Method for isolation, maintenance, proliferation and differentiation of monoclonal cell from human salivary gland epithelial stem cell or progenitor cell and method for production of extracellular vesicles for treating salivary gland diseases

Abstract

The present invention relates to a method for isolating, maintaining, proliferating, and differentiating monoclonal cells derived from human salivary gland epithelial stem cells or progenitor cells, and a method for producing extracellular vesicles for treating salivary gland diseases. More specifically, conditions for isolating monoclonal salivary gland epithelial cells were established through an optimized stratified culture method from a small amount of major or minor salivary gland tissue obtained during surgery or histological examination, and culture medium conditions were established to enable long-term 2D culture of salivary gland epithelial stem cells or progenitor cells. It was confirmed that the cultured stem cells were monoclonal salivary gland basal stem or progenitor cells, and that they exhibited excellent yield and cell proliferation ability. The method for isolating epithelial stem cells was verified through characterization analysis, and a method for differentiating them into various cell types under a three-dimensional culture environment was presented. Finally, the process of obtaining extracellular vesicles and their therapeutic efficacy were described. These high-purity human salivary gland epithelial stem cells or progenitor cell-derived monoclonal cells are expected to be utilized not only as candidates for cell therapies and extracellular vesicles for the fundamental treatment of salivary gland dysfunction, but also for a wide range of applications in salivary gland research, including disease modeling, pathology studies, drug screening, toxicity assessment, and genetic engineering.

Description

Technology Field

[0001] The present invention relates to a method for isolating epithelial basal stem cells or progenitor cells of human major and minor salivary glands in the form of monoclonal cells, maintaining long-term culture in vitro, and proliferating them, and a method for producing extracellular vesicles for treating salivary gland diseases using the same. Background Technology

[0002] "Stem cells" refer to undifferentiated cells that possess the ability to differentiate into various types of cells, as well as the capacity for self-replication to continuously proliferate into identical cells symmetrically or asymmetrically. Stem cells are present in all parts of the body during fetal development; in adulthood, mesenchymal stem cells (MSCs) are found in the bone marrow, adipose tissue, and the stroma within tissues, while epithelial stem cells are located in the parenchyma of each tissue. Research utilizing stem cells has been actively conducted in various fields since the concept of stem cells was established. In particular, studies are being carried out on tissue regeneration through the direct differentiation of stem cells, anti-inflammatory and anti-fibrotic effects through paracrine secretion, and extracellular vesicles, which are known to play important roles in intercellular signaling and microenvironment regulation. Stem cells can be broadly classified into embryonic stem cells, adult stem cells, and induced pluripotent stem cells. Among these, embryonic stem cells, extracted from human embryos, have the disadvantage of limited research expansion due to bioethical controversies and concerns regarding tumor induction. Induced pluripotent stem cells are stem cells created by artificially manipulating specific genes in adult stem cells to induce a reprogramming process that returns them to an undifferentiated state, thereby enabling them to acquire the pluripotency of embryonic stem cells. However, research involving induced pluripotent stem cells entails financial challenges related to facilities, equipment, and researcher expertise. Adult stem cells, also known as tissue-specific stem cells, have a defined range of differentiation capabilities compared to the aforementioned two types; they are relatively free from ethical controversies, allow for autologous transplantation, and can resolve immune rejection issues that may arise when using stem cells from other donors. Furthermore, they offer the advantage of not requiring high levels of cost or expertise for research.

[0003] Stem cell therapies are currently garnering attention as promising candidates for treating intractable and degenerative diseases for which no cure has yet been discovered. However, several limitations also exist. The widely used method for isolating adult stem cells for stem cell therapies is the Density-Gradient Centrifugation (GCM). This method involves separating cells via centrifugation, sorting them using antibodies or Fluorescence-Activated Cell Sorting (FACS)—a type of flow cytometry analysis—and then separating and mass-cultured cells that adhere to the bottom of the culture vessel. This method has a clear limitation in terms of stem cell purity, specifically the inability to exclude non-stem cells. It has been pointed out that the limitations of existing cell therapies may stem from stem cell purity, a situation that clearly requires improvement. To address this, the Subfractionation Culturing (SCM) method has been devised. SCM is a technology that selects only stem cells by moving them through multiple stages, offering the advantage of isolating stem cells of higher purity compared to GCM. This suggests that it can contribute to fundamentally improving the efficacy of stem cell therapy.

[0004] Existing applications of SCM have focused on 'mesenchymal stem cells (MSCs)' found in bone marrow, fat, and umbilical cord blood. By secreting various signaling molecules or growth factors, such as cytokines and chemokines, into the surrounding environment, they not only induce the regeneration or differentiation of specific tissues but also exert immune tolerance or immunosuppressive effects, leading to the exploration of various applications.

