KR20230073997A - 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 - Google Patents

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, and in detail, a small amount obtained during surgery or biopsy Conditions for isolating monoclonal salivary gland epithelial cells from main salivary gland or minor salivary gland tissue were established through an optimized layered culture method, and culture medium conditions for long-term culture of salivary gland epithelial stem cells or progenitor cells in 2D were established. It was confirmed that the cultured stem cells were monoclonal salivary gland basal stem or progenitor cells, and it was confirmed that the yield and cell proliferation ability were excellent. The separation method of epithelial stem cells was verified by characterization analysis, and a method for differentiating into various cells in a 3-dimensional culture environment was presented. Finally, the process of obtaining extracellular endoplasmic reticulum and its therapeutic effect were described. These high-purity human salivary gland epithelial stem cell or progenitor cell-derived monoclonal cells can be used as candidates for cell therapy and extracellular vesicles for fundamental treatment of salivary gland dysfunction, as well as for disease modeling, pathology research, drug screening, It is expected to be widely used in salivary gland research, such as toxicity evaluation and genetic manipulation.

Description

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

The present invention provides a method for isolating epithelial basal stem cells or progenitor cells of human main and minor salivary glands in the form of monoclonal cells and proliferating while maintaining long-term culture in vitro and a method for producing extracellular vesicles for treatment of salivary gland diseases using the same It is about.

"Stem cell" refers to an undifferentiated cell having the ability to differentiate into various cells and the self-renewal ability to continuously proliferate symmetrically or asymmetrically into the same cell as itself. Stem cells exist in all parts of the fetal development process, and in adulthood, mesenchymal stem cells (MSC) exist in the bone marrow, adipose tissue, and interstitium within tissues, and epithelial stem cells ( Epithelial stem cells) are located in the parenchyme of each tissue. Research using stem cells has been actively conducted in various fields since the concept of stem cells was established until now. In particular, tissue regeneration by direct differentiation of stem cells, anti-inflammation and anti-fibrosis by paracrine secretion , and studies on extracellular vesicles, which are known to play an important role in intercellular signaling and microenvironmental regulation, are being conducted. Stem cells can be largely divided into embryonic stem cells, adult stem cells, and induced pluripotent stem cells. Among these, embryonic stem cells are extracted from human embryos and have the disadvantage of limiting the expansion of research due to bioethical controversy and limitations on tumor induction. Induced pluripotent stem cells are stem cells that acquire the pluripotency of embryonic stem cells by artificially manipulating specific genes of adult stem cells to induce a dedifferentiation phenomenon that returns to an undifferentiated state. However, induced pluripotent stem cell research entails cost issues such as facilities, equipment, and researcher skills. Compared to the above two types of stem cells, adult stem cells are also called tissue-specific stem cells because the range of cells that can be differentiated is set. In this case, possible immune rejection reactions can also be resolved. In addition, it has the advantage that it does not require a high level of cost or skill in research.

Stem cell therapy is in the limelight as a candidate for treatment of incurable and degenerative diseases for which no cure has been found. However, several limitations also exist. Currently, the widely used method for isolating adult stem cells for stem cell therapy is 'Density-Gradient Centrifugation Method (GCM)', which is a type of antibody or flow cytometry. After selection using Fluorescence-activated Cell Sorting (FACS), cells that attach to the bottom of the incubator and grow are separated and cultured in large quantities. The disadvantage of this method is that it cannot exclude cells other than stem cells, that is, the limitation in terms of purity of stem cells is clear. It is pointed out that the limitations of existing cell therapy products may be due to the purity of stem cells, and this is clearly a situation that needs to be improved. As a way to compensate for this, the ‘Subfractionation Culturing Method (SCM)’ was devised. SCM is a technology that selects only stem cells while moving cells through several stages. Compared to GCM, it has the advantage of being able to isolate stem cells with a higher purity. This suggests that it can affect the improvement of the fundamental effect of stem cell therapy.

