Composition for culture medium for bat tissue organoids and method for producing bat tissue organoids

Abstract

To provide a medium composition optimal for an organoid prepared from intestinal or lung tissue of bats.SOLUTION: A composition for a bat tissue organoid medium contains TGF-α. A composition for a bat lung organoid medium contains TGF-α and / or FGF7.SELECTED DRAWING: Figure 6

Description

[Technical Field]

[0001] The present invention relates to a medium composition and medium for culturing organoids prepared from tissues such as the intestinal tract and lungs of bats, as well as a method for culturing and producing bat tissue organoids using the medium.The present invention also relates to bat lung organoids. [Background technology]

[0002] The three-dimensional organoid culture method (Non-Patent Document 1: Sato et al., Nature, 2009) was developed as a method for reproducing three-dimensional epithelial tissue structures in a culture dish by mixing epithelial cells isolated from various organs with Matrigel and culturing them in a special medium containing factors that enhance stemness, such as Wnt, Noggin, and R-spondin. In recent years, organoid culture methods have been established using surgical specimens from human patients with colon cancer or pancreatic cancer, and structural similarities with tissue immediately after removal and correlations with genetic mutations have been demonstrated (Non-Patent Document 2: Wetering et al., Cell, 2015 and Non-Patent Document 3: Boj et al., Cell, 2015), making it expected to become a useful tool for personalized medicine.

[0003] In addition, a technology has been developed to non-invasively cultivate bladder cancer organoids using urine samples from dogs with bladder cancer. The organoids thus produced have been shown to three-dimensionally reproduce the characteristics of bladder cancer in vivo and can be used to test the ability of tumors to regenerate in immunocompromised mice and to test the anticancer drug sensitivity of individual patients (Non-Patent Document 4: Elbadawy and Usui et al., Cancer Sci. 2019).

[0004] Bats are known to be hosts for the novel coronavirus (SARS-CoV-2), which causes COVID-19, as well as other viruses that cause infectious diseases. Other viruses known to use bats as hosts and cause infectious diseases include Menangle virus, Nipah virus, Australian-European lyssavirus, Hendra virus, Ebola virus, Marburg virus, and rabies virus. These bat-originated viruses infect other animals and eventually humans.

[0005] Research into bats' defense mechanisms against viruses has focused on "viral tolerance mediated by intracellular factors." Research primarily based on whole-genome analysis using next-generation sequencers has accumulated knowledge about bat-specific diversity and mutations in subtypes of factors involved in innate immunity, such as type 1 interferon (IFN) genes, MHC class I genes, and NKG2 genes, as well as novel antiviral mechanisms mediated by IFN-ω (Non-Patent Document 5: Stephanie SP, et al., 2018; Non-Patent Document 6: Zhou P, et al., 2016; Non-Patent Document 7: Pavlovich SS, et al., 2020). Infection experiments using bat kidney-derived cell lines have shown that Niemann-Pick C1 is involved in filovirus susceptibility (Non-Patent Document 8: Takadate Y, et al., 2020), and that artificial suppression of IRF3 and MAPK pathway expression in persistently MERS-CoV-infected cells induces viral proliferation and cell death (Non-Patent Document 9: Benerjee A, et al., 2020).

[0006] Additionally, a Hendra virus infection experiment using individual Japanese flying foxes reported significant increases in the chemokine CXCL10 along with IFN gene suppression in the lungs and spleen (Non-Patent Document 10: Woon AP, et al., 2020). In vivo analysis of mallards, another natural host of low-pathogenic influenza A viruses, reported that rapid and transient expression of RIG-I and Mx may be involved in tolerance (Non-Patent Document 11: Helin AS, et al., 2018). However, the link between the reported suppression of viral replication by intracellular factors and avoidance of cellular damage associated with viral replication remains unknown.

[0007] Organoids using human intestinal and lung tissue obtained by the above-mentioned three-dimensional organoid culture method have been used in viral infection experiments. Regarding organoids derived from bats, a method using intestinal tissue has recently been reported (Non-Patent Document 12: Zhou J, et al., Nat Med., 26(7):1077-1083, 2020), but no reports have been published on bat organoid culture methods using lung tissue. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Sato et al., Nature, 459(7244):262-5, 2009 [Non-patent document 2] Wetering et al., Cell, 161(4):933-45, 2015 [Non-patent document 3] Boj et al., Cell, 160(1-2):324-38, 2015 [Non-patent document 4] Elbadawy and Usui et al., Cancer Sci. 110(9):2806-2821, 2019 [Non-patent document 5] Stephanie SP, et al., Cell, 173(5):1098-1110, 2018 [Non-patent document 6] Zhou P, et al., PNAS, 113(10):2696-2701, 2016 [Non-Patent Document 7] Pavlovich SS, et al., Front Immunol., 11:435, 2020 [Non-patent document 8] Takadate Y, et al., Cell Rep., 30(2);308-319, 2020 [Non-Patent Document 9] Benerjee A, et al., Sci Rep., 10(1):7257, 2020 [Non-Patent Document 10] Woon AP, et al., PLoS Pathog., 16(3):e1008412, 2020 [Non-Patent Document 11] Helin AS, et al., Mol Immunol., 95:64-72, 2018 [Non-Patent Document 12] Zhou J, et al., Nat Med., 26(7):1077-1083, 2020 Summary of the Invention [Problem to be solved by the invention]

[0009] Although there have been reports of the creation of bat intestinal organoids, they only have a lifespan of about one month, making them difficult to use in viral infection experiments, etc. Furthermore, for experiments involving viruses that use bats as hosts, organoids created from bat lung tissue are desirable.

