Modeling of neural sialadenosis and testing method of drug efficacy using co-culture of human salivary gland organoid and neuroblastoma

Abstract

The present invention relates to a co-culture of salivary gland organoids and neuroblastomas, a method for preparing the same, and uses. The present invention provides a model of neurosalivary gland disease by culturing epithelial cell-based organoids derived from human salivary gland tissue and co-culturing them with neuroblastomas to establish a cell-cell direct interaction environment. Furthermore, by utilizing the said neurosalivary gland disease model to screen for novel therapeutic agents and verify the responsiveness of botulinum toxin drugs, it can contribute to providing personalized treatment methods for patients.

Description

Technology Field

[0001] The present invention relates to a co-culture of salivary gland organoids and neuroblastomas, a method for preparing the same, and uses. Background Technology

[0002] Sialosis (sialadenosis) is a non-inflammatory, non-neoplastic, parenchymal lesion clinically characterized by recurrent bilateral swelling of the salivary glands. It typically manifests as bilateral and symmetrical lesions and is often painless, though tenderness may be present in some cases. Known causes of sialosis include nutritional deficiencies, various types of endocrine abnormalities, or autonomic nervous system disorders; however, neurogenic sialosis also exists, resulting from nervous system abnormalities or hyperactivity caused by factors such as unclear causes or uncontrollable stress. Generally, botulinum toxin is used to treat such neurogenic sialosis.

[0003] Botulinum toxin refers to Clostridium botulinum ( Clostridium botulinum It is a neurotoxic protein produced by bacteria. Functionally, it inhibits the secretion of the neurotransmitter acetylcholine at the presynapse of the neuromuscular junction, thereby inducing flaccid paralysis of the muscles. Recently, therapeutic attempts have been made to utilize the paralyzing mechanism of botulinum toxin for various autonomic nervous system dysfunctions and indications. However, while botulinum toxin has therapeutic effects in various diseases, it is highly toxic and can be fatal even in minute amounts; therefore, precise control of concentration is essential when using it in vivo.

[0004] Currently, to confirm the potency of Botox, LD50 is obtained through intraperitoneal injection in mice.50 Measurement methods and the CBPA method using neuroblastoma cell lines are widely used, and other methods using ELISA are also being developed. However, existing mouse-based methods are scheduled to be banned in the near future due to ethical issues, and there are already countries where they have been banned. Furthermore, since the CBPA method is patented by multinational pharmaceutical companies, there is a problem of reduced price competitiveness due to royalty payments.

[0005] Accordingly, the inventors completed the present invention by manufacturing a neurogenic salivary gland disease model based on human salivary gland organoids and confirming that it can be used to screen for novel therapeutic agents and measure the responsiveness of botulinum toxin drugs. The problem to be solved

[0006] One objective of the present invention is to provide a co-culture of salivary gland organoids and neuroblastomas and a method for preparing the same.

[0007] Another objective of the present invention is to provide a neurogenic salivary gland disease model comprising a co-culture of salivary gland organoids and neuroblastoma, and a method for preparing the same.

[0008] Another objective of the present invention is to provide a screening method for a treatment for neurogenic salivary gland disease.

[0009] Another objective of the present invention is to provide a method for confirming the responsiveness of a botulinum toxin drug to a neurogenic salivary gland model. means of solving the problem

[0010] In the following, redundant details will be omitted to prevent clutter. In other words, the content of the invention is not limited solely to the following, and should be interpreted in accordance with the overall context of the invention.

[0011] The present invention will be described in detail below.

[0012] The present invention provides a method for preparing a co-culture of salivary gland organoids and neuroblastomas.

[0013] The above method for preparing a co-culture of salivary gland organoids and neuroblastomas is carried out through the following steps:

[0014] (a) A step of culturing salivary gland organoids from salivary gland tissue-derived epithelial cells in a culture medium containing a TGF-β inhibitor, a Wnt activator, a BMP inhibitor, an FGF family, a receptor tyrosine kinase ligand, and a ROCK inhibitor;

[0015] (b) a step of culturing a neuroblastoma; and

[0016] (c) A step of preparing a co-culture by mixing and culturing the salivary gland organoid and neuroblastoma in a culture medium containing a TGF-β inhibitor, a Wnt activator, a BMP inhibitor, an FGF family, a receptor tyrosine kinase ligand, a ROCK inhibitor, and a cAMP activator.

[0017] In the present invention, "co-culture" refers to the result of co-culturing homologous or heterologous cells maintained under conditions suitable for mutual proliferation, and collectively refers to the entirety including the co-cultured cells, extracellular materials used in the co-culture, and materials produced during the co-culture.

[0018] In the present invention, "salivary gland" refers to an organ that produces and secretes saliva. The salivary glands are classified into major salivary glands, such as the parotid gland, submaxillary gland, and sublingual gland, and minor salivary glands, such as mucous glands present in the mucous membrane of the oral cavity, which are distributed in various parts of the oral mucosa.

[0019] In the present invention, "organoid" refers to a cell aggregate formed by aggregating and recombining cells isolated from stem cells or organ-derived cells through culture, and may include an organoid or cell cluster formed from a suspension cell culture.

[0020] In the present invention, "neuroblastoma" is a tumor of the nervous system originating from nerve tissue; when it occurs in the central nervous system, it mainly develops in the lateral ventricles of the brain and is known to cause symptoms such as headache and reduced reaction speed due to increased intracranial pressure.

[0021] The culture medium used in step (a) of the method for preparing a co-culture of a salivary gland organoid and a neuroblastoma according to the present invention is provided for culturing a salivary gland organoid from epithelial cells derived from salivary gland tissue and essentially comprises a TGF-β inhibitor, a Wnt activator, a BMP inhibitor, an FGF family, a receptor tyrosine kinase ligand, and a ROCK inhibitor.