[0005] The primary cells responsible for salivary gland function are epithelial cells. These epithelial cells are classified into acinar epithelial cells, ductal epithelial cells, myoepithelial cells (which stimulate saliva secretion by squeezing acinar epithelial cells), and neuronal cells (which secrete neurotransmitters to myoepithelial and acinar epithelial cells), each possessing distinct functions and characteristics. Salivary gland hypofunction is a condition that occurs when epithelial cells—specifically acinar epithelial cells, which play a major role in salivary secretion, and epithelial stem cells (ESCs), which play a primary role in salivary gland damage repair—are damaged and depleted due to various factors, such as drug therapy, radiation therapy, aging, and autoimmune diseases. This leads to a decline in quality of life and a frequent occurrence of oral diseases due to reduced salivary secretion and regenerative capacity. To address this, temporary symptom-relieving methods such as artificial saliva are being explored, but a fundamental cure has not yet been presented. Stem cell therapy is emerging as a potential fundamental treatment; mesenchymal stem cells are known to secrete tissue growth factors or cytokines that can alleviate damage to or regenerate epithelial cells within tissues. Research results are continuously being published indicating that acinar epithelial cells and tubular epithelial cells possess progenitor and stem cell characteristics and play a role in tissue regeneration. However, directly developing these epithelial progenitor cells, stem cells, or stem cell-derived substances such as exosomes into therapeutic agents remains challenging.

[0006] The primary reason why it is difficult to develop epithelial stem cells as therapeutic agents is that, to date, it has been challenging to isolate tissue-specific epithelial stem cells derived from small amounts of tissue and to culture monoclonal epithelial stem cells. Above all, long-term culturing of epithelial stem cells in 2D is a highly difficult technical task. Therefore, the technology to isolate high-purity monoclonal cells from small amounts of salivary gland tissue-derived epithelial stem cells obtained through patient biopsies, and to maintain and proliferate them under 2D culture conditions, is expected to be highly useful. Furthermore, if epithelial stem cells with excellent proliferative capacity, superior extracellular vesicle secretion, tissue specificity, and excellent differentiation ability into tissue cells can be isolated and proliferated and utilized as cell therapies or extracellular vesicle therapies, they could serve not only as a fundamental treatment for salivary gland dysfunction but also be applicable to various aspects of salivary gland research, such as embryology using stem cells. To achieve this, the main challenges include isolating and culturing monoclonal epithelial stem cells from a small amount of salivary gland tissue, maintaining proliferative capacity for long-term in vitro culture and mass production of extracellular vesicles, and ensuring that heterologous substances are not included in the culture medium for clinical application. Prior art literature

[0007] Korean Registered Patent No. 10-2168088 (Registered on Oct. 14, 2020) The problem to be solved

[0008] The objective of the present invention is to provide a method for specifically isolating high-purity human salivary gland epithelial stem or progenitor cell-derived monoclonal cells from a small amount of salivary gland tissue by optimizing layer separation culture technology, and then minimizing the content of heterologous substances (bovine serum, bovine pituitary extract, porcine-derived materials) through culture using a combination of a chemically formulated culture medium and low-molecular-weight compounds, ultimately maintaining the characteristics of salivary gland epithelial stem cells for a long time in an animal-free environment, thereby enabling long-term culture and proliferation, reproduction of differentiation ability, and obtaining a large amount of extracellular vesicles. means of solving the problem

[0009] To achieve the above objective, the present invention provides a culture medium composition for isolating, maintaining, proliferating, or differentiating salivary gland epithelial stem cells or progenitor cell-derived monoclonal cells, comprising a medium containing Y-27632, A83-01, and a bone morphogenetic protein (BMP) inhibitor as an active ingredient.

[0010] In addition, the present invention provides a method for isolating, maintaining, and proliferating salivary gland epithelial stem cells or progenitor cells derived from salivary gland tissue, comprising: (1) obtaining salivary gland epithelial stem cells or progenitor cells from salivary gland tissue; (2) culturing the obtained salivary gland epithelial stem cells or progenitor cells in the culture medium composition to isolate salivary gland epithelial monoclonal cells; and (3) culturing the isolated salivary gland epithelial monoclonal cells in the culture medium composition to maintain or proliferate salivary gland epithelial cells.

[0011] In addition, the present invention provides a method for producing extracellular vesicles from monoclonal cells derived from salivary gland epithelial stem cells or progenitor cells, comprising the steps of: (1) obtaining salivary gland epithelial stem cells or progenitor cells from salivary gland tissue; (2) culturing the obtained salivary gland epithelial stem cells or progenitor cells in the culture medium composition to isolate salivary gland epithelial monoclonal cells; (3) culturing the isolated salivary gland epithelial monoclonal cells in the culture medium composition to maintain or proliferate salivary gland epithelial cells; and (4) isolating extracellular vesicles from the maintained or proliferated salivary gland epithelial cells.

