Human salivary gland organ-based salivary adenitis modeling and drug curative effect testing method

Abstract

The invention relates to a preparation method and application of a salivary adenitis model based on human salivary gland organs. The present invention provides a salivary adenitis model prepared by culturing an organoid based on epithelial cells derived from human salivary gland tissue and treating the organoid with an inflammation-inducing substance. Furthermore, by using the salivary adenitis model, a novel therapeutic agent can be screened and a main effective target (target cell) of the novel therapeutic agent can be determined, thereby contributing to providing a personalized treatment method for a patient.

Description

Technical Field

[0001] The present invention relates to a method for preparing a salivary gland inflammation model based on human salivary gland organoids and its uses. Background Art

[0002] Salivary gland infections (or sialadenitis) mainly occur in the parotid gland and submandibular gland among the salivary glands, and their causes are diverse, including infections caused by bacteria such as Staphylococcus aureus, viruses (such as mumps virus), fungi, as well as autoimmune diseases such as salivary stones, salivary gland obstruction, and Sjogren's syndrome. Most are acute infections, but if some progress to chronic, it will be accompanied by a significant decline in salivary gland function. If it spreads from salivary gland infection to other organs in the head and neck, it may pose a fatal risk to the human body.

[0003] The main symptoms include pain, swelling, high fever, xerostomia, etc. The treatment methods include injecting antibiotics in case of bacterial infection. Common treatment methods include rehydration and using glucocorticoid anti-inflammatory drugs such as Dexamethasone (Dex). However, it is reported that long-term use of dexamethasone will cause side effects. Typically reported in animal models, it has a negative effect on saliva secretion due to the reduction and decline in the function of acinar cells in the salivary gland. In addition, since the exact mechanism of action of dexamethasone in the salivary gland is not clear, it is essential to establish a model system for evaluating it.

[0004] On the other hand, the typical immune system signaling system caused by pathogen infection is the Toll-like receptor (TLR) signaling pathway. This starts with the recognition of a specific molecular structure (PAMP; pathogen-associated molecular pattern) of an external pathogen different from host cells, and the result of signal transduction is an increase in the expression of inflammatory cytokines, type I interferons, chemokines, anti-microbial peptides, etc. It is reported that it mainly plays a role and is activated in innate immune cells such as neutrophils and macrophages, but also participates in the activation of adaptive immunity, and acts not only on immune cells but also on the immune-related functions of non-immune cells such as epithelial cells. In addition, in addition to the signal transduction caused by external pathogens, components derived from dead cells also activate signal transduction, so TLR also has the characteristic of being activated as a means to respond to abnormal homeostasis in the body such as tissue damage, immune diseases, and cancer.

[0005] However, due to the severe shortage of cell culture models necessary for in vitro experiments, there is a lack of comparative studies on other tissues regarding the association between salivary gland inflammation or related diseases and TLRs. Therefore, the present inventors completed the present invention by preparing a salivary gland inflammation model based on human salivary gland organoids and using this model to screen for novel therapeutic agents and confirm the main target (target cells) to which the novel therapeutic agents can be assigned to be effective. Summary of the Invention

[0006] Technical Problem

[0007] An object of the present invention is to provide a salivary gland inflammation model and a method for preparing the same.

[0008] Another object of the present invention is to provide a method for screening a therapeutic agent for salivary gland inflammation.

[0009] Means for Solving the Problem

[0010] To avoid confusion caused by repeated content, the description of repeated content will be omitted below. That is, the content of the present invention is not limited to the following content and should be interpreted based on the overall content of the invention.

[0011] Hereinafter, the present invention will be described in detail.

[0012] The present invention provides a method for preparing a salivary gland inflammation model.

[0013] The method for preparing the salivary gland inflammation model is carried out through the following steps:

[0014] Step (a), culturing salivary gland organoids 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; and

[0015] Step (b), treating the salivary gland organoids with a culture medium containing LPS, TNF, or a combination thereof.

[0016] In the present invention, unless otherwise specified, the "TNF" refers to TNF-α. That is, according to the present invention, "TNF" and "TNF-α" have the same meaning.

[0017] In the present invention, "salivary gland" refers to an organ that generates and secretes saliva. The salivary glands are divided into major salivary glands such as the parotid gland (subparotid gland), submaxillary gland (submandibular gland), and sublingual gland, and minor salivary glands such as mucous glands that are distributed in multiple parts of the mucous membrane of the oral cavity.

[0018] In the present invention, "organoid" refers to a cell aggregate formed by aggregating and reorganizing cells isolated from stem cells or organ-derived cells through re-culture, and may include organoids or cell clusters formed from suspension cell cultures.

[0019] In the present invention, "sialoadenitis" refers to a condition in which the salivary gland becomes inflamed due to various reasons. It is divided into parotitis, submaxillitis, and sublinguitis according to the location of occurrence. It is known that acute onset usually occurs through hematogenous dissemination during systemic infections such as typhoid fever, pneumonia, and measles, or due to decreased saliva secretion caused by postoperative dehydration, or due to epidemic parotitis caused by the virus commonly known as mumps, or due to oral infections. It is known that the causes of chronic onset are the formation of stones (salivary gland stones) in the salivary gland, stenosis of the salivary gland duct, and infectious foci in the oral cavity or pharynx. The affected area has swelling, redness, and a feeling of tension, with spontaneous pain and tenderness locally. Systemic symptoms such as fever, chills, headache, and general fatigue may also be present, and in extremely rare cases, pain on opening the mouth may be complained of.

[0020] The culture medium used in step (a) of the method for preparing a salivary gland inflammation model according to the present invention is for culturing salivary gland organoids from epithelial cells derived from salivary gland tissue, and it must contain a TGF-β inhibitor, a Wnt activator, a BMP inhibitor, the FGF family, a receptor tyrosine kinase ligand, and a ROCK inhibitor.

[0021] In the present invention, "culture medium" refers to a solution containing nutrients that maintain the viability of cells and support cell proliferation, and is used to maintain a cell population or culture a cell population.