[0022] In the present invention, "culture medium" refers to a solution for maintaining or culturing a cell population containing nutrients that maintain cell viability and support proliferation.

[0023] The culture medium used in the present invention comprises a basic medium. The basic medium is any basic medium suitable for the culture of animal or human cells.

[0024] The above basic medium typically contains a number of components necessary to support the maintenance of cultured cells. A suitable combination of components can be easily formulated by a skilled person with consideration of the following. Additionally, it includes a nutrient solution containing common standard cell culture components, e.g., amino acids, vitamins, lipid supplements, inorganic salts, carbon energy sources, and buffers.

[0025] The above basic media may be commercially available and include, but are not limited to, Dulbecco's Modified Eagles Media (DMEM), Minimum Essential Media (MEM), KnockOut-DMEM (KO-DMEM), Glasgow's Minimum Essential Media (G-MEM), Eagle's Minimum Essential Medium (EMEM), Basal Medium Eagle (BME), DMEM / Ham's F12, Advanced DMEM / Ham's F12, Iscove's Modified Dulbecco's Media and Minimum Essential Media (MEM), Ham's F-10, Ham's F-12, Medium 199, RPMI 1640 medium, and KnockOut Serum replacement XenoFree medium. For example, the above basic media may be Advanced DMEM / F12 medium.

[0026] The above TGF-β inhibitor is any substance that inhibits the function of the TGF-β receptor, for example, a protein, peptide, or small molecule, and may be any one selected from the group consisting of A83-01, SB-431542, SB-505124, SB-525334, SD-208, LY-36494, and SJN-2511. Preferably, it may be A83-01. More preferably, it may be A83-01 in an amount of 0.5 μM to 15 μM. More preferably, it may be A83-01 in an amount of 0.5 μM to 5 μM.

[0027] The above Wnt activator may be any one selected from the group consisting of R-spondin 1, R-spondin 2, R-spondin 3, and R-spondin 4. Preferably, it may be R-spondin 3.

[0028] The above BMP inhibitor is an agent that binds to a BMP molecule to form a complex, and may be, for example, an antibody that binds to said receptor, an agent that binds to a BMP receptor and prevents the binding of a BMP ligand to said receptor. The above BMP inhibitor may be a protein or a small molecule and may be natural, modified, and / or partially or entirely synthetic. Additionally, the above BMP inhibitor may be any one selected from the group consisting of Noggin, Dorsomorphin, DMH1, and LDN-193189. Preferably, it may be Noggin.

[0029] The above-mentioned FGF (fibroblast growth factor) family is a potent factor that regulates cell proliferation and differentiation, playing a particularly important role in the normal development of stem cells, tissue maintenance, wound healing, and angiogenesis. The above-mentioned FGF family may be one or more selected from the group consisting of FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, and FGF10. Preferably, it may be FGF2 and FGF10. More preferably, it may be FGF2 and / or FGF10 in an amount of 5 ng / mL to 20 ng / mL. More preferably, it may be FGF2 and / or FGF10 in an amount of 5 ng / mL to 10 ng / mL.

[0030] The receptor tyrosine kinase ligand may be any one selected from the group consisting of Neuregulin β1 (NRG1), Heregulin β1 (HRG1), epidermal growth factor (EGF), transforming growth factor-α (TGF-α), basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF), hepatocyte growth factor (HGF), and keratinocyte growth factor (KGF). Preferably, it may be Neuregulin β1 (NRG1). More preferably, it may be Neuregulin β1 (NRG1) at a concentration of 5 ng / mL to 20 ng / mL. More preferably, it may be Neuregulin β1 (NRG1) at a concentration of 5 ng / mL to 10 ng / mL.

[0031] The above ROCK (Rho-associated protein kinase) inhibitor acts to inhibit the activity of serine / threonine kinases that act as target proteins for Rho (Rho A, Rho B, and Rho C), and may be any one selected from the group consisting of R-(+)-trans-4-(1-Aminoethyl)-N-(4-pyridyl)cyclohexane carboxamide dihydrochloride monohydrate (Y-27632), Fasudil, and H-1152. Preferably, it may be Y-27632. More preferably, it may be Y-27632 at a concentration of 5 μM to 20 μM. More preferably, it may be Y-27632 at a concentration of 5 μM to 15 μM.

[0032] In addition to the essential composition described above, the culture medium of the present invention may further comprise one or more additional components selected from the group consisting of Glutamax, HEPES, Primocin, Prostaglandin E2 (PGE2), N-acetylcysteine ​​(NAC), B27, and Nicotinamide.

[0033] The above B27 may be replaced with a generic formulation containing one or more of the ingredients selected from the following list: biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinyl acetate, sodium selenite, tri-iodothyronine (T3), DL-alpha-tocopherol (vitamin E), albumin, insulin, and transferrin.

[0034] The concentration of each of these additional components can be appropriately controlled within the general range typically used in culture media.

[0035] More specifically, the culture medium used in step (a) of the method for preparing a co-culture of salivary gland organoids and neuroblastomas according to the present invention may comprise A83-01, R-spondin 3, Noggin, FGF2, FGF10, Neuregulin β1 (NRG1), and Y-27632.

[0036] The salivary gland tissue-derived epithelial cells according to the present invention may be derived from the salivary gland tissue of a subject. Such salivary gland tissue may be normal salivary gland tissue or the salivary gland tissue of a patient. Additionally, they may be derived from commercially available salivary gland cells and / or tissue.

[0037] The culture medium used in step (b) of the method for preparing a co-culture of salivary gland organoids and neuroblastomas according to the present invention may further include common antibiotics and / or serum components under a commonly used culture medium. For example, it may include antibiotics such as P / S or Primocin, and serum components such as FBS.

[0038] These neuroblastomas can be used by differentiating commercially available Neuro-2a cells, SK-N-SH cells, SH-SY5Y cells, B35 cells, IMR-32 cells, or NTERA-2 cells that produce teratomas. Additionally, cells obtained from and cultured from a patient's neuroblastoma can be used.