[0010] Therefore, the present invention aims to provide a culture medium composition that is optimal for organoids prepared from tissues such as the intestinal tract and lung tissue of bats, a bat tissue organoid culture medium using the culture medium composition, and a method for culturing and producing bat tissue organoids. [Means for solving the problem]

[0011] As a result of intensive research conducted by the present inventors to achieve the above-mentioned object, they discovered that long-term culture of organoids prepared from tissues such as the intestinal or lung tissues of bats is possible when certain components are present, and thus they have completed the present invention.

[0012] [1] A composition for bat tissue organoid culture medium containing TGF-α. [2] The composition for bat tissue organoid culture medium described in [1], wherein the tissue is the intestine or lung. [3] A composition for bat tissue organoid culture medium according to [1] or [2], further comprising at least one component selected from the group consisting of a Wnt agonist, a BMP inhibitor, an EGF and a TGFβ inhibitor. [4] A composition for a bat lung organoid culture medium containing TGF-α and / or FGF7. [5] The composition for a bat lung organoid culture medium according to [4], further comprising at least one component selected from the group consisting of a Wnt agonist, a BMP inhibitor, an EGF, and a TGFβ inhibitor. [6] Bat tissue organoid medium containing TGF-α. [7] The bat tissue organoid culture medium according to [6], wherein the tissue is the intestine or lung. [8] The bat tissue organoid culture medium according to [6] or [7], further comprising at least one component selected from the group consisting of a Wnt agonist, a BMP inhibitor, an EGF, and a TGFβ inhibitor. [9] Bat lung organoid culture medium containing TGF-α and / or FGF7.

[10] The bat lung organoid culture medium according to [9], further comprising at least one component selected from the group consisting of a Wnt agonist, a BMP inhibitor, an EGF, and a TGFβ inhibitor.

[11] A method for culturing bat tissue organoids, comprising culturing bat tissue organoids using the bat tissue organoid culture medium described in any one of [6] to [8].

[12] A method for culturing bat lung organoids, comprising culturing bat lung organoids using the bat lung organoid culture medium described in [9] or

[10] .

[13] collecting cells or tissues from a bat; combining the cells or tissue with an extracellular matrix; [6] to [8], using the bat tissue organoid culture medium described in any one of [6] to [8], a step of culturing the cells or tissue mixed with the extracellular matrix. A method for producing bat tissue organoids, comprising:

[14] collecting lung cells or lung tissue from a bat; combining the lung cells or lung tissue with an extracellular matrix; [9] or

[10] , using the bat lung organoid culture medium described above, the lung cells or lung tissue mixed with the extracellular matrix is ​​cultured. A method for producing bat lung organoids, comprising:

[15] Bat lung organoids derived from bat lungs and expressing at least one protein selected from the group consisting of MUC5AC, CK5, and SFTPC. [Effects of the Invention]

[0013] The bat tissue organoid culture medium composition and bat lung organoid culture medium composition of the present invention contain specific components, making it possible to prepare media that enable the production and long-term culture of bat tissue organoids and bat lung organoids. That is, by using the bat tissue organoid culture medium composition and bat lung organoid culture medium composition, it becomes possible to produce and long-term culture bat tissue organoids and bat lung organoids that can be used for basic research on viruses that use bats as hosts.

[0014] Furthermore, the bat tissue organoid culture method and bat lung organoid culture method according to the present invention use a medium containing specific components, allowing bat tissue organoids and bat lung organoids to be grown over a long period of time. Therefore, by using the bat tissue organoid culture method and bat lung organoid culture method, bat tissue organoids and bat lung organoids that can be used for basic research on viruses that use bats as hosts can be cultured over a long period of time.

[0015] Furthermore, the method for producing bat tissue organoids and the method for producing bat lung organoids according to the present invention can efficiently produce bat tissue organoids and bat lung organoids by using a medium containing specific components.

