model (SARS-CoV-2), and a virus-infected hBO 3D culture model in the presence of 10 μM Camostat (SARS-CoV+Camostat).

[0115] Figure 25 shows a heatmap of genes related to type I IFN signaling based on the results of RNA-seq in the non-infected hBO 3D culture model (control) and the infected hBO 3D culture model (SARS-CoV-2).

[0116] The results in Figures 23 to 25 reveal that the expression of innate immune response-related genes was slightly enhanced by virus infection.

[0117] These results suggest that the virus had little ability to infect basal stem cells present at the outer edge of hBOs, and that viral replication was limited.

[0118] [Experimental Example 6] SARS-CoV-2 infection and replication in human body at the ALI To mimic viral infection from the luminal side of the bronchi, we used hBO-ALI. Expanded hBO 3D culture models were dissociated and seeded onto Transwell inserts and cultured in differentiation medium for 5 days. Figure 26 shows a schematic diagram of the experimental schedule.

[0119] Figures 27 and 28 are photographs showing the results of immunofluorescence staining of non-virus-infected hBO-ALI. In Figure 27, acetylated α-tubulin and KRT5 were detected. Nuclei were counterstained with DAPI. In Figure 28, acetylated α-tubulin and ACE2 were detected. Nuclei were counterstained with DAPI. The results confirmed the presence of acetylated α-tubulin-positive cells and KRT5-positive cells in hBO-ALI. ACE2 was also found to colocalize with acetylated α-tubulin. These results indicate that hBO-ALI contains ciliated cells that strongly express the viral receptor ACE2.

[0120] Figure 29 shows the results of quantitative real-time PCR analysis of the expression levels of ACE2, TMPRSS2, FURIN, NGFR, MCIDAS, MUC5B, and SCGB1A genes in the hBO 3D culture model, hBO-ALI, and bronchial basal stem cells (basal stem cells). Statistical significance was assessed by one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test. In Figure 29, "hBO" indicates the results for the hBO 3D culture model, "hBO-ALI" indicates the results for the hBO-ALI, and "Basal cells" indicates the results for the basal stem cells. Additionally, "*" indicates a significant difference at p<0.05, and "**" indicates a significant difference at p<0.01.

[0121] The results showed that there were no differences in the expression levels of SARS-CoV-2-related marker genes and bronchial epithelial cell marker genes, except for the multiciliate differentiation and DNA synthesis associated cell cycle protein (MCIDAS) gene, between the hBO 3D culture model and the hBO-ALI. The enhanced expression level of the MCIDAS gene in the hBO-ALI compared with the hBO 3D culture model suggests that the maturation of ciliated cells is promoted in the hBO-ALI.

[0122] The hBO 3D culture model and hBO-ALI were then infected with SARS-CoV-2 and cultured in differentiation medium for two days. Figure 30 shows the results of measuring infectious virus in the culture supernatants of the hBO 3D culture model and hBO-ALI by TCID50 assay. Statistical significance was assessed by one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test. In Figure 30, "*" indicates a significant difference at p<0.05. The results revealed significantly greater infectious virus replication in the hBO-ALI compared to the hBO 3D culture model.

[0123] Figure 31 shows the results of immunofluorescence staining of hBO-ALI cells 2 days after virus infection. SARS-CoV-2 SP, acetylated α-tubulin, and KRT5 were detected. Nuclei were counterstained with DAPI. The results confirmed that SP colocalized with acetylated α-tubulin but not with KRT5.

[0124] These results indicate that SARS-CoV-2 replicates efficiently in ciliated cells but not in basal stem cells in hBO-ALI, possibly due to the low expression of ACE2 in basal stem cells, as shown in Figure 29 above.

[0125] [Experimental Example 7] FGF10 is essential for regenerating the bronchial epithelial cell layer from remaining basal stem cells. Figure 32 shows the 7.0 x 10 4 hBO-ALI cells were infected with SARS-CoV-2 at a concentration of TCID50 / well. Seven days after infection, acetylated α-tubulin, SARS-CoV-2 SP, and KRT5 were detected by immunofluorescence staining. Nuclei were counterstained with DAPI. No acetylated α-tubulin- or SP-positive cells were observed. This indicates that ciliated cells were killed by virus infection. However, KRT5-positive basal stem cells remained even seven days after virus infection.