[0039] The culture medium used in step (c) of the method for preparing a co-culture of salivary gland organoids and neuroma according to the present invention is provided for culturing a mixture of salivary gland organoids and neuroma and essentially comprises a TGF-β inhibitor, a Wnt activator, a BMP inhibitor, an FGF family, a receptor tyrosine kinase ligand, a ROCK inhibitor, and a cAMP activator.

[0040] The details regarding the above TGF-β inhibitors, Wnt activators, BMP inhibitors, FGF family, receptor tyrosine kinase ligands, and ROCK inhibitors are as previously mentioned.

[0041] The above cAMP (cyclic adenosine monophosphate) activator may be any one selected from the group consisting of forskolin, aminophylline, pentoxifylline, theophylline, isobutyl-methylxanthine (IBMX), and dehydroabietic acid (DAA). Preferably, it may be forskolin. More preferably, it may be forskolin at a concentration of 5 μM to 15 μM.

[0042] More specifically, the culture medium used in step (c) of the method for preparing a co-culture of salivary gland organoids and neuroblastomas according to the present invention may include A83-01, R-spondin 3, Noggin, FGF2, FGF10, Neuregulin β1 (NRG1), Y-27632, and Forskolin.

[0043] The present invention provides a culture medium composition for co-culture of salivary gland organoids and neuroblastomas comprising the above-mentioned TGF-β inhibitor, Wnt activator, BMP inhibitor, FGF family, receptor tyrosine kinase ligand, ROCK inhibitor, and cAMP activator.

[0044] More specifically, the culture medium composition for the above-mentioned co-culture may essentially include A83-01, R-spondin 3, Noggin, FGF2, FGF10, Neuregulin β1 (NRG1), and Y-27632.

[0045] The present invention provides a co-culture of salivary gland organoids and neuroblastomas prepared according to the above-described manufacturing method.

[0046] The present invention provides a method for manufacturing a neurogenic salivary gland model.

[0047] The method for manufacturing the above-mentioned neurosalivary gland model is carried out through the following steps:

[0048] (a) A step of culturing salivary gland organoids from salivary gland tissue-derived epithelial cells in a culture medium containing a TGF-β inhibitor, a Wnt activator, a BMP inhibitor, an FGF family, a receptor tyrosine kinase ligand, and a ROCK inhibitor;

[0049] (b) Step of culturing the neuroblastoma;

[0050] (c) a step of preparing a co-culture by mixing and culturing the salivary gland organoid and neuroblastoma in a culture medium containing a TGF-β inhibitor, a Wnt activator, a BMP inhibitor, an FGF family, a receptor tyrosine kinase ligand, a ROCK inhibitor, and a cAMP activator; and

[0051] (d) A step of treating the above co-culture with a neurotransmitter.

[0052] In this invention, "sialosis" refers to a non-inflammatory, non-neoplastic, parenchymal lesion clinically characterized by recurrent bilateral edema of the salivary glands. It primarily occurs in the parotid gland (below the ear) and, rarely, in the submandibular and sublingual glands. The edema is bilateral and symmetrical, and while often painless, tenderness may be present in some cases. The causes of sialosis are known to include nutritional deficiencies, various types of endocrine abnormalities, or autonomic nervous system disorders.

[0053] In the present invention, "neurogenic salivary gland syndrome" refers to salivary gland syndrome caused by abnormalities or hyperactivity of the nervous system resulting from factors such as unclear causes or uncontrollable stress.

[0054] In the present invention, "neurotransmitter" refers to a signaling substance secreted by a nerve cell, which moves through a synapse and plays a role in raising or lowering the electrical potential of an adjacent nerve cell. The neurotransmitter may be any one selected from the group consisting of acetylcholine, dopamine, norepinephrine, serotonin, and histamine. Preferably, it may be acetylcholine.

[0055] According to one embodiment of the present invention, the co-culture prepared according to steps (a) to (c) may be treated with acetylcholine, a neurotransmitter, and / or neostigmine, an inhibitor of its degradation. In this case, a neurogenic salivary gland disease model can be prepared by increasing the amount of acetylcholine in the neuroblastoma by neostigmine and stimulating the parasympathetic nerves by acetylcholine, thereby expanding the lumen of the salivary gland organoid.

[0056] Preferably, the method according to the present invention may include any one selected from the group consisting of acetylcholine, dopamine, norepinephrine, serotonin, and histamine; and treating neostigmine.

[0057] The present invention provides a culture medium composition for co-culture of salivary gland organoids and neuroblastomas.

[0058] The above medium composition may include a TGF-β inhibitor, a Wnt activator, a BMP inhibitor, an FGF family, a receptor tyrosine kinase ligand, a ROCK inhibitor, and a cAMP activator, and preferably may include A83-01, R-spondin 3, Noggin, FGF2, FGF10, Neuregulin β1 (NRG1), and Y-27632.

[0059] The present invention provides a neurogenic salivary gland model manufactured according to the above manufacturing method.

[0060] The present invention provides a screening method for a treatment for neurogenic salivary gland disease.

[0061] Specifically, the present invention provides a screening method for a neurosalivary gland disease treatment comprising the step of treating a neurosalivary gland disease model prepared according to the above manufacturing method with a test substance.

[0062] More specifically, the screening method for a neurosalivary gland disease treatment may further include the step of selecting a test substance as a neurosalivary gland disease treatment if, compared to a control group not treated with the test substance, the expression level of one or more selected from the group consisting of KRT7, KRT19, AchE, and Aqp5 is reduced in a neurosalivary gland disease model treated with the test substance.

[0063] In the present invention, "screening" refers to selecting a substance having a specific desired property from a candidate group of various substances using a specific operation or evaluation method.