[0022] The culture medium used in the present invention contains a basal medium. The basal medium is any basal medium suitable for culturing animal or human cells.

[0023] The basal medium usually contains various components required to support the maintenance of cells in typical culture. Considering the following, a person skilled in the art can easily formulate a suitable combination of components. In addition, it also includes a nutrient solution containing conventional standard cell culture components, such as amino acids, vitamins, lipid supplements, inorganic salts, carbon energy sources, and buffers.

[0024] The basal medium can be commercially purchased and includes, without limitation, 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 basal medium can be Advanced DMEM / F12 medium.

[0025] The TGF-β inhibitor is any substance that inhibits the function of the TGF-β receptor, such as a protein, peptide, or small molecule, and can 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 can be A83-01. More preferably, it can be A83-01 at a concentration of 0.5 μM to 10 μM.

[0026] The Wnt activator can be any one or more selected from the group consisting of R-spondin 1, R-spondin 2, R-spondin 3, R-spondin 4, and CHIR99021. Preferably, it can be R-spondin 3 and CHIR99021.

[0027] The BMP inhibitor is an agonist that binds to the BMP molecule to form a complex, or an agonist that binds to the BMP receptor and prevents the BMP ligand from binding to the receptor. For example, it can be an antibody that binds to the receptor. The BMP inhibitor can be a protein or a small molecule, and can be natural, modified, and / or partially or fully synthetic. In addition, the BMP inhibitor can be any one selected from the group consisting of Noggin, Dorsomorphin, DMH1, and LDN-193189. Preferably, it can be Noggin.

[0028] The fibroblast growth factor (FGF) family is a powerful factor that regulates cell proliferation and differentiation, and plays a very important role especially in the normal development of stem cells, tissue maintenance, wound healing, and angiogenesis. The FGF family can be any one or more selected from the group consisting of FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, and FGF10. Preferably, it can be FGF2 and FGF10. More preferably, it can be FGF2 and FGF10 at 5 ng / mL to 20 ng / mL.

[0029] The receptor tyrosine kinase ligand can be any one selected from the group consisting of NRG1 (Neuregulinβ1), HRG1 (Heregulinβ1), 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 can be NRG1 (Neuregulinβ1). More preferably, it can be NRG1 (Neuregulinβ1) at 5 ng / mL to 10 ng / mL.

[0030] The Rho-associated protein kinase (ROCK) inhibitor is a substance that inhibits the serine / threonine kinase activity of the target protein of Rho (Rho A, Rho B, and Rho C), and can 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 can be Y-27632. More preferably, it can be Y-27632 at a concentration of 5 μM to 15 μM.

[0031] In addition to the above essential components, the culture medium of the present invention may further contain one or more additional components selected from the group consisting of Glutamax, HEPES, Primocin, Prostaglandin E2 (PGE2), N-acetylcysteine (NAC), B27, and Nicotinamide.

[0032] The B27 can be replaced by a universal formulation containing one or more components 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.

[0033] In addition, the culture medium of the present invention may further contain antibiotics such as Penicillin-Streptomycin (P / S) and / or Primocin in addition to the above essential components.

[0034] The concentration of each of these additional components can be appropriately adjusted within the general range commonly used in the culture medium.

[0035] More specifically, the culture medium used in step (a) of the method for preparing a sialadenitis model according to the present invention may contain A83-01, R-spondin 3, Noggin, FGF2, FGF10, NRG1 (Neuregulinβ1), Y-27632, and CHIR99021.

[0036] The culture medium used in step (b) of the method for preparing a sialadenitis model according to the present invention may not contain a ROCK inhibitor.

[0037] More specifically, the culture medium used in step (b) of the method for preparing a sialadenitis model according to the present invention may contain A83-01, R-spondin 3, Noggin, FGF2, FGF10, NRG1 (Neuregulinβ1), CHIR99021, and an inflammation inducer. At this time, the inflammation inducer may be LPS, TNF, or a combination thereof. At this time, LPS may preferably contain 5 μg / mL to 15 μg / mL, and TNF may contain 5 ng / mL to 50 ng / mL.

[0038] In a specific embodiment of the present invention, it was confirmed through single-cell transcriptome analysis results that the distribution or frequency of the LPS receptor TLR4, which causes salivary gland inflammation, in major salivary gland organoids varies among different salivary glands ( Figure 1 ). In order to compensate for the differential expression of TLR4 among different salivary glands and patients, an attempt was made to micro-treat the inflammatory cytokine TNF increased by LPS to induce an additional effect unrelated to the LSP-TLR4 negative feedback and a synergistic effect of amplifying the signal. In particular, when LPS and TNF were treated simultaneously, it was confirmed that there were no differences in tissues or subjects, the gene expression of acinar cells decreased, the gene expression of ductal cells increased, and the inflammatory cytokines increased. Therefore, it was confirmed that the simultaneous treatment of LPS and TNF is particularly suitable for preparing a sialadenitis model based on salivary gland organoids.

[0039] Accordingly, the present invention provides a culture medium composition for preparing a sialadenitis model, which contains a TGF-β inhibitor, a Wnt activator, a BMP inhibitor, an FGF family, a receptor tyrosine kinase ligand, and an inflammation inducer, wherein the inflammation inducer is LPS, TNF, or a combination thereof.

[0040] The method for preparing a sialadenitis model according to the present invention may perform three-dimensional culture both in the step of culturing salivary gland organoids and in the step of treating the salivary gland organoids with a culture medium containing LPS, TNF, or a combination thereof.

[0041] When cells of most organs, including salivary glands, are cultured two-dimensionally, they tend to selectively culture only progenitor cells or stem cells, while salivary glands can maintain acinar or myoepithelial cells, etc. only for a very short time after being isolated from tissues. However, in the case of three-dimensional culture, due to factors such as concentration gradients of extracellular matrix and growth factors, acinar cells or myoepithelial cells, etc. will develop. This means that phenomena dominated by acinar or myoepithelial cells cannot be observed in two-dimensional culture, but can be observed in three-dimensional culture.