[0016] Furthermore, the bat lung organoids according to the present invention can be used for basic research on viruses that use bats as hosts. [Brief explanation of the drawings]

[0017] [Figure 1] Photographs showing phase-contrast microscopic images of bat intestinal organoids (BIO) and lung organoids (BLO). [Figure 2] Photographs showing the results of tissue structure analysis of each organoid by HE staining. [Figure 3]Photographs showing the results of ultrastructural analysis of each organoid using an electron microscope. [Figure 4] 1 shows photographs showing the results of analyzing the expression of various cell markers in bat intestinal organoids (BIO). [Figure 5] 1 shows photographs showing the results of analyzing the expression of various cell markers in bat lung organoids (BLOs). [Figure 6] FIG. 1 shows photographs of bat intestinal organoids (BIO) cultured using various supplements, as well as a characteristic diagram showing the results of measuring the proliferation rate. [Figure 7] FIG. 1 shows photographs of bat lung organoids (BLOs) cultured using various supplements and a characteristic diagram showing the results of measuring the proliferation rate. [Figure 8] FIG. 1 is a characteristic diagram showing the results of analyzing coronavirus-related receptor expression in bat intestinal organoids (BIO). [Figure 9] This is a characteristic diagram showing the results of analyzing coronavirus-related receptor expression in bat lung organoids (BLOs). DETAILED DESCRIPTION OF THE INVENTION

[0018] The present invention will be described in detail below. In the present invention, "organoid" refers to a self-organized, three-dimensional cell tissue or organ obtained by culturing cells or tissues. Furthermore, "bat tissue organoid" refers to an organoid derived from a bat, which exhibits a morphology and / or function similar to that of a bat tissue or organ. Bat tissue organoids include, for example, bat intestinal organoids and bat lung organoids.

[0019] The bat tissue organoid culture medium composition of the present invention contains TGF-α. That is, TGF-α enables the production and long-term culture of all bat tissue organoids, including bat intestinal organoids and bat lung organoids. The bat tissue organoid culture medium composition of the present invention can be particularly suitably used as a culture medium composition for bat epithelial tissue organoids, and particularly as a culture medium composition for bat intestinal organoids. Here, epithelial tissues include, but are not limited to, the intestine, stomach, uterus, lungs, nasal cavity, epidermis, esophagus, etc. That is, the bat tissue organoid culture medium composition can be used for at least one organoid selected from the group consisting of bat intestinal organoids, bat stomach organoids, bat uterine organoids, bat lung organoids, bat nasal cavity organoids, bat epidermal organoids, and bat esophageal organoids, for example.

[0020] Furthermore, the composition for a culture medium for bat lung organoids according to the present invention contains TGF-α and / or FGF7. That is, FGF7 enables the production and long-term culture of bat lung organoids.

[0021] Therefore, TGF-α is utilized in the culture medium for bat tissue organoids, and FGF7 is utilized in the culture medium for bat lung organoids.

[0022] Furthermore, the bat tissue organoid culture medium composition of the present invention and the bat lung organoid culture medium composition of the present invention may further contain some or all of the medium components described below. When the bat tissue organoid culture medium composition of the present invention and the bat lung organoid culture medium composition of the present invention contain some of the medium components described below, they can be used together with the remaining medium components as the bat tissue organoid culture medium or bat lung organoid culture medium described below. Furthermore, when the bat tissue organoid culture medium composition of the present invention and the bat lung organoid culture medium composition of the present invention contain all of the medium components described below, they can be used as is as a bat tissue organoid culture medium or bat lung organoid culture medium.

[0023] In the bat tissue organoid culture medium composition or bat lung organoid culture medium composition of the present invention, the concentration of TGF-α or FGF7 is not particularly limited, and can be appropriately determined according to the dilution ratio when used as a culture medium.In addition, when the bat tissue organoid culture medium composition or bat lung organoid culture medium composition further contains some or all of the medium components described below, the concentration of these medium components is also not particularly limited, and can be appropriately determined according to the dilution ratio when used as a culture medium.

[0024] TGF-α is a transforming growth factor-α (or transforming growth factor-α), a known cytokine produced by monocytes, keratinocytes, and various tumor cells. There are no particular limitations on the TGF-α, and TGF-α derived from any animal can be used. For example, commercially available TGF-α derived from various animals, such as human TGF-α, mouse TGF-α, or rat TGF-α, can be used. Alternatively, bat TGF-α produced as a recombinant by isolating the bat TGF-α gene can be used. Furthermore, modified TGF-α can also be used in the present invention, as long as it has TGF-α activity.

[0025] The concentration of TGF-α contained in the medium is not particularly limited, but can be, for example, 4 ng / mL to 1000 ng / mL, preferably 10 ng / mL to 1000 ng / mL, more preferably 10 ng / mL to 800 ng / mL, more preferably 10 ng / mL to 600 ng / mL, more preferably 10 ng / mL to 400 ng / mL, more preferably 10 ng / mL to 200 ng / mL, and more preferably 10 ng / mL to 100 ng / mL. More specifically, the concentration of TGF-α contained in the medium can be 20 ng / mL.