[0064] The term "test substance" used in reference to the screening method of the present invention refers to an unknown substance used in screening to test whether it affects the expression levels of KRT7, KRT19, AchE, and Aqp5. The test substance includes, but is not limited to, chemical substances, nucleotides, antisense RNA, shRNA, miRNA, siRNA (small interference RNA), and natural product extracts.

[0065] The term "selection" as used in referring to the screening method of the present invention means selecting a substance that reduces the expression level of one or more selected from the group consisting of specific selection markers, e.g., KRT7, KRT19, AchE, and Aqp5.

[0066] In the present invention, "KRT7 (Keratin 7)" is a member of the keratin gene family and corresponds to Type II cytokeratin. Type II cytokeratin consists of a basic neutral protein arranged in pairs of heteromorphic keratin chains that are co-expressed during the differentiation of simple and stratified epithelial tissues. Such Type II cytokeratin is specifically expressed in internal organs, specifically in the simple epithelial layer, glandular ducts, and blood vessels.

[0067] In the present invention, "KRT19 (Keratin 19)" is a member of the keratin gene family and corresponds to type I cytokeratin, and specifically is known to be expressed in the outer epidermis, which is a transient superficial layer covering the epidermis during development.

[0068] In the present invention, "AchE (acetylcholinesterase)" plays a role in terminating neurotransmission at synapses by breaking down the neurotransmitter acetylcholine into acetate and choline, and is also known to be involved in various stress responses.

[0069] In the present invention, "Aqp5 (Aquaporin 5)" is one of the membrane protein families associated with major intrinsic proteins and is a factor that plays an important role in the production of saliva, tears, and lung secretions.

[0070] The present invention provides a method for confirming the responsiveness of a botulinum toxin drug to a neurogenic salivary gland disease model.

[0071] Specifically, the present invention provides a method for confirming the responsiveness of a botulinum toxin drug to a neurosalivary gland disease model, comprising the step of treating a neurosalivary gland disease model prepared according to the above manufacturing method with a botulinum toxin drug.

[0072] More specifically, the method for confirming the responsiveness of a botulinum toxin drug to the above-mentioned neurosalivary gland disease model may further include the step of comparing the expression level of one or more selected from the group consisting of KRT7, KRT19, AchE, and Aqp5 in the neurosalivary gland disease model treated with the botulinum toxin drug with a control group not treated with the botulinum toxin drug.

[0073] In the present invention, "botulinum toxin" refers to a toxin secreted by anaerobic bacteria such as Clostridium botulinum, C. butyricum, C. baratii, and C. argentinense, of which there are a total of seven types; among these, botulinum toxin types A and B are purified and used medically. The toxin inhibits the secretion of acetylcholine at motor nerve endings, thereby paralyzing the corresponding muscles.

[0074] The above botulinum toxin drug may be Botox®, Meditoxin, Dysport®, Botulax, Myobloc®, BTX, or Xeomin®. Preferably, it may be Botox®. More preferably, it may be Botox® at a concentration of 1 U / mL to 200 U / mL.

[0075] According to one embodiment of the present invention, a botulinum toxin receptor, such as GT1b, can be administered to a neurogenic salivary gland disease model together with the botulinum toxin drug.

[0076] The salivary gland tissue-derived epithelial cells according to the present invention may be derived from the salivary gland tissue of a subject. Such salivary gland tissue may be normal salivary gland tissue or the salivary gland tissue of a patient.

[0077] According to one embodiment of the present invention, the expression levels of lumen-related genes (KRT7 and KRT19), AchE, and Aqp5 in a neurogenic salivary gland disease model can be significantly reduced by treating with the botulinum toxin drug. Effects of the invention

[0078] The present invention provides a model of neurosalivary gland disease by culturing epithelial cell-based organoids derived from human salivary gland tissue and co-culturing them with neuroblastomas to establish a cell-cell direct interaction environment. Furthermore, by utilizing the said neurosalivary gland disease model to screen for novel therapeutic agents and verify the responsiveness of botulinum toxin drugs, it can contribute to providing personalized treatment methods for patients. Brief explanation of the drawing

[0079] Figure 1 is a figure showing the culture time and initial cell number at which gene expression of ChAT, which provides acetylcholine to salivary gland organoids, is high during suspension culture of N2a cells alone, confirmed by qPCR. Figure 2 is a bright-field microscope image showing whether the lumen expands depending on the presence or absence of co-culture of human salivary gland organoids and N2a cells. Figure 3 is a figure showing the results of immunofluorescence staining for the factors expressed in salivary gland organoids co-cultured with N2a cells and salivary gland organoids not co-cultured. Figure 4 is a bright-field microscope view of a group treated with a neurotransmitter (Acetylcholine+Neostigmine) and a group treated with Botox to block the neurotransmitter (GT1b+BoNT / A). Figure 5 shows the expression levels of luminal-related genes (KRT7, KRT19) for a group treated with a neurotransmitter (Acetylcholine+Neostigmine) and a group treated with Botox to block the neurotransmitter (GT1b+BoNT / A). Figure 6 is a schematic diagram showing the experimental schedule to confirm the effect of Botox on salivary gland organoids. Figure 7 shows the changes that occur when mouse salivary gland organoids are treated with various concentrations of acetylcholine. Figure 8 shows the changes that occur when various concentrations of neostigmine are treated to mouse salivary gland organoids. Figure 9 shows the changes that occur when 25 μg / mL of GT1b and various concentrations of Botox are treated to mouse salivary gland organoids. Figure 10 is a schematic diagram showing the experimental schedule for preparing and verifying a co-culture system of mouse salivary gland organoids and N2a cells. Figure 11 is a bright-field microscope view of a co-culture system of mouse salivary gland organoids and N2a cells. Figure 12 shows the results of a single-cell transcriptome analysis performed on a co-culture system of mouse salivary gland organoids and N2a cells. Figure 13 shows the results of confirming factors expressed in a co-culture system of mouse salivary gland organoids and N2a cells by immunofluorescence staining. Figure 14 shows the expression levels of the ChAT gene and AchE gene for the Untreated (-Stim-Botox), Stimulated (+Stim-Botox), and Stimulated+Botox (+Stim+Botox) groups. Figure 15 shows the expression levels of Krt5, Krt7, Aqp5, and Bhlha15 for the Untreated (-Stim-Botox), Stimulated (+Stim-Botox), and Stimulated+Botox (+Stim+Botox) groups. Specific details for implementing the invention

[0080] The following experimental examples are intended to provide experimental examples that are commonly applied to each embodiment according to the present invention.