[0042] That is, when performing three-dimensional culture, it shows an excellent development level compared to two-dimensional culture at the developmental level related to glandular cells. More specifically, there may be differences in aspects such as the expression of AQP5, which is highly related to salivary gland function. In addition, there may be differences in aspects such as the expression of STATH. That is to say, according to the three-dimensional culture method of the present invention, salivary gland tissue can be made more suitable for the construction of disease models.

[0043] For such a three-dimensional culture method, for example, the specific conditions of three-dimensional culture are not particularly limited, and methods commonly used in the art can be adopted. For example, three-dimensional culture can be carried out together with a biocompatible scaffold in a culture medium. Here, the biocompatible scaffold refers to a support made of a material that has affinity for cells and has a so-called "cell adhesion" surface, and can adhere and culture cells three-dimensionally. In the present invention, examples of natural source scaffolds include alginate, protein, collagen, fibrin, hyaluronic acid, cellulose, etc., and examples of synthetic polymer scaffolds include poly(α-hydroxy acid) types, poly(vinyl alcohol), polyanhydrides, etc., but are not limited thereto. In addition, the hanging drop culture method can also be used to prepare a culture body and perform three-dimensional culture.

[0044] The present invention provides a method for screening a therapeutic agent for sialadenitis.

[0045] Specifically, the present invention provides a method for screening a therapeutic agent for sialadenitis, which includes the step of treating a test substance with a sialadenitis model prepared by the above preparation method.

[0046] More specifically, the method for screening a therapeutic agent for sialadenitis may further include: compared with a sialadenitis model group not treated with a test substance, if the expression level of any one or more selected from the group consisting of CXCL8, CCL2, CXCL5, CXCL12, IL-6 and TNF is reduced, then the step of screening the test substance as a therapeutic agent for sialadenitis.

[0047] The CXCL8, CCL2, CXCL5, CXCL12, IL-6 and TNF are inflammation-related cytokine genes, and the reduction of the expression levels of these genes is an indicator indicating the improvement or treatment of inflammation.

[0048] CXCL8, also known as IL-8, is a major mediator related to inflammation and plays an important role in neutrophil recruitment and neutrophil degranulation. The secretion of IL-8 is increased due to oxidative stress, leading to the recruitment of inflammatory cells and further increasing oxidative stress mediators, which becomes a key parameter for local inflammation.

[0049] Chemokine (C-C motif) ligand 2 (CCL2) also known as MCP1 recruits monocytes, memory T cells and dendritic cells to the sites of inflammation caused by tissue damage or infection by strictly regulating cell kinetics.

[0050] It is known that C-X-C motif chemokine ligand 5 (CXCL5) is generated after cells are stimulated by inflammatory cytokines IL-1 or TNF-α, stimulates the chemotaxis of neutrophils with angiogenic properties, and regulates neutrophil homeostasis.

[0051] It is known that C-X-C motif chemokine ligand 12 (CXCL12) is involved in embryonic development, immune surveillance, inflammatory response, tissue homeostasis, tumor growth and metastasis in a variety of cells.

[0052] It is known that IL-6 is a cytokine produced by a variety of cells such as T lymphocytes, B lymphocytes, macrophages, fibroblasts, etc. The overproduction of IL-6 is closely related to the occurrence of various immune abnormalities, inflammatory diseases and lymphatic system tumors.

[0053] It is known that TNF is a protein produced by macrophages in the body and is one of the substances that cause inflammatory reactions; it is known to be involved in the occurrence of autoimmune diseases or inflammation-mediated diseases such as rheumatoid arthritis, ankylosing spondylitis, inflammatory bowel disease, psoriasis, hidradenitis suppurativa, refractory asthma, etc.

[0054] In addition, more specifically, the method for screening a salivary gland inflammation therapeutic agent may further include: compared with the salivary gland inflammation model group not treated with the test substance, if the expression level of any one or more selected from the group consisting of KRT7, SLPI and FDCSP is decreased, the step of screening the test substance as a salivary gland inflammation therapeutic agent.

[0055] The aforementioned KRT7, SLPI and FDCSP are duct cell-related genes. According to an example of the present invention, in the salivary gland inflammation model of the present invention, it was confirmed that the gene expressions of KRT7, SLPI and FDCSP were increased.

[0056] Therefore, compared with the sialadenitis model group not treated with the test substance, a test substance in which the expression level of any one or more selected from the group consisting of KRT7, SLPI, and FDCSP is confirmed to be decreased can be judged to target duct cells.

[0057] KRT7 (Keratin 7) is a member of the keratin gene family and belongs to type II cytokeratins. Type II cytokeratins are composed of basic neutral proteins arranged in pairs of heterologous keratin chains that are co-expressed during the differentiation of simple and stratified epithelial tissues. This type II cytokeratin is specifically expressed in the common simple epithelial layer of internal organs or glandular ducts and blood vessels.

[0058] Secretory leukocyte protease inhibitor (SLPI) is also known as antilekoproteinase. The SLPI gene encodes a secretory inhibitor that protects epithelial tissues from serine proteases and is expressed in many mucosal surface cells in the lung, cervix, seminal vesicle, and parotid duct tissues.

[0059] FDCSP is a follicular dendritic cell secreted protein, and its structure is very similar to statherin contained in saliva. It was initially discovered in follicular dendritic cells isolated from the tonsils and was later found to be specifically expressed within the periodontal ligament. FDCSP is highly expressed in the junctional epithelium, tonsils, prostate, and lymph nodes.

[0060] In addition, more specifically, the method for screening a sialadenitis therapeutic agent may further include: compared with the sialadenitis model group not treated with the test substance, if the expression of AQP5 or STATH increases, the step of screening the test substance as a sialadenitis therapeutic agent.

[0061] The aforementioned AQP5 and STATH are gland cell-related genes. According to an embodiment of the present invention, in the sialadenitis model of the present invention, a decrease in the expression of the AQP5 and STATH genes was confirmed.

[0062] Therefore, compared with the sialadenitis model group not treated with the test substance, a test substance in which the expression of AQP5 or STATH is confirmed to be increased can be judged to target acinar cells.