[0026] FGF7 is a fibroblast growth factor, also known as keratinocyte growth factor (KGF). FGF7 is not particularly limited, and FGF7 derived from any animal can be used. For example, commercially available FGF7 derived from various animals, such as human FGF7, mouse FGF7, or rat FGF7, can be used. Alternatively, bat FGF7, which is prepared as a recombinant by isolating the bat FGF7 gene, can also be used. Furthermore, modified FGF7 can also be used in the present invention, as long as it has FGF7 activity.

[0027] The concentration of FGF7 contained in the medium is not particularly limited, but can be, for example, 0.4 ng / mL to 100 ng / mL, preferably 1 ng / mL to 100 ng / mL, more preferably 1 ng / mL to 80 ng / mL, more preferably 1 ng / mL to 60 ng / mL, more preferably 1 ng / mL to 40 ng / mL, more preferably 1 ng / mL to 20 ng / mL, and more preferably 1 ng / mL to 10 ng / mL. More specifically, the concentration of FGF7 contained in the medium can be 5 ng / mL.

[0028] The medium components used with the bat tissue organoid culture medium composition or bat lung organoid culture medium composition of the present invention include components contained in a medium typically used to culture three-dimensional organoids. Specifically, the medium used to culture three-dimensional organoids is not particularly limited, but a serum-free basal cell culture medium can be used. Examples of serum-free basal cell culture media include synthetic media adjusted to a pH of approximately 7.0 to 7.6 using a carbonate buffer. More specifically, Dulbecco's Modified Eagle Medium (Nutrient Mixture F-12; DMEM / F12) supplemented with glutamine, insulin, penicillin or streptomycin, and transferrin can be used. Another example is Roswell Park Memorial Institute 1640 medium (RPMI 1640 medium) supplemented with glutamine, insulin, penicillin or streptomycin, and transferrin. Other examples include Advanced-DMEM / F12 supplemented with glutamine and penicillin or streptomycin, and Advanced RPMI medium supplemented with glutamine and penicillin or streptomycin.

[0029] The serum-free basal cell culture medium may also be supplemented with purified natural, semi-synthetic, or synthetic growth factors, such as B-27 Supplement (Thermo Fisher Scientific), N-acetyl-L-cysteine ​​(Sigma), and N-2 Supplement (Thermo Fisher Scientific).

[0030] In addition to these basic media, the organoid culture medium can also contain a Wnt agonist that activates Wnt signaling, a BMP inhibitor that inhibits BMP signaling, epidermal growth factor (EGF), and a TGFβ inhibitor.

[0031] Examples of Wnt agonists include Wnt, Wnt-3a, Noggin, GSK inhibitors, and R-spondins such as R-spondin1, R-spondin2, R-spondin3, and R-spondin4.

[0032] Examples of BMP inhibitors include Noggin, Chordin, Chordin-like proteins containing a Chordin domain, Follistatin, Follistatin-related proteins containing a Follistatin domain, DAN, DAN-like proteins containing a DAN cysteine-knot domain, sclerostin / SOST, decorin, and α-2 macroglobulin.

[0033] EGF is a 6045 Da protein consisting of 53 amino acid residues and three intramolecular disulfide bonds, and binds as a ligand to the epidermal growth factor receptor (EGFR) present on the cell surface. The concentration of EGF contained in the culture medium is not particularly limited, but can be, for example, 2 ng / mL to 500 ng / mL, preferably 5 ng / mL to 500 ng / mL, more preferably 10 ng / mL to 400 ng / mL, more preferably 20 ng / mL to 300 ng / mL, more preferably 30 ng / mL to 200 ng / mL, and even more preferably 40 ng / mL to 100 ng / mL. More specifically, the concentration of EGF can be 50 ng / mL.

[0034] Examples of TGFβ (transforming growth factor β) inhibitors include A83-01 (3-(6-methylpyridin-2-yl)-1-phenylthiocarbamoyl-4-quinolin-4-ylpyrazole), ALK5 inhibitor I (3-(pyridin-2-yl)-4-(4-quinonyl)-1H-pyrazole), LDN193189 (4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline), SB431542 (4-[4-(1,3-benzodioxol-5-yl)-5-pyridin-2-yl-1H-imidazol-2-yl]benzamide), SB-505124 (2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine hydrochloride hydrate), SD-20 8 ((2-(5-chloro-2-fluorophenyl)pteridin-4-yl)pyridin-4-yl-amine), SB-525334 (6-[2-(1,1-dimethylethyl)-5-(6-methyl-2-pyridinyl)-1H-imidazol-4-yl]quinoxaline), LY-364947 (4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]quinoline), LY2157299 (4-[2-(6-methyl-pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]quinoline-6-carboxylic acid amide), TGF-β RI Kinase Inhibitor II 616452 (2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine), TGF-β RI Kinase Inhibitor III 616453 (2-(5-benzo[1,3]dioxol-4-yl-2-tert-butyl-1H-imidazol-4-yl)-6-methylpyridine, HCl), TGF-β RI Kinase Inhibitor IX 616463 (4-((4-((2,6-dimethylpyridin-3-yl)oxy)pyridin-2-yl)amino)benzenesulfonamide), TGF-β RI Kinase Inhibitor VII 616458 (1-2-((6,7-dimethoxy-4-quinolyl)oxy)-(4,5-dimethylphenyl)-1-ethanone), naphthyridine (6-(2-tert-butyl-5-(6-methyl-pyridin-2-yl)-1H-imidazol-4-yl)-quinoxaline), AP12009 (TGF-β2 antisense compound "Trabedersen"), Belagenpumatucel-L (TGF-β2 antisense gene-modified allogeneic tumor cell vaccine), CAT-152 (Glaucoma-lerdelimumab (anti-TGF-β-2 monoclonal antibody)), CAT-192 (Metelimumab (human IgG4 monoclonal antibody that neutralizes TGF-β1)), GC-1008 (anti-TGF-β monoclonal antibody), etc.