[0012] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of salivary gland inflammatory diseases comprising, as an active ingredient, a culture medium of salivary gland epithelial stem cells or progenitor cell-derived monoclonal cells isolated, maintained, and proliferated according to the above method; or an extracellular vesicle produced according to the above method. Effects of the invention

[0013] The present invention relates to a method for isolating, maintaining, proliferating, and differentiating monoclonal cells derived from human salivary gland epithelial stem cells or progenitor cells, and a method for producing extracellular vesicles for treating salivary gland diseases. More specifically, conditions for isolating monoclonal salivary gland epithelial cells were established through an optimized stratified culture method from a small amount of major or minor salivary gland tissue obtained during surgery or histological examination, and culture medium conditions were established to enable long-term 2D culture of salivary gland epithelial stem cells or progenitor cells. It was confirmed that the cultured stem cells were monoclonal salivary gland basal stem or progenitor cells, and that they exhibited excellent yield and cell proliferation ability. The method for isolating epithelial stem cells was verified through characterization analysis, and a method for differentiating them into various cell types under a three-dimensional culture environment was presented. Finally, the process of obtaining extracellular vesicles and their therapeutic efficacy were described. These high-purity human salivary gland epithelial stem cells or progenitor cell-derived monoclonal cells are expected to be utilized not only as candidates for cell therapies and extracellular vesicles for the fundamental treatment of salivary gland dysfunction, but also for a wide range of applications in salivary gland research, including disease modeling, pathology studies, drug screening, toxicity assessment, and genetic engineering. Brief explanation of the drawing

[0014] Figure 1 is a schematic diagram of obtaining monoclonal cells of salivary gland epithelial cells through SCM. Figure 2 shows the appearance of salivary gland epithelial cells at the growth limit without a combination of small molecules in each CDM (Chemically defined medium). Figure 3 shows the results of concentration screening of low molecular weight compounds using Dermacult. Figure 4 shows the concentration screening results under low molecular weight compound complex conditions. Figure 5 shows the results of the growth limit of salivary gland epithelial cells increased by small molecule compound combinations in each CDM. Figure 6 shows the results of population doubling time shortened by low molecule compound complex conditions. Figure 7 shows the concentration screening results under low molecular weight compound complex conditions. Figure 8 shows the results of promoting cell growth by adding WNT during cell separation with initial SCM. Figure 9 shows the results of characterizing cultured cells through flow cytometry. Figure 10 shows the results of identifying basal cell gene expression markers through parotid gland unicellular transcriptome analysis. Figure 11 shows the results of identifying salivary gland basal (progenitor) stem cells through immunofluorescence staining. Figure 12 shows the results of verifying salivary gland epithelial stem cell markers through flow cytometry. Figure 13 shows the differential results of gene expression in Bulk-RNA seq. Figure 14 shows the results of the analysis of cell characteristics that differ by colony. Figure 15 shows the results of verifying salivary gland epithelial stem cell markers through flow cytometry. Figure 16 shows a comparison of exosome production between K-SFM and Dermacult and the results of increased exosome production by small molecule compounds. Figure 17 shows the results of exosome extraction and verification from salivary gland epithelial stem cells. Figure 18 shows the results of the anti-inflammatory effect by exosomes in a salivary gland ligation model. Specific details for implementing the invention

[0015] The inventors tested four types of CDM (Chemically defined medium) as basal media capable of culturing epithelial cells, and optimized a culture medium composition for isolating, maintaining, proliferating, or differentiating salivary gland epithelial stem or progenitor cells in the form of a monoclonal cell population, comprising as active ingredients a medium containing the growth factor WNT3A, the small molecule compound Y-27632 or a similar ROCK I / II inhibitor, A83-01 or a similar TGF-beta pathway inhibitor, and a BMP pathway inhibitor such as DMH1 and LDN193189.

[0016] The present invention provides a culture medium composition for isolating, maintaining, proliferating, or differentiating salivary gland epithelial stem cells or progenitor cell-derived monoclonal cells, comprising a medium containing Y-27632, A83-01, and a bone morphogenetic protein (BMP) inhibitor as an active ingredient.

[0017] Preferably, the culture medium composition may include Y-27632 at a final concentration of 1 to 20 μM and A83-01 at 0.2 to 2 μM, but is not limited thereto.