[0063] Aquaporin 5 (AQP5) is one of the series of membrane proteins related to the major intrinsic protein and is a factor that plays an important role in the production of saliva, tears, and lung secretions. It is known that AQP5 is a marker related to the cells directly responsible for saliva secretion in the salivary gland.

[0064] Statherin is a factor that prevents calcium phosphate from precipitating in saliva, maintaining a relatively high calcium level in saliva available for enamel remineralization and a relatively high phosphate level for buffering. Statherin is a gene that interferes with calcium phosphate precipitation and may be associated with the inhibition of sialoliths or dental calculus in salivary gland function.

[0065] Effects of the Invention

[0066] The present invention provides a sialadenitis model prepared by culturing organoids based on epithelial cells derived from human salivary gland tissue and treating them with inflammatory inducers. Additionally, using the sialadenitis model, new therapeutic agents can be screened and the main targets (target cells) for which new therapeutic agents are effective can be determined, thus contributing to the provision of personalized treatment methods for patients. Brief Description of the Drawings

[0067] Figure 1 It is the result of confirming the difference in TLR4 expression of organoids based on major salivary glands.

[0068] Figure 2 It is a schematic diagram for evaluating inflammation regulation using LPS and an anti-inflammatory drug (Dexamethasone).

[0069] Figure 3 It is a graph confirming the changes in the expression of gland cell-related genes in the inflammation assessment modeling through three-dimensional culture of salivary gland-derived organoids.

[0070] Figure 4 It is a graph confirming the changes in the expression of gland cell-related genes in the inflammation assessment modeling through two-dimensional culture of salivary gland-derived epithelial cells.

[0071] Figure 5 It is a graph confirming the changes in the expression of duct cell-related genes in the inflammation assessment modeling through three-dimensional culture of salivary gland-derived organoids.

[0072] Figure 6 It is a graph confirming the changes in the expression of duct cell-related genes in the inflammation assessment modeling through two-dimensional culture of salivary gland-derived epithelial cells.

[0073] Figure 7 It is a graph confirming the changes in the expression of immune-related genes in the inflammation assessment modeling through three-dimensional culture of salivary gland-derived organoids.

[0074] Figure 8 It is a graph confirming the changes in the expression of immune-related genes in the inflammation assessment modeling through two-dimensional culture of salivary gland-derived epithelial cells.

[0075] Figure 9It is a figure observed with a bright-field microscope for the group treated with TNF alone.

[0076] Figure 10 It is a figure observed with a bright-field microscope for the group treated with Y-26732 and TNF.

[0077] Figure 11 It is a figure confirming the changes in the expression of immune and inflammation-related genes in the group treated with Y-27632 and TNF.

[0078] Figure 12 It is a figure confirming the changes in the expression of genes related to salivary gland organoids in the group treated with Y-26732 and TNF.

[0079] Figure 13 It is a figure observed with a bright-field microscope for the group treated with LPS and TNF.

[0080] Figure 14 It is a figure confirming the changes in the expression of immune and inflammation-related genes in the group treated with LPS and TNF.

[0081] Figure 15 It is a figure confirming the changes in the expression of inflammation-related genes after applying an anti-inflammatory drug (Dexamethasone) in a salivary gland inflammation model treated with LPS and TNF.

[0082] Figure 16 It is a figure confirming the changes in the expression of genes related to salivary gland epithelial cells after applying an anti-inflammatory drug (Dexamethasone) in a salivary gland inflammation model treated with LPS and TNF. Detailed implementation manners

[0083] Hereinafter, in order to help understand the present invention, preferred embodiments are presented.

[0084] However, the following embodiments are only provided for easier understanding of the present invention, and the content of the present invention is not limited thereto.

[0085] Example 1. Preparation of a lipopolysaccharide (LPS)-treated sialadenitis model

[0086] 1. Reagents used in the present invention

[0087] 24-well suspension culture plate (24well plate for suspension culture) (#662102, GreinerBio-one), 24-well cell culture plate (24well Cell Culture Plate) (#30024, SPL), RNeasy Micro Kit (#74004, QIAGEN), PrimeScript TMRT Reverse Transcription Kit (PrimeScript TM RT reagent Kit) (#RR037A, Takara), Bovine Serum Albumin (BSA) (#BSA - 68700, LSP), Hanks Balanced Salt Solution (1X HBSS) (#14025092, Gibco), Collagenase type Ⅱ (#4176, Worthington), TrypLE (#12605 - 010, Life technologies), Cell Freezing Medium 1 (CellBanker 1) (#621, Zenoaq), Matrigel (#356231, Corning), Dexamethasone (#D8893, Sigma), Lipopolysaccharide (LPS, #L3024, Sigma), Dulbecco's Phosphate Buffered Saline (DPBS) (#LB 001 - 02, Wellgene), Cell Recovery Solution (#354253, Corning), SensiFAST TM Lo - ROX Kit (SensiFAST TM Lo-ROX Kit)(#BIO-94020, Bioline), Advanced Dulbecco's Modified Eagle Medium / F12 (ADF12)(#12634010, Gibco), Penicillin-Streptomycin (P / S, #15140122, Gibco), GlutaMAX(#35050-061, Gibco), HEPES(#15630-080, Gibco), Primocin(#ant-pm, Invivogen), B-27 Supplement(#17504-044, Gibco), N-Acetyl-L-cysteine (NAC, #A9165, Sigma), Nicotinamide(#N0636, Sigma), A83-01(#2939, Tocris), PGE2 (Prostaglandin E2, #2296, Tocris), hRSPO3CM (RSPO3-Fc fusion protein conditioned medium, #R001, U-Protein Express BV), hNoggin (Noggin-Fc Fusion Protein conditioned medium, #N002, U-Protein Express BV), hNRG1(#100-03, Peprotech), hFGF2(#100-18B, Peprotech), hFGF10(#100-26, Peprotech), CHIR99021(#2520691, Biogems), Y-27632(#1254, Tocris), LDN193189(#6053, Tocris), DermaCult TM DermaCult TM Keratinocyte Expansion Medium)(#100-0500, Stemcell).