[0035] The concentration of the TGF-β inhibitor contained in the medium varies depending on the type and is not particularly limited. For example, when A83-01 is used as the TGF-β inhibitor, the concentration can be 0.1 μM to 5 μM, preferably 0.2 μM to 3 μM, more preferably 0.3 μM to 1 μM, and even more preferably 0.4 μM to 0.8 μM. More specifically, the concentration of A83-01 can be 0.5 μM.

[0036] The culture conditions for bat tissue organoids and bat lung organoids are not particularly limited, and reference can be made to, for example, Zhou J, et al., Nat Med., 26(7):1077-1083, 2020, Ameen A. Salahudeen et al., Nature volume 588, pages 670-675 (2020), Sato et al., Nature, 2009, and Sato T et al., Gastroenterology. 2011 Nov;141(5):1762-1772. When culturing bat tissue organoids and bat lung organoids with reference to these, the culture temperature can be set to 30-40°C, with approximately 37°C being most preferred.

[0037] By using the bat tissue organoid culture medium and bat lung organoid culture medium of the present invention, bat tissue organoids and bat lung organoids can be produced with excellent efficiency. Furthermore, by using the bat tissue organoid culture medium and bat lung organoid culture medium of the present invention, bat tissue organoids and bat lung organoids can be cultured for longer periods than with conventional culture methods. Thus, by applying the present invention, bat tissue organoids and bat lung organoids can be produced and grown and maintained for long periods, allowing for research such as virus infection experiments using bat tissue organoids and bat lung organoids.

[0038] In particular, some viruses that live in bats, such as the novel coronavirus and Ebola hemorrhagic fever virus, cause infectious diseases in humans. By applying the present invention, bat tissue organoids and bat lung organoids can be used to elucidate the defense mechanisms that bats have against these viruses. Furthermore, by applying the present invention, various applications are possible, such as the development of antiviral drugs using bat tissue organoids and bat lung organoids, the creation of infection simulation models by co-culturing them with human organoids, and the application of viral genome mutations to prediction models.

[0039] The bat tissue organoids and bat lung organoids to be cultured can be produced using the above-mentioned bat tissue organoid medium and bat lung organoid medium.In addition, the bat tissue organoids and bat lung organoids to be cultured can also be obtained by methods other than the above-mentioned bat tissue organoid medium and bat lung organoid medium, for example, Zhou J, et al., Nat Med., 26(7):1077-1083, 2020; Ameen A. Salahudeen et al., Nature volume 588, pages 670-675 (2020); Sato et al., Nature, 2009 and Sato T et al., Gastroenterology. 2011 Nov;141(5):1762-1772, etc.

[0040] In the method for producing bat tissue organoids according to the present invention, first, cells or tissues derived from bats are collected, and the cells or tissues are mixed with an extracellular matrix. By mixing the cells or tissues with the extracellular matrix, the cells or tissues can be held in the extracellular matrix. Then, the cells or tissues mixed with the extracellular matrix are cultured using the bat tissue organoid medium containing the above-mentioned TGF-α. This results in the production of bat tissue organoids. According to the method for producing bat tissue organoids according to the present invention, bat tissue organoids can be produced efficiently.

[0041] In the method for producing bat tissue organoids according to the present invention, it is preferable to use cells derived from bats. For example, tissue collected from a bat is mechanically suspended in a culture medium to prepare a cell suspension, and bat-derived cells can be obtained by repeatedly filtering, sedimenting, washing, etc. Bat-derived cells are preferably bat-derived stem cells.

[0042] Extracellular matrix (ECM) is a structure that serves as a scaffold for cell proliferation and is composed of water and various polysaccharides, proteins, glycoproteins, etc. Extracellular matrix can be prepared using ECM-producing cells such as fibroblasts and chondrocytes, and commercially available extracellular matrices such as Matrigel (registered trademark, Corning) can also be used.

[0043] In the method for producing bat tissue organoids, cells or tissues can be cultured by directly or indirectly contacting collected bat-derived cells or tissues or the extracellular matrix that holds them with the bat tissue organoid medium described above, which allows the cultured cells or tissues to proliferate and / or differentiate, producing bat tissue organoids.