[0018] Preferably, the BMP inhibitor may be DMH1 or LDN193189, but is not limited thereto. More preferably, the culture medium composition may include DMH1 at a final concentration of 0.1 to 2 μM or LDN193189 at a final concentration of 0.05 to 0.5 μM, but is not limited thereto.

[0019] Preferably, the medium may be applied to the Subfractionation Culturing Method (SCM), but is not limited thereto.

[0020] In addition, the present invention provides a method for isolating, maintaining, and proliferating salivary gland epithelial stem cells or progenitor cells derived from salivary gland tissue, comprising: (1) obtaining salivary gland epithelial stem cells or progenitor cells from salivary gland tissue; (2) culturing the obtained salivary gland epithelial stem cells or progenitor cells in the culture medium composition to isolate salivary gland epithelial monoclonal cells; and (3) culturing the isolated salivary gland epithelial monoclonal cells in the culture medium composition to maintain or proliferate salivary gland epithelial cells.

[0021] Preferably, WNT3A may be added to step (2) above to culture salivary gland epithelial stem cells or progenitor cells, but is not limited thereto.

[0022] Preferably, the salivary gland epithelial stem cells or progenitor cells of step (2) above may be cultured according to the Subfractionation Culturing Method (SCM), but are not limited thereto.

[0023] Preferably, the salivary gland epithelial stem cells or progenitor cell-derived monoclonal cells may be CD49f+ / CD26- salivary gland basal cells, but are not limited thereto.

[0024] Preferably, the salivary gland epithelial stem cells or progenitor cell-derived monoclonal cells may express salivary gland basal cell markers and salivary gland progenitor cell markers KRT14, KRT5, KRT19, SOX9, and TP63, and may express salivary gland epithelial cell marker CDH1, but are not limited thereto.

[0025] Preferably, the salivary gland epithelial stem cells or progenitor cell-derived monoclonal cells may have the ability to pluripotently differentiate into salivary gland epithelial tissue.

[0026] In addition, the present invention provides a method for producing extracellular vesicles from monoclonal cells derived from salivary gland epithelial stem cells or progenitor cells, comprising the steps of: (1) obtaining salivary gland epithelial stem cells or progenitor cells from salivary gland tissue; (2) culturing the obtained salivary gland epithelial stem cells or progenitor cells in the culture medium composition to isolate salivary gland epithelial monoclonal cells; (3) culturing the isolated salivary gland epithelial monoclonal cells in the culture medium composition to maintain or proliferate salivary gland epithelial cells; and (4) isolating extracellular vesicles from the maintained or proliferated salivary gland epithelial cells.

[0027] Preferably, the extracellular vesicle may be a salivary gland epithelial stem cell exosome, but is not limited thereto.

[0028] The "extracellular vesicle (EV)" of the present invention refers to a nano-sized vesicle derived from a cell, and is classified into exosomes, microvesicles, ectosomes, microparticles, membrane vesicles, nanovesicles, outer membrane vesicles, etc., depending on the form of secretion and size. Extracellular vesicles contain nucleic acids and proteins, which are major components of cells, and are a primary means of intercellular communication.

[0029] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of salivary gland inflammatory diseases comprising, as an active ingredient, a culture medium of salivary gland epithelial stem cells or progenitor cell-derived monoclonal cells isolated, maintained, and proliferated according to the above method; or an extracellular vesicle produced according to the above method.

[0030] The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier, said pharmaceutically acceptable carrier being one commonly used in formulations and including, but not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the composition for preventing or treating cancer metastasis of the present invention may further comprise a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, etc.

[0031] The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it may be administered via intravenous infusion, subcutaneous infusion, intramuscular infusion, intraperitoneal infusion, endothelial administration, local administration, direct intraductal infusion, intranasal administration, intrapulmonary administration, and rectal administration. When administered orally, since proteins or peptides are digested, the oral composition may be formulated to coat the active agent or protect it from degradation in the stomach, and the composition of the present invention may be administered by any device capable of transporting the active substance to target cells.

[0032] The suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, mode of administration, patient's age, body weight, gender, pathological condition, food, time of administration, route of administration, excretion rate, and response sensitivity, and a physician who is normally skilled can easily determine and prescribe a dosage effective for the desired treatment or prevention.

[0033] The pharmaceutical composition of the present invention may be prepared in a unit volume form or contained in a multi-dose container by formulation using pharmaceutically acceptable carriers and / or excipients according to methods that can be easily carried out by a person skilled in the art to which the invention belongs. In this case, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or in the form of an extract, powder, suppository, powder, granule, tablet, or capsule, and may additionally include a dispersant or a stabilizer.