[0088] 2. Treatment and Preservation of Human Salivary Gland Stem Cells

[0089] Human salivary gland samples were placed in 1X HBSS supplemented with 1% BSA and stored at 4°C until the experiment was conducted on the same day or the next day. The samples were minced with a blade so that each piece was less than 1 cm in length. A digestion solution was prepared by adding 5 mg / mL Collagenase type II and 10 μM Y-27632 to 4 mL of Advanced DMEM / F12. 1 mL of this digestion solution was added per 50 mg of salivary gland sample, and the reaction was carried out at 200 rpm for 1 hour in a shaking incubator at 37°C. Subsequently, centrifugation was performed at 500 g for 5 minutes.

[0090] After adding wash media (Advanced DMEM / F12 + HEPES (1X) + GlutaMAX (1X) + P / S (1X)) equal in volume to the digestion solution, the clumps were thoroughly dispersed and then centrifuged at 500 g for 5 minutes.

[0091] After removing the supernatant, 1 mL of TrypLE supplemented with 10 μM Y-27632 was added, mixed well, and the reaction was carried out in an incubator at 37°C for 10 minutes.

[0092] The reaction was stopped by adding wash media equal to or more than the volume of TrypLE, and then the samples were filtered through a 70-μm strainer to remove undigested tissue. The filtered solution was centrifuged at 500 g for 5 minutes to obtain only cells.

[0093] After counting the cells, calculate the number to ensure that each cryovial (1 mL) contains at least 2×10 5 cells, and centrifuge again at 500 g for 5 minutes. The calculated number of cell clumps was dissolved in CellBanker 1, and then aliquoted into cryovials, 1 mL per tube, stored at -80°C for 2 - 3 days and then transferred to a nitrogen tank for long-term storage.

[0094] 3. 3D culture of human salivary gland stem cell-derived organoids

[0095] The human salivary gland tissue samples stored in the nitrogen tank were thawed at 37°C and transferred to a conical tube. An equal or greater volume of wash media was added, mixed well, and then centrifuged at 500 g for 5 minutes. Calculate the cell number to ensure that each 24-well plate contains 5.0×10 3cells.

[0096] In a 24-well suspension culture plate (#662102, Greiner Bio-one) preheated at 37°C for more than 1 hour, the cell aggregates were thoroughly mixed with 40 μL of Matrigel per well and then dispensed into each well to form a dome shape.

[0097] After that, it was stored in an incubator at 37°C for 20 minutes, and then 500 μL of culture medium was added to each well for culturing. At this time, the conditions of the culture medium are shown in Table 1 below, and the culture medium was changed every 2 - 3 days.

[0098]

Table 1

[0099] Composition of salivary gland organoid culture medium

[0100]

[0101]

[0102] 4. 2D culture of human salivary gland epithelial stem cells

[0103] In the part of "2. Treatment and preservation of human salivary gland stem cells", the human salivary gland tissue sample stored in a nitrogen tank was thawed at 37°C and then transferred to a conical tube. After adding an equal amount or more of wash media (Advanced DMEM / F12 + HEPES(1X) + GlutaMAX(1X) + P / S(1X)) and mixing well, it was centrifuged at 500 g for 5 minutes.

[0104] Calculate the number of cells so that each 24-well plate contains 5.0×10 4 cells. The calculated number of cells was mixed with the culture medium and then added to a 24-well cell culture plate (#30024, SPL) for culturing. At this time, the conditions of the culture medium are shown in Table 2 below.

[0105] Before the cells adhered to the bottom of the plate, the medium was not removed and continued to be added. When about 70% of the cells adhered to the bottom of the plate, the medium was changed every 2 - 3 days.

[0106] After that, observe the cell growth, confirm whether the cells grow in a polygonal shape, and confirm under a microscope whether their size is approximately 25 μm. In addition, confirm that they are epithelial stem cells using EPCAM, KRT5, and KRT14 as markers before conducting the experiment.

[0107]

Table 2

[0108] Composition of the culture medium used for two-dimensional culture of salivary gland-derived epithelial cells

[0109]

[0110]

[0111] 5. Induction of inflammation by lipopolysaccharide (LPS) and treatment with an anti-inflammatory drug (dexamethasone, Dex)

[0112] Three days after embedding, change to the culture medium shown in Table 1 for salivary gland organoids, which has Y-27632 removed, and culture. After 2 hours, add LPS at a concentration of 10 μg / mL to the culture medium, mix well, and use. At this time, Y-27632 is removed in the drug treatment step because it can reduce the stress caused by drug treatment, etc. In addition, by removing Y-27632, the polarity of the organoid cells can be arranged to be similar to that of the tissue.

[0113] For 2D cells, when the 2D cells reach approximately 70% confluency, change to the culture medium shown in Table 2 for two-dimensional culture of salivary gland-derived epithelial cells, and culture. After 2 hours, add LPS at a concentration of 10 μg / mL to the culture medium, mix well, and use.

[0114] Experimental Example 1. Evaluation of gene expression changes in the LPS-treated sialadenitis model

[0115] In this experiment, in order to induce inflammation in epithelial cells, using TLR4, which is known to be expressed in salivary glands from the literature, as a target, after stimulation with the TLR4 ligand LPS, observe the differences in gene expression.

[0116] On the other hand, for the dexamethasone and LPS co - administration group used in this experiment, for organoids, after 3 days of embedding, the culture medium was changed to the culture medium shown in Table 1 for salivary gland organoids with Y - 27632 removed, which contained 1 μM dexamethasone for culture. After 2 hours, 10 μg / mL LPS was added to the culture medium and thoroughly mixed before use; for 2D cells, when the 2D cells reached approximately 70% confluency, the culture medium was changed to the culture medium composition used in the two - dimensional culture of salivary gland - derived epithelial cells shown in Table 2, which contained 1 μM dexamethasone for culture. After 2 hours, 10 μg / mL LPS was added to the culture medium and thoroughly mixed before use.