[0044] The medium used to produce bat tissue organoids may be a single medium, or multiple types of medium. For example, a growth medium (Expansion medium) that primarily promotes cell proliferation and a differentiation medium (Differentiation medium) that primarily promotes cell differentiation may be used. When using a growth medium and a differentiation medium, bat tissue organoids can be produced by first contacting the growth medium with the extracellular matrix and culturing, then replacing the medium and contacting the differentiation medium with the extracellular matrix and culturing. Alternatively, bat tissue organoids can also be produced by first contacting the differentiation medium with the extracellular matrix and culturing, then replacing the medium and contacting the growth medium with the extracellular matrix and culturing. The bat tissue organoid medium of the present invention containing the above-mentioned TGF-α can be used as both a growth medium and a differentiation medium, but it is particularly preferred to use it as a growth medium.

[0045] On the other hand, in the method for producing bat pulmonary organoids according to the present invention, first, bat pulmonary cells or lung tissue are collected, and the lung cells or lung tissue are mixed with an extracellular matrix. By mixing the lung cells or lung tissue with an extracellular matrix, the lung cells or lung tissue can be held in the extracellular matrix. Then, the lung cells or lung tissue mixed with the extracellular matrix are cultured using the bat pulmonary organoid medium containing the above-mentioned TGF-α and / or FGF7. This results in the production of bat pulmonary organoids. According to the method for producing bat pulmonary organoids according to the present invention, bat pulmonary organoids can be produced efficiently.

[0046] The present inventors are the first in the world to successfully produce bat lung organoids. The bat lung organoids of the present invention express at least one of the respiratory goblet cell marker MUC5AC, the airway basal cell marker CK5, and the lung cell marker surfactant-related protein C (SFTPC), thereby reproducing the constituent cells of bat lung tissue. Therefore, the bat lung organoids of the present invention can be suitably used for basic research, and in particular, for basic research on respiratory infectious viruses such as SARS-CoV (SARS coronavirus) and SARS-CoV2 (novel coronavirus). The bat lung organoids of the present invention preferably express all of MUC5AC, CK5, and SFTPC. In this case, they recapitulate the majority of the constituent cells of lung tissue, making them even more suitable for basic research. [Example]

[0047] The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention is not limited to the following examples.

[0048] Example 1 [Creation of bat intestinal organoids (BIO) and bat lung organoids (BLO)] To generate bat intestinal organoids (BIO) and bat lung organoids (BLO), intestinal and lung tissues were collected from bats that died naturally in zoos. Using the collected intestinal and lung tissues, bat intestinal organoids (BIO) and bat lung organoids (BLO) were generated using the following method.

[0049] First, the collected tissue was transferred to a 10 cm dish containing 2 ml of PBS to prevent drying. Then, using surgical scissors, tissue pieces approximately 1 cm square were cut into pieces within the dish. The resulting tissue pieces were transferred to another 10 cm dish and washed three times with 2 ml of PBS to remove any attached blood components. The dish was then placed on ice, and the tissue pieces were cut using ophthalmic scissors until they reached a viscous consistency. The tissue pieces were transferred to a 15 ml tube containing 500 μl of Liberase TH 1.25 mg / ml (Sigma) and 450 μl of Advanced DMEM medium (Gibco) and pipetted 10 times using a 1 ml pipetman. The tube was then placed in a 37°C incubator and shaken for 15 minutes. After 15 minutes, the tube was removed, pipetted 10 times using a 1 ml pipetman, and shaken for another 15 minutes. The tube was removed and pipetted 10 times again to confirm that the cell suspension was translucent, and then passed through a 100 μm cell strainer. If tissue fragments were still visible after 30 minutes of shaking, the tube was centrifuged at 600 × g for 3 minutes, after which the supernatant was removed. 1 ml of TrypLE (Gibco) was added and the tube was placed in an incubator for 5 minutes. The tube was removed and pipetted as described above, then passed through a cell strainer. The filtered cell suspension was centrifuged at 600 × g for 3 minutes. After removing the supernatant, 8 ml of PBS was added and pipetted 10 times using a 1 ml pipette. This procedure was repeated three times. The supernatant was removed, and 40 μl of Matrigel (Corning) was added per well of a 24-well plate, depending on the amount of cell sediment, and gently mixed several times using a 200 μl pipette. 40 μl of Matrigel containing cell components was seeded into each well and placed in an incubator at 37°C for 30 minutes. After that, 500 μl / well of medium warmed to 37°C was added and culturing was initiated.

[0050] Using the above method, we generated bat intestinal organoids (BIO) and bat lung organoids (BLO) (Figure 1). As shown in Figure 1, the bat intestinal organoids generated in this example had a structure mimicking the intestinal mucosal epithelial cells, and the bat lung organoids had a spherical morphology.