[0081] Experimental Example 1. Reagents used in the present invention

[0082] Advanced DMEM / F12 (# 12634010, Gibco), HEPES (#15630-080, Gibco), GlutaMAX (# 35050-061, Gibco), Penicillin / Streptomycin (# 15140122, Gibco), Primocin (# ant-pm-2, Invivogen), B-27 (50X) (# 12587-010, Gibco), N-acetyl-cysteine (# A9165, Sigma), A83-01 (# 2939, Tocris), Y-27632 dihydrochloride (# 1254, Tocris), Recombinant Murine R-Spondin-1 (# 315-32, Peprotech), Recombinant Human Heregulin beta-1 (Neuregulin-1; # 100-03, Peprotech), Recombinant Murine FGF- acidic (# 450-33A, Peprotech), Recombinant Murine KGF (# 450-60, Peprotech), Collagenase, type II (# 4176, Worthington), 마트리젤 (# 356231, Corning), Cell Recovery Solution (#354253, Corning), TrypLE express (# 12605-010, Gibco), CellBanker 1 (Zenoaq), 24 well plate for suspension culture (# 677102, Greiner Bio-one), 48 well plate for suspension culture (# 677102, Greiner Bio-one), 25cm 2cell culture plate (# 70025, SPL), Trypan Blue Solution, 0.4% (# 15250-061, Gibco), EMEM (#30-2003, ATCC), Fetal bovine serum (#16000-044, Gibco), PBS (# 14190144, Gibco), Strainer (# 93070, SPL), Falcon 5 mL Round Bottom Polystyrene Test Tube, with Cell Strainer Snap Cap (# 352235, Falcon), Protein LoBind Tube 1.5 mL (#022431081, Eppendorf AG), 5 mL conical Tube (# 51105, SPL), 15 mL conical Tube (# 51115, SPL), 50 mL conical Tube (# 51150, SPL), GT1b (# G3767, Sigma), 보툴렉스주 200 단위 (휴젤), TRIzol (# 15596018, Thermo Fisher Scientific), RT Reagent kit (# RR037A, Takara), SensiFAST SYBR Lo-ROX kit (# BIO-94020, BIOLINE), Acetylcholine chloride (# A2661, Sigma), Neostigmine bromide (# N2001, Sigma), PGE2 (# 2296, Tocris), 포스콜린(Forskolin) (# 1099, Tocris), Recombinant RSPO3-Fc fusion protein conditioned medium (hRSPO3-CM) (R001, U-Protein Express BV), Noggin-Fc Fusion Protein conditioned medium (hNoggin-CM) (# N002, U-Protein Express BV), Recombinant Human FGF-basic (154 a.a.) (# 100-18B, Peprotech), Recombinant Human FGF-10 (# 100-26, Peprotech), Hematoxylin (# 63534, Abcam), Eosin (# 64044, Abcam), 100% Ethanol (# 1.08543.0250, Merck), Periodic Acid Schiff (PAS) stain kit (# ab150680, Abcam), Permount mounting medium (# SP15-100, Fisherbrand).

[0083] Experimental Example 2. Processing and Storage of Human Salivary Gland Stem Cells

[0084] Salivary gland samples were placed in a solution containing 1x Glutamax and 1x HEPES (basal medium) in Advanced DMEM / F12 and stored at 4°C. A digestion solution was prepared by adding 5 mg / mL of Collagenase type II and 10 μM of Y-27632 to Advanced DMEM / F12, calculated at a rate of 1 mL per 50 mg of salivary gland sample. After crushing the salivary gland samples with a blade, they were transferred to a new conical tube along with the digestion solution.

[0085] After incubating at 200 rpm for 1 hour in a 37℃ shaker, the supernatant was removed by centrifugation at 300 g for 5 minutes. 2 mL of TrypLE express and 10 μM of Y-27632 were added to the supernatant-removed cells via pipetting, followed by incubation at 37℃ for 10 minutes.

[0086] Salivary gland samples were filtered through a 100 µm strainer to remove undigested tissue, 2 mL of Advanced DMEM / F12 was added to stop the reaction, and the cell pellet was obtained by centrifugation at 300 g for 5 minutes. At least 2 x 10⁶ cells were used per cryovial (1 mL). 5 After calculating the number of cells to fit into CellBanker 1, the cell pellet was released into 1 mL aliquots in frozen vials and stored at -80℃ for 2 to 3 days, then transferred to a nitrogen tank for long-term storage.

[0087] Experimental Example 3. Culture of salivary gland organoids derived from human salivary gland stem cells

[0088] The cell stock was removed from the nitrogen tank and incubated at 37°C until about half dissolved, then centrifuged at 300g for 5 minutes to remove the supernatant and obtain a cell pellet. 1 mL of organoid culture medium was added to thoroughly loosen the cell pellet, 10 μL was collected and transferred to a 1.5 mL Eppendorf tube, and 10 μL of Trypan Blue Solution 0.4% was added and mixed to check the number and viability of the cells.

[0089] 5.0 x 10 per well in a 48-well plate 3 Cells were dispensed into each well, and 20 μL of Matrigel was added per well and mixed with the cells. The 20 μL mixture of cells and Matrigel was embedded and solidified into a dome shape, after which 300 μL of culture medium was added and incubated at 37°C. The composition of the culture medium used is as shown in Table 1 below.