[0117] The control group used the dexamethasone single - treatment group. For organoids, after 3 days of embedding, the culture medium was changed to the culture medium shown in Table 1 for salivary gland organoids with Y - 27632 removed, which contained 1 μM dexamethasone and then used; for 2D cells, when the 2D cells reached approximately 70% confluency, the culture medium was changed to the culture medium composition used in the two - dimensional culture of salivary gland - derived epithelial cells shown in Table 2, which contained 1 μM dexamethasone and then used.

[0118] After 3 days of final reagent treatment and culture, all experimental groups collected samples for RNA extraction.

[0119] 1. RNA Extraction and Gene Expression Analysis Methods

[0120] Prepare cell samples for RNA extraction.

[0121] For organoids, (1) After removing the original media, wash twice with 500 μL DPBS. Add 500 μL of ice - cold Cell Recovery Solution to each well to collect the cells and transfer them to a 5 mL conical tube. Keep them at 4 °C on ice for 1 hour to dissolve Matrigel. Then, add an equal volume of wash media, mix well, and centrifuge at 500 g for 5 minutes to obtain only the cells.

[0122] For 2D cells, after removing the original medium, wash twice with 500 μL of DPBS. Add 1 mL of TrypLE and incubate in an incubator at 37 °C for 10 minutes. Then transfer the mixture of TrypLE and cells to a conical tube. After that, wash twice with 500 μL of DPBS and transfer to the conical tube, and then centrifuge at 500 g for 5 minutes to obtain only the cells.

[0123] From the organoid cells and 2D cell aggregates obtained by the above method, extract RNA using the RNeasy Micro Kit (#74004, QIAGEN), and quantify it using a Nanodrop.

[0124] Use the quantified RNA with PrimeScript TM RT reagent Kit (#RR037A, Takara) to synthesize cDNA. Add 4 μL of the synthesized cDNA, 0.5 μL each of the forward primer and reverse primer for the target gene, and 5 μL of SensiFAST TM Lo-ROX Kit, and perform accurate pipetting.

[0125] Seal the top of the culture plate with an Optical Adhesive Film, and then spin down. After that, place it in a PCR machine, set the volume to 10 μL, and then start the machine.

[0126] The primer base sequences of the target genes used at this time are shown in Table 3 below.

[0127]

Table 3

[0128]

[0129]

[0130] 2. Inflammatory regulation assessment

[0131] As Figure 2As shown, there are three cases of gene expression changes caused by LPS and anti-inflammatory drugs: increase, no change, and decrease. Therefore, there are a total of nine combinations. At this time, if the anti-inflammatory drug has no effect on the regulation of gene expression, it is classified as "(2) no effect" and can be excluded from the anti-inflammatory drug candidate group. In addition, if the direction of regulating gene expression is the same as that of LPS, it can be interpreted as "(1) instead causes inflammation". On the contrary, if no gene expression changes caused by LPS are observed, but changes are caused by the anti-inflammatory drug, it is "(4) side effect", and the gene expression changes related to the function of the anti-inflammatory drug will be the same as those in case "(3)" where the regulation direction is opposite to that of LPS. Through this strategy, candidate substances with a large number of "(3)" effects and few "(1)" effects can be considered the best anti-inflammatory drugs.

[0132] 3. Evaluation of changes in gene expression related to glandular cells in the inflammation assessment model through three-dimensional culture of salivary gland-derived organoids and two-dimensional culture of salivary gland-derived epithelial cells

[0133] As Figure 3 shown, after three-dimensional culture of organoids derived from human normal salivary gland tissue, LPS and Dex were treated and the results of confirming gene expression changes showed that the expression of the glandular cell-related gene AQP5 decreased due to LPS treatment and increased due to Dex treatment, belonging to case "(3)". AQP5 is a marker related to the cells directly involved in saliva secretion in the salivary gland, so it can be used as a clue for the restoration of salivary gland function.

[0134] The expression of another glandular cell-related gene, STATH, also decreased due to LPS treatment but showed no change due to Dex treatment, so it belongs to case "(2)". STATH is a gene that prevents calcium phosphate precipitation and is associated with the inhibition of salivary gland stones or dental calculus in salivary gland function.

[0135] On the contrary, as Figure 4 shown, this phenomenon did not occur in the two-dimensional culture of salivary gland-derived epithelial cells, providing evidence for the superiority of three-dimensional organoid culture.

[0136] 4. Evaluation of changes in gene expression related to duct cells in the inflammation assessment model through three-dimensional culture of salivary gland-derived organoids and two-dimensional culture of salivary gland-derived epithelial cells

[0137] As Figure 4As shown, after three-dimensional culture of human-derived normal salivary gland tissue-derived organoids, the results of treating with LPS and Dex and confirming gene expression changes showed that the expression of the duct cell-related gene KRT7 showed a trend of being increased by both LPS and Dex, belonging to the situation of "(1)". It seems that Dex instead exacerbated the marker changes caused by LPS. KRT7 is a duct-related marker that acts as a channel during saliva secretion. Therefore, the increase in overexpression can be explained as being related to the inhibition of salivary gland function.

[0138] KRT5 was not affected by LPS but increased due to Dex, belonging to the situation of "(4)". It is known that KRT5 acts as a progenitor cell in the salivary gland, which can be explained as Dex being related to enhancing the ability of salivary gland stem cells.

[0139] SLPI is an antiviral protein. If the expression level in saliva is insufficient, the risk of virus infection increases. Therefore, the decrease in expression may affect salivary gland inflammation and diseases.

[0140] It is known that FDCSP is expressed in cells activated by TNF and regulates the immune response through interactions with immune cells. FDCSP is also closely related to the immune function of saliva. Therefore, it can be explained that its expression level is related to salivary gland function.

[0141] These SLPI and FDCSP genes were increased by LPS, but SLPI was increased by Dex, belonging to the situation of "(1)", while FDCSP was not affected by Dex, belonging to the situation of "(2)".

[0142] On the contrary, as Figure 6 shown, this phenomenon did not occur in the two-dimensional culture of salivary gland-derived epithelial cells, and this result provides a basis for the superiority of three-dimensional organoid culture.