[0034] The “saliva” of the present invention is a mixture of fluids secreted from the parotid gland, submandibular gland, sublingual gland, and mucous glands present in the oral mucosa. Saliva is a core component of the human body, produced by the salivary glands and discharged into the oral cavity. As an essential component of the human body, saliva contains bioactive proteins, digestive enzymes, mucus, immunoglobulins, various salts, and the like.

[0035] Saliva plays a crucial role not only in oral health but also in maintaining homeostasis in the human body. For example, major components of saliva, such as mucin and immunoglobulins, serve as a primary defense against external infections and protect the oral mucosa and teeth through lubrication, moisture retention, and pH neutralization. Furthermore, saliva contains digestive enzymes, such as amylases like ptyalin, which facilitate digestion by breaking down starch into maltose units. Additionally, saliva secretion enables the regulation of water metabolism and body temperature, and facilitates the excretion of toxins (such as I, Hg, and Pb).

[0036] The “salivary gland” of the present invention is an organ that produces and secretes saliva, and is classified into major salivary glands such as the parotid gland, submaxillary gland, and sublingual gland, and minor salivary glands distributed in various parts of the oral mucosa, such as mucous glands existing in the oral mucosa.

[0037] The present invention will be explained in more detail below through the following examples. However, the present invention is not limited by these examples.

[0038] < Experimental Example >

[0039] The following experimental examples are intended to provide experimental examples that are commonly applied to each embodiment according to the present invention.

[0040] 1. Reagents

[0041] The following reagents were used in the present invention.

[0042] Keratinocyte SFM (1×) without calcium chloride (# 10725-018, Gibco), Epidermal growth factor (EGF) (# 10450-013, Gibco), Bovine pituitary extract (BPE) (# 13028-014, Gibco), Recombinant Human Wnt-3a Protein (# 5036-WN-010, R&D), EpiLife™ Medium with 60 uM Calcium (# MEPI500CA, Gibco), EpiLife™ Defined Growth Supplement Medium (# S0125, Gibco), DermaCult™ Keratinocyte Expansion Medium (# 100-0500, STEMCELL), DermaCult™ Keratinocyte Expansion Supplement (# 100-0502, STEMCELL), CnT-Prime Epithelial Proliferation Medium (# CnT-PR, CellnTec), Primocin (# anti-pm, Invivogen), Y-27632 dihydrochloride (# TB1254-GMP, Tocris), A83-01 (# TB2939-RMU, Tocris), DMH-1 (# 4126, Tocris), LDN 193189 dihydrochloride (# TB6053-GMP, Tocris), CHIR i99021 (# 4423, Tocris), PD0325901 (# 4192, Tocris), Isoproterenol hydrochloride (# 1741, Tocris), CTS™ DPBS, calcium, magnesium (# A1285801, Gibco), CTS™ DPBS (1×), without calcium chloride, without magnesium chloride (# A1285601, Gibco),Collagenase NB6 GMP grade (N0002779, Nordmark), CTS™ TrypLE™ Select Enzyme (# A1285901, Gibco), 100×20mm culture dish (# 150466, Thermo), 48 well culture plate (#30048, SPL), 24 well culture plate (#30024, SPL), 12 well culture plate (# 30012, SPL), 6 well culture plate (# 30006, SPL), 25cm, 2 cell culture plate (# 70025, SPL), 75cm 2 cell culture plate (# 70075, SPL), 175cm 2 cell culture plate (# 70175, SPL), STEM-CELLBANKER-GMP Grade (# 11924, AMSBIO), Scalpel blade (feather #FM.4 ), 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)

[0043] 2. Human salivary gland epithelial cells obtained from tissue

[0044] (1) Normal tissue of the major salivary gland that has not been invaded by the tumor is collected by means of a biopsy or other method during the surgical process such as tumor removal, or 1-2 lobes are collected from the minor salivary gland of a patient suspected of Sjögren's disease, and then placed in CTS™ DPBS, calcium, and magnesium and stored in a refrigerator at 4°C until the day or the next day before the experiment.

[0045] (2) Add collagenase to CTS™ DPBS, calcium, and magnesium to make 0.4 U / mL. Add Y27632 to the enzyme solution to a concentration of 10 μM. Prepare 1 mL per 10 mg of tissue, provided that if the tissue weight is less than 10 mg, prepare a minimum volume of 1 mL.

[0046] (3) Place the tissue in a petri dish and grind it finely using a scalpel blade. Add it to the prepared enzyme solution and react at 37°C for 1 hour. Invert the solution every 10 minutes to ensure the tissue and solution are well mixed.

[0047] (4) Centrifuge 300g at 4°C for 5 min to remove the enzyme solution, add CTS™ DPBS (1×), without calcium chloride, without magnesium chloride at twice the volume of the enzyme solution, centrifuge again at 4°C for 5 min to remove the DPBS.