[0090] Advanced DMEM / F12 Basal medium Glutamax 1 X HEPES 1 X Primocin 0.2% B27 vit A+ 1 X NAC 1 mM Nicotinamide 5 mM A83-01 5 μM PGE2 3 μM hRSPO3 CM 1% hNoggin CM 2% hNRG1 5 ng / mL hFGF2 5 ng / mL hFGF10 10 ng / mL Y-27632 10 μM

[0091] The existing culture medium was replaced with a new culture medium at intervals of 2 to 3 days, and Y-27632 was removed from the culture medium after the first 3 days. In addition, subculture was performed before 14 days had passed.

[0092] Experimental Example 4. Culture and subculture of N2a cells

[0093] Cell stock (total number of cells is at least 5.0 x 10 5 The cells were removed from the nitrogen tank and incubated at 37°C until about half dissolved. Then, a neutralizing solution containing 10% FBS was added to the EMEM, and the supernatant was removed by centrifuging at 300 g for 5 minutes. Afterward, the neutralizing solution was added again and the cell pellet was obtained by centrifuging at 300 g for 5 minutes. After adding 1 mL of culture medium to loosen the cell pellet, 10 μL was taken and transferred to an Eppendorf tube. 10 μL of 0.4% Trypan Blue Solution was added and mixed, after which the number of cells was checked.

[0094] 5.0 x 10 on T25 plate 5 The cells were divided and added along with the culture medium, and the composition of the culture medium used is as shown in Table 2 below.

[0095] EMEM Basal medium FBS 10% Primocin 0.2%

[0096] Subculturing was performed when the cells occupied 80–90% of the total area. The culture medium was removed and Ca 2+ Mg 2+ After lightly washing the surface with free PBS, 2 mL of TrypLE was added and incubated at 37℃.

[0097] Microscopic examination was performed every 5 minutes until most cells became a single-cell suspension. Once it was confirmed that the cells had formed a single-cell form, they were transferred to a conical tube, an equal volume of neutralizing solution was added, and the mixture was centrifuged at 300 g for 5 minutes to remove the supernatant and obtain a cell pellet. After adding 1 mL of culture medium to loosen the cell pellet, 10 μL was collected and transferred to an Eppendorf tube. 10 μL of 0.4% Trypan Blue Solution was added and mixed, the cell count was checked, and cell culture was repeated.

[0099] Preferred embodiments are presented below to aid in understanding the present invention. However, the following embodiments are provided merely to facilitate a better understanding of the invention and do not limit the scope of the invention.

[0100] Example 1. Suspension co-culture system of human salivary gland organoids and N2a cells

[0101] Prior to proceeding with suspension co-culture, the culture time and initial cell number at which gene expression of ChAT, which provides acetylcholine to salivary gland organoids, was high during suspension culture of N2a cells alone were confirmed via qPCR, and the culture time was 24 hours and the initial cell number was 1.0 x 10⁶ 4 The number was set at 1.0 x 10⁶ per well in a 48-well plate. Therefore, the organoids were subcultured at a rate of 1.0 x 10⁶ per well in a 48-well plate. 4 The procedure was performed with 300 μL of cells without Matrigel. The composition of the culture medium is as shown in Table 3 below.

[0102] Advanced DMEM / F12 Basal medium Glutamax 1 X HEPES 1 X Primocin 0.2% B27 vit A+ 1 X NAC 1 mM Nicotinamide 5 mM A83-01 5 μM PGE2 3 μM hRSPO3 CM 1% hNoggin CM 2% hNRG1 5 ng / mL hFGF2 5 ng / mL hFGF10 10 ng / mL Y-27632 10 μM Forskolin 10 μM

[0103] After 72 hours had elapsed, 100 μL of the above culture medium was added, and after another 72 hours had elapsed, 1.0 x 10⁶ N2a cells were added to 1 mL of culture medium. 5 100 μL of the diluted solution was added to each well for co-culture. The experiment was terminated after 24 hours, and a post-hoc analysis was performed.

[0104] Example 2. Characterization of a suspension co-culture system of human salivary gland organoids and N2a cells

[0105] 2.1. Lumen swelling

[0106] The extent of lumen expansion was compared through bright-field observation with and without co-culture of human salivary gland organoids and N2a cells (Fig. 2). As a result, it was confirmed that the lumen of the salivary gland organoid expanded only when co-cultured with N2a cells.

[0107] 2.2. Immunofluorescence staining

[0108] Immunofluorescence staining was performed to confirm that KU80, which labels the nucleus of human cells, and TUJ1, which is not expressed in salivary gland epithelial cells but is expressed only in N2a cells, were expressed in a co-culture system of human salivary gland organoids and N2a cells. Meanwhile, salivary gland epithelial cell markers such as CDH1, KRT7, KRT5, KRT14, and KRT19 were confirmed to be expressed in both the group co-cultured with N2a cells and the group not co-cultured (Fig. 3).

[0109] Example 3. Model of neurogenic salivary gland disease using a suspension co-culture system of human salivary gland organoids and N2a cells and drug efficacy test using the same

[0110] To determine the effects of treatment with or without neurotransmitters such as acetylcholine and neostigmine on the co-culture system, brightfield observation was performed on the group treated with neurotransmitters (Acetylcholine+Neostigmine) and the group with neurotransmitter blockade by treatment with Botox (GT1b+BoNT / A), and the expression levels of luminal-related genes (KRT7, KRT19) were measured.

[0111] Observation with a bright-field microscope confirmed that even though the organoid's lumen expanded due to co-culture with N2a cells and neurotransmitters, the lumen contracted when the neurotransmitters were blocked (Fig. 4). In addition, an examination of the expression of lumen-related genes revealed that the expression levels of KRT7 and KRT19 increased when neurotransmitters were supplied and decreased when the neurotransmitters were blocked (Fig. 5).