[0143] 5. Evaluation of changes in the expression of immune-related genes in the inflammation assessment model through three-dimensional culture of salivary gland-derived organoids and two-dimensional culture of salivary gland-derived epithelial cells

[0144] As Figure 7 shown, after three-dimensional culture of human-derived normal salivary gland tissue-derived organoids, the results of treating with LPS and Dex and confirming gene expression changes showed that the expression of the inflammatory cytokine gene CXC8 increased due to LPS treatment and decreased due to Dex treatment, belonging to the situation of "(3)".

[0145] It is known that TNF is a cytokine accompanying the acute inflammatory response that regulates immune cells. Its expression increased due to LPS treatment and decreased due to Dex treatment, belonging to the situation of "(3)".

[0146] Considering that CXCL8 is a chemokine and TNF is an inflammatory cytokine, the function of Dex can be interpreted as reducing immune cell infiltration by alleviating inflammation.

[0147] In contrast, as Figure 8 shown, this phenomenon did not occur in the two-dimensional culture of salivary gland-derived epithelial cells, and this result provides a basis for the superiority of three-dimensional organoid culture.

[0148] 6. Evaluation of the anti-inflammatory function of dexamethasone (Dex) based on salivary gland organoids

[0149] Based on the comprehensive results, the evaluation results of the anti-inflammatory function of Dex based on salivary gland organoids are shown in Table 4 below.

[0150]

Table 4

[0151]

[0152]

[0153] That is, the anti-inflammatory function of Dex is supported by the expression changes of AQP5, CXCL8 and TNF. However, in terms of the inability to restore the increased expression of KRT7 and SLPI and the expression changes of STATH and FDCSP affected by LPS, it cannot be called a good anti-inflammatory drug; and the stem cell ability of the salivary gland can be confirmed to be incidentally improved through the increase in KRT5 expression. The results of evaluating the anti-inflammatory function of the common clinical drug Dex through this gene combination can be applied to the screening of other future anti-inflammatory drugs for salivary gland inflammation, etc., and can be used as a basis index for evaluating anti-inflammatory drugs superior to the existing Dex.

[0154] Example 2. Preparation of a tumor necrosis factor (TNF)-treated sialadenitis model

[0155] Using the organoids prepared by the same method as in Example 1 as the object, after 3 days of embedding, the culture was changed to the composition of the salivary gland organoid culture medium shown in Table 1 or the culture medium with Y-27632 removed from this culture medium composition, and after 2 hours, TNF at a concentration of 10 or 50 ng / mL was added to the culture medium and thoroughly mixed before use.

[0156] Experimental Example 2. Evaluation of organoid growth and gene expression changes in the TNF-treated sialadenitis model

[0157] 1. Evaluation of organoid growth changes

[0158] As Figure 9 shown, in the organoids treated with TNF at a concentration of 10 ng / mL, there was no significant change in the growth of the organoids; at high magnification, no significant difference was found in the appearance of the organoids.

[0159] On the other hand, Y-27632 is a substance that inhibits apoptosis such as anoikis and stress. To understand the appropriate culture medium combination during TNF treatment, TNF at 10 and 50 ng / mL was treated in the presence and absence of 10 μM Y-27632, respectively. The results are as Figure 10 shown. In the absence of Y-27632, organoid growth inhibition was observed with 50 ng / mL of TNF; in the presence of Y-27632, organoid growth inhibition caused by TNF addition was observed at all concentrations of 10 and 50 ng / mL.

[0160] 2. Evaluation of gene expression changes

[0161] The RNA extraction and gene expression analysis methods for evaluating gene expression changes were performed in the same manner as in Experimental Example 1.

[0162] As Figure 11 shown, when treated with 50 ng / mL of TNF, the expression of immune and inflammation-related markers increased regardless of the presence or absence of Y-27632. However, in terms of the expression of IL-6, etc., it was considered more beneficial in the absence of Y-27632. Therefore, Y-27632 was excluded from the culture medium during TNF treatment, considering organoid growth and the expression of immune and inflammation-related markers.

[0163] On the other hand, as Figure 12 shown, the confirmed expression results of genes such as basal cells (KRT5), luminal cells (KRT7), myoepithelial cells (ACTA2), and acinar cells (AQP) of salivary gland organoids showed that, similar to the Figure 11 results, in the absence of Y-27632, the changes caused by TNF were statistically significant compared to the presence.

[0164] Example 3. Preparation of an LPS- and TNF-treated sialadenitis model

[0165] Using the organoids prepared in the same method as in Example 1 as the object. Three days after organoid embedding, the culture medium was changed to the culture medium shown in Table 1 for salivary gland organoids with Y-27632 removed, and after 2 hours, LPS at a concentration of 10 μg / mL and TNF at concentrations of 5, 10, 20, and 40 ng / mL were added to the culture medium and thoroughly mixed before use.

[0166] Experimental Example 3. Evaluation of organoid growth and gene expression changes in the LPS- and TNF-treated sialadenitis model

[0167] In this experiment, the control group used the dexamethasone single-treatment group. Three days after the organoid embedding, the culture medium was changed to the culture medium shown in Table 1 for salivary gland organoids with Y-27632 removed, which contained dexamethasone at a concentration of 1 μM and was then used.

[0168] As the LPS single-treatment group in the control group, three days after the organoid embedding, the culture medium was changed to the culture medium shown in Table 1 for salivary gland organoids with Y-27632 removed for culturing. After 2 hours, LPS at a concentration of 10 μg / mL was added to the culture medium and thoroughly mixed before use.

[0169] In addition, as the TNF single-treatment group in the control group, three days after the organoid embedding, the culture medium was changed to the culture medium shown in Table 1 for salivary gland organoids with Y-27632 removed for culturing. After 2 hours, TNF at concentrations of 5, 10, 20, and 40 ng / mL was respectively added to the culture medium and thoroughly mixed before use.