[0048] (5) Add 1 mL of CTS™ TrypLE™ Select Enzyme per 50 mg of original tissue weight. Add Y27632 to achieve a concentration of 10 μM. (For tissue weights of 50 mg or less, add 1 mL.) React at 37°C for 20 minutes, and invert every 10 minutes to ensure even mixing.

[0049] (6) After mixing with an equal amount of culture medium, pass through a 70 μm strainer and then through a Falcon tube with strainer, centrifuge at 300g for 5 min at 4°C, and remove the supernatant. Then, add culture medium again and centrifuge at 300g for 5 min at 4°C to remove the supernatant and count the cells.

[0050] 3. Human salivary gland epithelial cells using the SCM culture method monoclonal cells separation

[0051] (1) Homogenize the cells in 10 ml of culture medium and spread them evenly in a 100×20 mm culture dish, then store them in an incubator at 37°C with 5% CO2. At this time, the composition of the culture medium is as shown in Table 1.

[0052] (2) After 2 hours, transfer the culture medium containing the cells to a new 100×20mm culture dish to perform the layered culture method (SCM) for isolating epithelial monoclonal cells, and repeat this 2 more times.

[0053] (3) After 24 hours, the dish resulting from (2) is cultured, and the existing culture medium is removed and replaced with new culture medium at intervals of 2-3 days.

[0054] (4) After confirming that a colony has formed, the size of the cell cluster forming a single colony is 1 cm 2 After moving on, remove the culture medium, mark with a hydrophobic pen, and remove the residual culture medium using 1×PBS. Add 50–100 μL of TrypLE + Y27632 and store in an incubator at 37°C with 5% CO2 for 15 minutes.

[0055] (5) Place the cell clusters separated from the dish by TrypLE into separate 1.5 ml tubes, and add 1 ml of culture medium.

[0056] (6) After centrifuging 300 g at 4°C for 5 minutes, remove the supernatant and homogenize the cells into 250 μL of fresh culture medium for each well of a 48-well plate.

[0057] [Table 1]

[0058]

[0059] 4. Proliferation of salivary gland epithelial cells isolated into single cell populations

[0060] (1) Remove the culture medium at intervals of 2-3 days and replace it with new culture medium.

[0061] (2) When the well is filled with about 80 to 90 percent cells, subculture is performed.

[0062] (3) At this time, the composition of the culture medium used for subculturing is as shown in Table 2.

[0063] (4) After removing the culture medium, remove any remaining culture medium using PBS. Then, add TrypLE express (about 250 μl) to cover the bottom of the well, and store in an incubator at 37°C with 5% CO2 for 15 minutes.

[0064] (5) Cells separated from the culture plate by TrypLE express are placed into separate 5 ml tubes, and 1 ml of the culture medium of Table 2 is added to neutralize them.

[0065] (6) After centrifuging 300 g at 4°C for 5 minutes, remove the supernatant and homogenize 1 ml of the culture solution from Table 2 into each well of a 12-well plate for each cell colony.

[0066] (7) Remove the existing culture medium at intervals of 2-3 days and replace it with a new culture medium.

[0067] (8) The above subcultures are scaled up as follows, using a 24-well culture plate (0.5 mL), a 12-well culture plate (1 mL), a 6-well culture plate (2 mL), and a 25 cm 2 cell culture flask (5 ml), 75cm 2 cell culture flask (15 ml), 175cm 2 Proceed with a cell culture flask (35 ml). Refer to parentheses for the volume of culture medium to be added.

[0068] (9) From 6 wells onwards, it can be used for various experiments, and when subculture, it is done at a ratio of 1:2 to 1:10.

[0069] (10) Check the population doubling, and if the proliferation rate is not exponential, determine that the cells have aged and discard them.

[0070] [Table 2]

[0071]

[0072] 5. Analysis of the characteristics of proliferated epithelial cells

[0073] (1) When the successfully proliferated clone and culture medium conditions reach 70-80% confluency, the following experiments are initiated.

[0074] (2) The characteristics of isolated and cultured human salivary gland epithelial stem cells or progenitor cell-derived monoclonal cells are analyzed by immunofluorescence staining.

[0075] (3) Isolate RNA using TRIzol or an RNA extraction kit, and check for changes in gene expression using real-time PCR and bulk-RNA seq.

[0076] (4) Use flow cytometry to check for the expression of stem cell-related proteins on the cell surface.

[0077] (5) Differentiation ability test is performed through 3D culture using Matrigel.

[0078] (6) By observing that extracellular vesicles reflect the characteristics of the cell that secreted them, extracellular vesicles are harvested and Western blot is used to check whether CD9 and others are highly expressed.