[0112] In other words, it was confirmed that a neurogenic salivary gland disease model can be effectively prepared by treating a co-culture system of human salivary gland organoids and N2a cells with neurotransmitters. This appears to be because the treatment with acetylcholine and neostigmine increased the production of acetylcholine in the neuroblastoma, thereby mimicking stimulation by the parasympathetic nervous system. Furthermore, the efficacy of the drug against the disease was evaluated by treating the aforementioned neurogenic salivary gland disease model with Botox.

[0113] Example 4. Mouse salivary gland organoid-based neurosialadenopathy model and Botox potency test using the same

[0114] 4.1. Determination of concentrations of acetylcholine, neostigmine, GT1b, and Botox that do not affect mouse salivary gland organoids

[0115] Mouse salivary gland organoids were obtained using the same method as in Experimental Example 3 above, and the composition of the culture medium used is as shown in Table 4 below.

[0116] Advanced DMEM / F12 Basal medium Glutamax 1 X HEPES 1 X P / S 1 X Primocin 1 X B27 1 X NAC 1.25 mm A83-01 0.5 μM mRSPO1 1 % hNRG1 37.5 ng / ml mFGF1 79.15 ng / ml mFGF7 18.8 ng / ml Y-27632 10 μM

[0117] To prevent contamination by mesenchymal cells, the effect of Botox on organoids was confirmed using mouse salivary gland organoids from passage 2 onwards through observation of cell morphology and measurement of cell viability (Fig. 6).

[0118] As a result of treating mouse salivary gland organoids with various concentrations of acetylcholine, it was confirmed that increasing the concentration of acetylcholine up to 100 nM had no effect on the morphology and growth of the organoids (Fig. 7). Even when neostigmine was treated at 0, 50, 100, and 200 μM, no significant changes were observed in the morphology and growth of the organoids (Fig. 8). In addition, it was confirmed that treatment with 25 μg / mL of GT1b and various concentrations of botox had no effect on the organoids (Fig. 9).

[0119] 4.2. Co-culture system of mouse salivary gland organoids and N2a cells

[0120] N2a cells were cultured and subcultured using the same method as in Experimental Example 4 above, and the composition of the culture medium used is as shown in Table 5 below.

[0121] EMEM Basal medium P / S 1X FBS 10%

[0122] Co-culture was carried out by mixing 4 μL of organoid culture medium containing 300 cells constituting mouse salivary gland organoids and 4 μL of organoid culture medium containing 3,000 N2a cells with 32 μL of Matrigel to form a 40 μL dome and dispensing it into a 24-well plate (Fig. 10).

[0123] Figure 11 shows the salivary gland organoid (black arrow) and N2a cells (red arrow) observed under a bright-field microscope 7 days after the start of co-culture. Single-cell transcriptome analysis revealed that the Tubb3 gene was not expressed in epithelial cells but was expressed in N2a cells, confirming that the gene could serve as a good indicator for distinguishing between salivary gland organoids and N2a cells (Figure 12). Based on this, immunofluorescence staining of KRT5 (basal cells), KRT7 (lumen cells), AQP5 (adenocyte cells), ACTA2 (myoepithelial cells), and TUBB3 confirmed that a co-culture system between salivary gland organoids and N2a cells had been established (Figure 13).

[0124] 4.3. Botox Potency Test Using a Co-culture System of Mouse Salivary Gland Organoids and N2a Cells

[0125] On day 6 of co-culture of salivary gland organoids and N2a cells, the experiment was conducted by dividing the cells into three groups: Untreated (-Stim-Botox), Stimulated (+Stim-Botox), and Stimulated+Botox (+Stim+Botox). +Stim was indicated when 1 nM of acetylcholine and 200 μM of neostigmine were treated simultaneously, and +Botox was indicated when 25 μg / mL of GT1b and 200 U / mL of Botox were treated simultaneously.

[0126] After 24 hours, the co-cultured wells were dissolved in Trizol, and mRNA was reverse transcribed into cDNA to perform qPCR. In the case of the ChAT gene, which is required for the synthesis of acetylcholine and is mainly expressed in N2a cells, the expression level did not change even after 24 hours had passed since treatment with Botox, whereas the AchE gene, which is required for the degradation of acetylcholine, increased with treatment with acetylcholine and neostigmine (+Stim) and decreased again with treatment with Botox (+Botox) (Fig. 14).

[0127] In addition, Krt5 (basal cells), Krt7 (lumen cells), Aqp5 (aqueous cells), and Bhlha15 (aqueous cells) expressed in salivary gland organoids were increased by treatment with acetylcholine and neostigmine, and decreased again by treatment with Botox (+Botox), and a similar trend was observed in Bhlha15 (Fig. 15).