[0051] [Evaluation of bat intestinal organoids (BIO) and bat lung organoids (BLO)] Histopathological analysis was performed on the BIO and BLO tissues prepared as described above. First, HE staining was performed according to standard methods. HE staining images of BIO, BLO, and the original tissues (intestinal tissue, lung tissue) are shown in Figure 2. As shown in Figure 2, organoids derived from each tissue of BIO and BLO were successfully prepared. As can be seen from Figure 2, BIO and BLO mimicked the multicellular structure of the bat small intestine and lung epithelium, respectively, and reproduced the histological characteristics of the original tissues.

[0052] Next, electron microscopy images of the prepared BIO and BLO organoids are shown in Figure 3 (top row: BIO, bottom row: BLO). In Figure 3, C indicates villi, E indicates epithelial cells, G indicates goblet cells, P indicates Paneth cells, V indicates microvilli, LB indicates lamellae, ER indicates rough endoplasmic reticulum, TJ indicates tight junctions, m indicates mitochondria, and n indicates nuclei. As shown in Figure 3, observation of the ultrastructure of each organoid revealed that they mimicked intestinal and pulmonary tissues. In BIO, absorptive epithelial cells with characteristic microvilli, goblet cells, and Paneth cells were observed. In BLO, organoids consisted of well-differentiated pseudoairway epithelium, including basal cells, secretory cells, and multiciliated cells.

[0053] [Analysis and evaluation of BIO and BLO constituent cells] In addition, to identify the constituent cells of the constructed BIO and BLO, immunofluorescence staining was used to confirm the expression of specific markers in intestinal and lung tissues.

[0054] In this example, we evaluated the expression of E-cadherin, an epithelial cell marker; CK20, a mature intestinal marker; MUC2, an intestinal goblet cell marker; LGR5, an intestinal stem cell marker; MUC5AC, a respiratory goblet cell marker; CK5, an airway basal cell marker; and surfactant-associated protein C (SFTPC), a lung cell marker.

[0055] First, we prepared frozen sections from organoids and excised tissues. The slides were washed with PBS for 5 minutes with shaking. After removing them from the PBS and drying, 50 μl of 1.5% normal goat serum (NGS) was added per section and allowed to stand in a humidified box for 30 minutes at room temperature. After removing the NGS, the primary antibody was diluted 1:100 in PBS and added at 50 μl per section, as described above, and allowed to stand overnight at 4°C. The next day, the slides were washed three times for 5 minutes each with PBS. 50 μl of fluorescent secondary antibody and Hexst, each diluted in PBS, was added per section and allowed to stand for 60 minutes in the dark. The slides were then washed three times for 5 minutes each with PBS, dried, mounted with a coverslip, and then observed under a confocal laser scanning microscope (Zeiss).

[0056] Figure 4 shows the results of marker expression in BIO and the original intestinal tissue, and Figure 5 shows the results of marker expression in BLO and the original lung tissue. Expression of the epithelial cell marker E-cadherin was confirmed in BIO, BLO, and their original tissues (Figures 4 and 5). Furthermore, expression levels of the mature intestinal marker CK20 and the intestinal goblet cell marker MUC2 were similar in BIO and bat intestinal tissue, while expression levels of the intestinal stem cell marker LGR5 were higher in organoids compared to the original tissue (Figure 4). These data indicate that BIO recapitulates the constituent cells of intestinal tissue with high stemness. Furthermore, expression of the respiratory goblet cell marker MUC5AC, the airway basal cell marker CK5, and the pneumocyte cell marker surfactant-related protein C (SFTPC) was observed in BLO and bat lung tissue, but expression of the Clara cell marker SCGB1A1 was not observed in BLO (Figure 5). These data indicate that BLO recapitulates most of the constituent cells of lung tissue.

[0057] [Searching for optimal supplements for long-term BIO and BLO culture] When culturing the BIO and BLO prepared as described above, we searched for medium components that would further promote cell proliferation and organoid formation. Specifically, we prepared culture solutions with the medium composition shown in Table 1 to which various medium components were added, and compared the cell proliferation rate due to the addition of culture components, with the control set at 100%.

[0058] [Table 1]

[0059] The medium components examined in this example are shown in Table 2.

[0060] [Table 2]

[0061] BIO and BLO were cultured for 7 days using culture media supplemented with the medium components shown in Table 2. Images of the organoids after culture and the results of measuring the cell proliferation rate are shown in Figures 6 and 7. The cell proliferation rate was calculated as follows: Culture media were prepared by adding the various medium components shown in Table 2 to the medium composition shown in Table 1. Fluorescence intensity was measured using a plate reader (TECAN), and the cell proliferation rate of the control group containing only the medium composition shown in Table 1 was set at 100%, and the other culture media components were used to compare the results.

[0062] Figure 6 shows photographs of BIO cells cultured with supplements and the results of measuring the proliferation rate of BIO cells (day 7). As shown in Figure 6, there were significant differences in the proliferation rate of BIO cells depending on the supplements tested. Specifically, when WNR, TGF-α, or EGF was added to the basal medium (Cont), the proliferation rate of BIO cells was significantly improved compared to when the basal medium (Cont) was used. On the other hand, FGF2, FGF7, FGF10, and IGF were not found to have an effect of improving the proliferation rate of BIO cells. These results demonstrate that the addition of at least one component selected from the group consisting of WNR, EGF, and TGF-α is preferable for long-term BIO cell culture.