[0079] < Examples 1> Human salivary gland epithelial stem cells or progenitor cells origin monoclonal cells Screening of optimal culture medium compositions for isolation and long-term in vitro culture

[0080] High-purity human salivary gland epithelial stem cells or progenitor cell-based monoclonal cells were isolated using a modified layered culture method (SCM) and a mixture of various types of chemically defined medium (CDM) and small molecules (Fig. 1). During the SCM process The experiment was conducted using the culture medium of [Table 1], and after passing the first subgeneration, the culture medium of [Table 2] was used.

[0081] First, we evaluated the extent to which culture is possible using only CDM, which is capable of culturing epithelial cells. When K-SFM, EpiLife, and CnT-PR were subcultured at a ratio of 1:2 in the absence of small molecule compounds, it was confirmed that growth stopped and senescence occurred after an average of 6, 2, and 3 subcultures, respectively, whereas Dermacult was able to undergo 20 to 30 subcultures even without small molecule compounds (Fig. 2).

[0082] Dermacult, which can stably culture human salivary gland epithelial cells among CDMs, was used as the keratinocyte expansion medium (KEM), and low molecule compounds such as ROCK I / II inhibitor (Y-27632), TGF-beta pathway inhibitor (A83-01), and BMP pathway inhibitor (LDN193189) were added to determine the optimal concentrations for stable long-term culture while maintaining characteristics (Fig. 3).

[0083] Finally, through combination experiments at various concentrations under conditions where low molecular weight compounds were mixed, it was confirmed that salivary gland epithelial cell growth was maintained at its maximum when Y-27632 at 10 μM, A83-01 at 1 μM, and LDN193189 at 0.1 μM were added to KEM (Fig. 4).

[0084] When the optimal combination of small molecule compounds was added, K-SFM could undergo 9 to 18 passages, EpiLife 3 times, CnT-PR more than 30 passages, and Dermacult exceeded 50 passages (Fig. 5).

[0085] In particular, it was confirmed that when Dermacult added a low-molecular-weight compound, the cells in the experimental group cultured with the addition proliferated faster and could be cultured for a longer period compared to the control group cultured without the addition, and the population doubling time of the cells was also drastically reduced (Fig. 6).

[0086] In the case of K-SFM among CDMs, when various combinations of small molecule compounds were examined, conditions containing Y-27632 and A83-01 generally showed good results, while conditions containing Chir99021 or PD0325901 showed poor results. Isoproterenol had no effect. Additionally, when LDN193189 was replaced with DMH-1, another type of BMP pathway inhibitor, it showed an effect similar to Dermacult's YAL, but required a higher concentration of 1 uM (Fig. 7).

[0087] When using K-SFM in CDM, rapid cell growth was observed when WNT was added initially, but this was not essential under small molecule culture conditions; on the contrary, changes in cell morphology were observed when added for a long time. If WNT3A is to be used, it is advisable to use it only during the SCM isolation process and exclude it during subsequent subcultures (Fig. 8).

[0088] < Examples 2> Flow cytometry and Immunofluorescence staining through human salivary gland epithelial stem cells or progenitor cells origin monoclonal cells Phylogenetic analysis

[0089] To determine which of the basal, luminal, myoepithelial, and alveolar cells the isolated cultured cells were, cells from parotid tissue were classified and cultured using a flow cytometer with CD49f and CD26 antibodies, and the cultured cells were analyzed again using flow cytometry, which revealed that they consisted of CD49f+ / CD26- basal cells (Fig. 9).

[0090] To further verify that only salivary gland basal progenitor and stem cells can be selectively grown under the culture conditions established as above, a candidate group of markers specifically expressed in salivary gland basal cells was selected by unicellular transcriptome analysis (Fig. 10).

[0091] Subsequently, basal cell expression markers were confirmed in cultured cells through immunofluorescence staining. KRT14, KRT5, KRT19, SOX9, and TP63, which are used as both salivary gland basal cell markers and progenitor cell markers, were identified, while KRT7, a marker for differentiated tubular epithelial cells, and SOX2, a progenitor cell marker for acinar cells, were not identified. Furthermore, the expression of CDH1 and the deficiency of VIM confirmed that the cells did not lose their epithelial characteristics even after prolonged culture (Fig. 11).

[0092] Next, to verify that they were epithelial stem cells, flow cytometry was performed to compare with mesenchymal stem cell markers, and CD34 and CD90, which are known to be positive in mesenchymal stem cells, were found to be negative (Fig. 12).