Claims

Claim 1 (a) a step of culturing a salivary gland organoid from epithelial cells derived from salivary gland tissue in a culture medium containing a TGF-β inhibitor, a Wnt activator, a BMP inhibitor, an FGF family, a receptor tyrosine kinase ligand, and a ROCK inhibitor; (b) a step of culturing a neuroma; and (c) a step of preparing a coculture by mixing and culturing the salivary gland organoid and the neuroma in a culture medium containing a TGF-β inhibitor, a Wnt activator, a BMP inhibitor, an FGF family, a receptor tyrosine kinase ligand, a ROCK inhibitor, and a cAMP activator; comprising a method for preparing a coculture of a salivary gland organoid and a neuroma. Claim 2 A method for preparing a co-culture of salivary gland organoids and neuroblastomas according to claim 1, wherein the TGF-β inhibitor is any one selected from the group consisting of A83-01, SB-431542, SB-505124, SB-525334, SD-208, LY-36494 and SJN-2511. Claim 3 A method for preparing a co-culture of salivary gland organoids and neuroblastomas according to claim 1, wherein the Wnt activator is any one selected from the group consisting of R-spondin 1, R-spondin 2, R-spondin 3 and R-spondin 4. Claim 4 A method for preparing a coculture of salivary gland organoids and neuroblastomas according to claim 1, wherein the BMP inhibitor is any one selected from the group consisting of Noggin, Dorsomorphin, DMH1, and LDN-193189. Claim 5 A method for preparing a co-culture of salivary gland organoids and neuroblastomas according to claim 1, wherein the FGF family is one or more selected from the group consisting of FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, and FGF10. Claim 6 A method for preparing a co-culture of salivary gland organoids and neuromas according to claim 1, wherein the receptor tyrosine kinase ligand is any one selected from the group consisting of Neuregulin β1 (NRG1), Heregulin β1 (HRG1), epidermal growth factor (EGF), transforming growth factor-α (TGF-α), basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF), hepatocyte growth factor (HGF), and keratinocyte growth factor (KGF). Claim 7 A method for preparing a coculture of salivary gland organoids and neuroblastomas according to claim 1, wherein the ROCK inhibitor is any one selected from the group consisting of Y-27632, Fasudil, and H-1152. Claim 8 A method for preparing a co-culture of salivary gland organoids and neuroblastomas according to claim 1, wherein the cAMP activator is any one selected from the group consisting of forskolin, aminophylline, pentoxifylline, theophylline, isobutyl-methylxanthine (IBMX), and dehydroabietic acid (DAA). Claim 9 A method for preparing a co-culture of a salivary gland organoid and a neuroma according to claim 1, wherein the salivary gland organoid from salivary gland tissue-derived epithelial cells is cultured in a culture medium containing A83-01, R-spondin 3, Noggin, FGF2, FGF10, Neuregulin β1 (NRG1), and Y-27632. Claim 10 A method for preparing a co-culture of salivary gland organoids and neuroblastomas according to claim 1, wherein the co-culture of the salivary gland organoids and neuroblastomas is prepared by mixing and culturing them in a culture medium containing A83-01, R-spondin 3, Noggin, FGF2, FGF10, Neuregulin β1 (NRG1), Y-27632, and Forskolin. Claim 11 A culture medium composition for co-culture of salivary gland organoids and neuroblastomas comprising a TGF-β inhibitor, a Wnt activator, a BMP inhibitor, an FGF family, a receptor tyrosine kinase ligand, a ROCK inhibitor, and a cAMP activator. Claim 12 A co-culture of salivary gland organoids and neuroblastomas prepared according to any one of the methods of claims 1 to 10. Claim 13 A method for preparing a model of neurogenic salivary gland disease comprising the following steps: (a) culturing salivary gland organoids from salivary gland tissue-derived epithelial cells in a culture medium containing a TGF-β inhibitor, a Wnt activator, a BMP inhibitor, an FGF family, a receptor tyrosine kinase ligand, and a ROCK inhibitor; (b) culturing neuroblastomas; (c) preparing a co-culture by mixing and culturing the salivary gland organoids and neuroblastomas in a culture medium containing a TGF-β inhibitor, a Wnt activator, a BMP inhibitor, an FGF family, a receptor tyrosine kinase ligand, a ROCK inhibitor, and a cAMP activator; and (d) treating the co-culture with a neurotransmitter. Claim 14 A method for preparing a neurosalivary gland disease model according to claim 13, wherein the salivary gland organoid from salivary gland tissue-derived epithelial cells is cultured in a culture medium containing A83-01, R-spondin 3, Noggin, FGF2, FGF10, Neuregulin β1 (NRG1), and Y-27632. Claim 15 A method for preparing a neurogenic salivary gland disease model according to claim 13, wherein the co-culture prepared by mixing and culturing the salivary gland organoid and the neuroblastoma is cultured in a culture medium containing A83-01, R-spondin 3, Noggin, FGF2, FGF10, Neuregulin β1 (NRG1), Y-27632, and Forskolin. Claim 16 A method for preparing a neurogenic salivary gland model according to claim 13, wherein the neurotransmitter is any one selected from the group consisting of acetylcholine, dopamine, norepinephrine, serotonin, and histamine. Claim 17 A method for manufacturing a neurogenic salivary gland disease model, wherein, in paragraph 16, the neurotransmitter is acetylcholine. Claim 18 A neurogenic salivary gland model manufactured according to the method of any one of paragraphs 13 to 17. Claim 19 A screening method for a neurosalivary gland disease treatment comprising the step of treating a neurosalivary gland disease model prepared according to any one of claims 13 to 17 with a test substance. Claim 20 A screening method for a neurosalivary gland treatment, further comprising the step of screening the test substance as a neurosalivary gland treatment when, in a neurosalivary gland model treated with the test substance compared to a control group not treated with the test substance, the expression level of any one or more selected from the group consisting of KRT7, KRT19, AchE, and Aqp5 is reduced. Claim 21 A method for confirming the responsiveness of a botulinum toxin drug to a neurosalivary gland model, comprising the step of treating a neurosalivary gland model prepared according to any one of claims 13 to 17 with a botulinum toxin drug. Claim 22 A method for confirming the responsiveness of a botulinum toxin drug to a neurosalivary gland disease model according to claim 21, further comprising the step of confirming that the expression level of one or more selected from the group consisting of KRT7, KRT19, AchE, and Aqp5 is reduced in a neurosalivary gland disease model treated with the botulinum toxin drug compared to a control group not treated with the botulinum toxin drug. Claim 23 A method for confirming the responsiveness of a botulinum toxin drug to a neurogenic salivary gland disease model, wherein, in claim 21, the botulinum toxin drug is Botox®, Meditoxin, Dysport®, Botulax, Myobloc®, BTX, or Xeomin®. Claim 24 A method for confirming the responsiveness of a botulinum toxin drug to a neurosalivary gland disease model, wherein the botulinum toxin drug is Botox® in claim 23.

Images