[0170] In addition, for the group with combined administration of dexamethasone, LPS, and TNF, three days after the organoid embedding, the culture medium was changed to the culture medium shown in Table 1 for salivary gland organoids with Y-27632 removed, which contained dexamethasone at a concentration of 1 μM for culturing. After 2 hours, LPS at a concentration of 10 μg / mL and TNF at concentrations of 5, 10, 20, and 40 ng / mL were added to the culture medium and thoroughly mixed before use.

[0171] After the final reagent treatment and culturing for 3 days in all experimental groups, samples were collected for RNA extraction.

[0172] 1. Evaluation of organoid growth changes

[0173] As Figure 13 shown, no obvious differences in organoid growth were observed between the control group and the experimental groups.

[0174] 2. Evaluation of gene expression changes

[0175] The methods for RNA extraction and gene expression analysis used to evaluate gene expression changes were carried out in the same way as in Experimental Example 1.

[0176] Based on Experimental Example 1, the primer base sequences of the genes further confirmed in this experiment are shown in Table 5 below.

[0177]

Table 5

[0178]

[0179] As Figure 14As shown, when LPS was treated alone, there was no change in gene expression (TNF, IL-6) or negative feedback (TLR4, CXCL12), or only a slight increase in gene expression (CXCL5, CCL2). When TNF was treated at a low dose (5 ng / mL, 10 ng / mL, 20 ng / mL, 40 ng / mL) simultaneously, an increase or amplification in its expression was confirmed. Referring to the results of TLR4 and CXCL12, simultaneous treatment with 10 ng / mL of TNF and LPS was considered more effective.

[0180] In addition, to verify the inflammatory organoids, dexamethasone, which is commonly used clinically in salivary gland inflammation, was applied. The results are as Figure 15 shown. Dexamethasone effectively reduced the expression of inflammation-related genes such as TNF, IL6, CCL2, CXCL5, and CXCL12.

[0181] On the other hand, the results of confirming the gene expression changes in salivary gland epithelial cells are as Figure 16 shown. No significant differences were observed in ductal cells of KRT5 and KRT7 or myoepithelial cells such as ACTA2. However, the expression levels of acinar cell genes such as AQP5 and STATH decreased significantly under simultaneous treatment with LPS and TNF and recovered under dexamethasone treatment.

Claims

1. A method for preparing a salivary gland inflammation model, characterized in that, Comprising the following steps: Step (a), culturing salivary gland organoids 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; and Step (b), treating the salivary gland organoids with a culture medium containing LPS, TNF, or a combination thereof.

2. The method for preparing a salivary gland inflammation model according to claim 1, characterized in that, The culture medium in step (b) does not contain a ROCK inhibitor.

3. The method for preparing a salivary gland inflammation model 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.

4. The method for preparing a salivary gland inflammation model according to claim 1, wherein, The Wnt activator is any one or more selected from the group consisting of R-spondin 1, R-spondin 2, R-spondin 3, R-spondin 4, and CHIR99021.

5. The method for preparing a parotitis model according to claim 1, wherein, The BMP inhibitor is any one selected from the group consisting of Noggin, Dorsomorphin, DMH1, and LDN-193189.

6. The method for preparing a salivary gland inflammation model according to claim 1, characterized in that, The FGF family is any one or more selected from the group consisting of FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, and FGF10.

7. The method for preparing a salivary gland inflammation model according to claim 1, wherein, The receptor tyrosine kinase ligand is any one selected from the group consisting of Neuregulinβ1 (NRG1), Heregulinβ1 (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).

8. The method for preparing a salivary gland inflammation model according to claim 1, wherein, The ROCK inhibitor is any one selected from the group consisting of Y-27632, Fasudil, and H-1152.

9. The method for preparing a salivary gland inflammation model according to claim 1, characterized in that, The culturing of salivary gland organoids from epithelial cells derived from salivary gland tissue in step (a) is carried out in a culture medium containing A83-01, R-spondin 3, Noggin, FGF2, FGF10, Neuregulinβ1, and Y-27632.

10. The method for preparing a salivary gland inflammation model according to claim 1, wherein The culture medium for treating the salivary gland organoids in step (b) is a culture medium containing A83-01, R-spondin 3, Noggin, FGF2, FGF10, NRG1 (Neuregulinβ1), CHIR99021, and an inflammation inducer, and the inflammation inducer is LPS, TNF, or a combination thereof.

11. A salivary gland inflammation model, characterized in that, Obtained by the method according to any one of claims 1 to 10.

12. A culture medium composition for preparing a model of sialadenitis, characterized in that, Comprising a TGF-β inhibitor, a Wnt activator, a BMP inhibitor, an FGF family, a receptor tyrosine kinase ligand, and an inflammation inducer, and the inflammation inducer is LPS, TNF, or a combination thereof.

13. A method for screening a therapeutic agent for sialadenitis, characterized in that, Including: The step of treating the parotitis model according to claim 11 with a test substance.

14. The screening method of a therapeutic agent for sialadenitis according to claim 13, wherein It further includes the following steps: compared with the parotitis model group not treated with the test substance, if the expression level of any one or more selected from the group consisting of CXCL8, CCL2, CXCL5, CXCL12, IL-6, and TNF decreases, then the test substance is screened out as a parotitis therapeutic agent.

15. The screening method of a therapeutic agent for sialadenitis according to claim 13, characterized in that, It further includes the following steps: compared with the parotitis model group not treated with the test substance, if the expression level of any one or more selected from the group consisting of KRT7, SLPI, and FDCSP decreases, then the test substance is screened out as a parotitis therapeutic agent.

16. The screening method of a therapeutic agent for sialadenitis according to claim 15, wherein, The test substance screened as the therapeutic agent targets duct cells.

17. The screening method of a therapeutic agent for sialadenitis according to claim 13, wherein, It further includes the following steps: compared with the parotitis model group not treated with the test substance, if the expression of AQP5 or STATH increases, then the test substance is screened out as a parotitis therapeutic agent.

18. The screening method of a therapeutic agent for sialadenitis according to claim 17, characterized in that, The test substance screened as the therapeutic agent targets acinar cells.

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