[0093] < Examples 3> Analysis of the characteristics of human salivary gland epithelial stem cells or progenitor cell-derived monoclonal cells using bulk-RNA sequencing

[0094] These characteristics of the cells were also expressed in bulk RNA-seq analysis of each of the five colonies, confirming that SOX2 and VIM were not expressed, KRT14, KRT5, KRT19, SOX9, TP63, and CDH1 were highly expressed, and KRT7 was expressed less in comparison (Fig. 13).

[0095] In addition, it was reconfirmed that monoclonal epithelial stem cells can be isolated using the SCM method by observing that each colony has slightly different characteristics (Fig. 14).

[0096] < Examples 4> Analysis of the differentiation potential of human salivary gland epithelial stem cells or progenitor cell-derived monoclonal cells via immunofluorescence staining

[0097] When organoids were prepared by embedding the cells in Matrigel, it was confirmed whether luminal cells formed from cells composed mainly of basal cells. After embedding, the growth of the organoid size could be observed in the bright field, and simultaneously, the expression and increase of KRT7 were confirmed by immunofluorescence staining and real-time PCR (Fig. 15).

[0098] < Examples 5> Extracellular vesicles Isolation, characterization, and efficacy verification

[0099] When comparing the production of exosomes in K-SFM and Dermacult, it was found that fewer exosomes were produced in K-SFM than in Dermacult when a combination of small molecules was added, and that more exosomes were produced when a combination of small molecules was added to Dermacult (Fig. 16).

[0100] Extracellular vesicles generated from epithelial stem cells were confirmed to have the characteristics of epithelial stem cell exosomes through Western blot, NTA, and transmission scanning microscopy (Fig. 17).

[0101] In a model in which the rat submandibular duct was ligated for 2 weeks and then released, superior anti-inflammatory, anti-fibrotic, and tissue-protective effects were observed compared to the control group injected with PBS (Fig. 18).

[0102] According to the present invention, high-purity human salivary gland epithelial stem cell culture is possible using a combination of small molecule compounds under serum-free and BPE-excluded conditions (Dermacult). Furthermore, it shows potential for success even under animal-free conditions (CnT-PR). Additionally, it was confirmed that it can be used as a therapeutic agent for inflammatory salivary gland diseases by increasing the secretion of extracellular vesicles under these conditions.

[0103] Foregoing, specific parts of the present invention have been described in detail. It will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

Claim 1 A culture medium composition for the proliferation of salivary gland epithelial stem cells or progenitor cell-derived monoclonal cells, comprising as active ingredients a medium containing Y-27632, A83-01, and DMH1 or LDN193189 as a bone morphogenetic protein (BMP) inhibitor. Claim 2 delete Claim 3 A culture medium composition according to claim 1, characterized in that the culture medium composition comprises Y-27632 at a final concentration of 1 to 20 μM and A83-01 at a final concentration of 0.2 to 2 μM. Claim 4 A culture medium composition according to claim 1, characterized in that the culture medium composition comprises DMH1 at a final concentration of 0.1 to 2 μM or LDN193189 at a final concentration of 0.05 to 0.5 μM. Claim 5 A culture medium composition characterized in that, in any one of claims 1, 3 and 4, the medium is for application to the Subfractionation Culturing Method (SCM). Claim 6 (1) a step of obtaining salivary gland epithelial stem cells or progenitor cells from salivary gland tissue; (2) a step of isolating salivary gland epithelial monoclonal cells by culturing the obtained salivary gland epithelial stem cells or progenitor cells in a culture medium composition according to any one of claims 1, 3 and 4; and (3) a step of maintaining or proliferating salivary gland epithelial cells by culturing the isolated salivary gland epithelial monoclonal cells in a culture medium composition according to any one of claims 1, 3 and 4. Claim 7 A method according to claim 6, characterized by adding WNT3A to step (2) above to culture salivary gland epithelial stem cells or progenitor cells. Claim 8 A method according to claim 6, wherein the salivary gland epithelial stem cells or progenitor cells of step (2) above are cultured according to the Subfractionation Culturing Method (SCM). Claim 9 A method according to claim 6, wherein the salivary gland epithelial stem cell or progenitor cell-derived monoclonal cell is a CD49f+ / CD26- salivary gland basal cell. Claim 10 A method according to claim 6, wherein the salivary gland epithelial stem cell or progenitor cell-derived monoclonal cell expresses salivary gland basal cell markers and salivary gland progenitor cell markers KRT14, KRT5, KRT19, SOX9, and TP63, and expresses salivary gland epithelial cell marker CDH1. Claim 11 A method according to claim 6, characterized in that the salivary gland epithelial stem cells or progenitor cell-derived monoclonal cells have the ability to multiply into salivary gland epithelial tissue. Claim 12 delete Claim 13 delete Claim 14 delete Claim 15 delete

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