[0126] Figure 33 shows the results of immunofluorescence staining of acetylated α-tubulin and KRT5 15 days after viral infection. Nuclei were counterstained with DAPI. The results revealed that the remaining basal stem cells differentiated into acetylated α-tubulin-positive ciliated cells, forming the bronchial epithelial layer. These results suggest that basal stem cells play an important role in repairing the bronchial epithelial layer after viral infection.

[0127] Next, the effect of human FGF on viral infection and subsequent regeneration of the bronchial epithelial layer was examined. 4 This graph shows the results of infecting hBO-ALI cells with TCID50 / well of SARS-CoV-2 and culturing them for two days in differentiation medium with or without FGF2, FGF7, and FGF10. The infectious virus content in the culture supernatant was measured by TCID50 assay. Statistical significance was assessed by one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test. In Figure 34, "*" indicates a significant difference at p<0.05. "w / o" indicates no FGF10. The results demonstrated that omission of FGF10 from the differentiation medium significantly increased infectious virus production.

[0128] Figure 35 shows photographs of the detection of acetylated α-tubulin and KRT5 by fluorescent immunostaining 15 days after viral infection. Nuclei were counterstained with DAPI. In Figure 35, "w / o" indicates absence. The results revealed that when FGF10 was removed from the differentiation medium, the remaining basal stem cells did not proliferate or differentiate into acetylated α-tubulin-positive ciliated cells. In contrast, even when FGF2 or FGF7 was removed from the differentiation medium, the remaining basal stem cells were able to proliferate and differentiate into acetylated α-tubulin-positive ciliated cells.

[0129] These results demonstrated that FGF10 is essential for the regeneration of the bronchial epithelial layer by the remaining basal stem cells.

[0130] [Experimental Example 8] (Examination of differences in viral replication efficiency depending on the donor) We investigated the difference in viral replication efficiency depending on the donor of NHBE used to generate hBO-ALI. hBO-ALI was generated from NHBE obtained from four donors. Information on each donor is summarized in Table 4.

[0131] Figure 36 shows the 7.0 x 10 4 This graph shows the results of infecting each hBO-ALI with TCID50 / well of SARS-CoV-2 and culturing them in differentiation medium for two days, followed by measuring the infectious virus content in the culture supernatant by TCID50 assay. Statistical significance was assessed by one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test. In Figure 36, "*" indicates a significant difference at p<0.05, and "**" indicates a significant difference at p<0.01.

[0132] As a result, it was revealed that virus production in hBO-ALI derived from donor 4 was significantly higher than that in hBO-ALI derived from donor 1 and hBO-ALI derived from donor 2.

[0133] These results indicate that hBO-ALI can reflect individual differences in viral replication efficiency, including gender differences.

[0134] [Table 4] [Industrial Applicability]

[0135] According to the present invention, a respiratory organoid technology can be provided that not only can reproduce the life cycle of pathogens of respiratory infections, but also can screen preventive or therapeutic agents for respiratory infections.

Claims

1. A method for producing a respiratory organoid-derived air-liquid interface cell culture model, (1-1) A step of culturing bronchial epithelial cells collected from a living body in an expansion culture medium containing fibroblast growth factor (FGF), a BMP signal inhibitor, a Wnt signal activator, and a p38 inhibitor to obtain respiratory organoids; (1-2) Culturing the obtained respiratory organoids in a differentiation medium containing FGF and a TGF-β inhibitor; and Before or after the step (1-2), the obtained respiratory organoids are dissociated and cultured at an air-liquid interface; The respiratory organoid-derived air-liquid interface cell culture model comprises ciliated cells and one or more cells from a cell group consisting of basal stem cells, goblet cells, club cells, pulmonary neuroendocrine cells, type I alveolar epithelial cells, and type II alveolar epithelial cells, and the apical surface where the ciliated cells are present is exposed; the FGF is FGF2, FGF7, or FGF10; In the step (1-1), the bronchial epithelial cells are embedded in a gel and then cultured in the expansion culture medium.

2. The method according to claim 1 , wherein the BMP signal inhibitor is Noggin.

3. The method according to claim 1 or 2, wherein the Wnt signal activator is R-spondin.

4. The method according to any one of claims 1 to 3, wherein the p38 inhibitor is SB202190.

5. The method according to any one of claims 1 to 4, wherein the TGF-β inhibitor is A83-01.

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