[0063] Photographs of BLOs cultured with supplements and the results of measuring the proliferation rate of BLOs (day 7) are shown in Figure 7. When TGF-α, EGF, WNR, or FGF7 was added to the basal medium (Cont), the proliferation rate of BLOs was significantly improved compared to when the basal medium (Cont) was used. In particular, the use of TGF-α significantly improved the proliferation rate of BLOs. These results demonstrate that the addition of at least one component selected from the group consisting of WNR, EGF, FGF7, and TGF-α is preferable for long-term BLO culture.

[0064] [Confirmation of the expression of coronavirus infection-related proteins in BIO and BLO] The novel coronavirus (SARS-CoV-2), which causes the novel coronavirus disease (COVID-19), has been confirmed to enter cells using the transmembrane cellular proteases TMPRSS2 and ACE2 receptors. Therefore, we analyzed the expression of ACE2 and TMPRSS2 in BIO and BLO cells using immunofluorescence staining.

[0065] In this example, the expression of E-cadherin, an epithelial cell marker, CK20, a mature intestinal marker, TMPRSS2, a transmembrane cellular protease and ACE2 receptor, which are intestinal goblet cell markers, was evaluated.

[0066] First, we prepared frozen sections from organoids and excised tissues. The slides were washed with PBS for 5 minutes with shaking. After removing them from the PBS and drying, 50 μl of 1.5% normal goat serum (NGS) was added per section and allowed to stand in a humidified box for 30 minutes at room temperature. After removing the NGS, the primary antibody was diluted 1:100 in PBS and added at 50 μl per section, as described above, and allowed to stand overnight at 4°C. The next day, the slides were washed three times for 5 minutes each with PBS. 50 μl of fluorescent secondary antibody and Hexst, each diluted in PBS, was added per section and allowed to stand for 60 minutes in the dark. The slides were then washed three times for 5 minutes each with PBS, dried, mounted with a coverslip, and then observed under a confocal laser scanning microscope (Zeiss).

[0067] The results of observing ACE2 and TMPRSS2 expression in BIO and the original intestinal tissue are shown in Figure 8, and the results of observing ACE2 and TMPRSS2 expression in BLO and the original lung tissue are shown in Figure 9. As shown in Figures 8 and 9, ACE2 was clearly expressed on the apical surface or basement membrane of both organoids, in the epithelial layer of the intestinal tissue, and in the alveolar cells of the lung tissue. Also, as shown in Figures 8 and 9, TMPRSS2 was expressed not only in the basal layer but also in various regions of both organoids and their original tissues. These data suggest that long-term culture of BIO and BLO may enable basic research, such as infection experiments with various bat-hosted viruses.

Claims

1. At least one component selected from the group consisting of TGF-α, FGF7, and EGF, and Wnt agonists, BMP inhibitors, growth factors, and TGFβ inhibitors A composition for a bat tissue organoid culture medium comprising:

2. The composition for bat tissue organoid culture medium according to claim 1, wherein the tissue is the intestine or lung.

3. A bat tissue organoid culture medium comprising the composition for a bat tissue organoid culture medium described in claim 1 or 2, and a serum-free basic cell culture medium.

4. The bat tissue organoid culture medium described in claim 3, wherein the concentration of the Wnt agonist is 400 ng / mL to 600 ng / mL.

5. A bat tissue organoid culture medium as described in claim 3 or 4, wherein the concentration of the BMP inhibitor is 50 ng / mL to 150 ng / mL.

6. A bat tissue organoid culture medium described in any one of claims 3 to 5, wherein the concentration of the growth factor is 0.5 mM to 15 mM.

7. A bat tissue organoid culture medium described in any one of claims 3 to 6, wherein the concentration of the TGFβ inhibitor is 0.4 μM to 0.8 μM.

8. A bat tissue organoid culture medium described in any one of claims 3 to 7, wherein the concentration of TGF-α is 10 ng / mL to 100 ng / mL.

9. A bat tissue organoid culture medium described in any one of claims 3 to 8, wherein the concentration of FGF7 is 1 ng / mL to 10 ng / mL.

10. A bat tissue organoid culture medium described in any one of claims 3 to 9, wherein the concentration of the EGF is 40 ng / mL to 100 ng / mL.

11. A method for culturing bat tissue organoids, comprising culturing bat tissue organoids using the bat tissue organoid culture medium according to any one of claims 3 to 10.

12. combining bat-derived cells or tissue with an extracellular matrix; Culturing the cells or tissue mixed with the extracellular matrix using the bat tissue organoid culture medium according to any one of claims 3 to 10; A method for producing bat tissue organoids, comprising:

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