The use of existing SCM is focused on mesenchymal stem cells (MSC), which exist in bone marrow, fat, and umbilical cord blood. It not only induces regeneration or differentiation of specific tissues by secreting various signaling substances or growth factors such as 'cytokine' or 'chemokine' into the surroundings, but also exhibits immune tolerance or immunosuppressive action. are being sought

The main cell responsible for the function of the salivary gland is the epithelial cell. It is divided into myoepithelial cells, which squeeze epithelial cells to secrete saliva, and neuroanl cells, which secrete neurotransmitters to myoepithelial cells and acinar epithelial cells. Salivary gland hypofunction is mainly caused by various factors such as drug treatment, radiation treatment, aging, autoimmune disease, etc., to repair damage to epithelial cells, especially acinar epithelial cells, which play a major role in salivary secretion function, and salivary glands. Epithelial stem cells (ESCs), which play a major role, are damaged and depleted. This leads to a decrease in salivary secretion and a decrease in resilience, resulting in a decrease in quality of life and frequent oral diseases. To this end, temporary symptom relief methods such as artificial saliva are being sought, but a fundamental treatment has not yet been proposed. Stem cell therapy is emerging as a fundamental treatment for this, and it is known that mesenchymal stem cells can secrete tissue growth factors or cytokines to relieve or regenerate damage to epithelial cells in tissues. Studies have been published one after another showing that acinar epithelial cells or ductal epithelial cells have characteristics of progenitors and stem cells and play a role in tissue regeneration. However, it is still not easy to directly develop these epithelial progenitor cells or stem cells, or stem cell-derived products such as exosomes as therapeutic agents.

The biggest reason why it is difficult to develop epithelial stem cells as a therapeutic agent is that it is not easy to isolate tissue-specific epithelial stem cells derived from a small amount of tissue and culture monoclonal epithelial stem cells. Above all, long-term culture of epithelial stem cells in 2D is a very difficult technique. Therefore, a technique for isolating a small amount of salivary gland tissue-derived epithelial stem cells obtained through a patient's biopsy process into high-purity monoclonal cells, and maintaining and proliferating them in two-dimensional culture conditions would be very useful. In addition, if epithelial stem cells with excellent proliferative ability, excellent secretion of extracellular vesicles, tissue specificity, and excellent ability to differentiate into cells within the tissue can be isolated and proliferated and used as cell therapy or extracellular vesicle therapy, salivary gland function decline It is expected that it can be used not only as one of the fundamental treatments for the disease, but also as an embryology using stem cells throughout salivary gland related research. To this end, the main challenges include isolation and cultivation of monoclonal epithelial stem cells from a small amount of salivary gland tissue, long-term in vitro culture and maintenance of proliferative ability capable of mass-producing extracellular vesicles, and preventing heterogeneous substances from being included in the culture medium for clinical application. am.

Korean Registered Patent No. 10-2168088 (registered on October 14, 2020)

The purpose of the present invention is to optimize the layer separation culture technology to specifically isolate high-purity human salivary gland epithelial stem or progenitor cells-derived monoclonal cells from a small amount of salivary gland tissue, and chemically Minimize the content of heterogeneous substances (bovine serum, bovine pituitary extract, porcine derived materials) through cultivation using a combination of the prepared culture medium and low-molecular compound, and ultimately maintain the characteristics of salivary gland epithelial stem cells for a long time in an animal-free environment for a long time It is to provide a method for obtaining a large amount of extracellular vesicles and reproduction of culture and proliferation, differentiation potential.

In order to achieve the above object, the present invention provides a monoclonal cell derived from salivary gland epithelial stem cells or progenitor cells containing a medium containing Y-27632, A83-01 and a bone morphogenetic protein (BMP) inhibitor as an active ingredient. A culture medium composition for cell isolation, maintenance, proliferation or differentiation is provided.

In addition, the present invention comprises the steps of (1) obtaining salivary gland epithelial stem cells or progenitor cells from salivary gland tissue; (2) isolating salivary gland epithelial monoclonal cells by culturing the obtained salivary gland epithelial stem cells or progenitor cells in the culture composition; and (3) maintaining or proliferating the isolated salivary gland epithelial cells by culturing the isolated salivary gland epithelial monoclonal cells in the culture composition, isolating and maintaining the monoclonal cells derived from salivary gland epithelial stem cells or progenitor cells. and propagation methods.

In addition, the present invention comprises the steps of (1) obtaining salivary gland epithelial stem cells or progenitor cells from salivary gland tissue; (2) isolating salivary gland epithelial monoclonal cells by culturing the obtained salivary gland epithelial stem cells or progenitor cells in the culture composition; (3) maintaining or proliferating the salivary gland epithelial cells by culturing the isolated salivary gland epithelial monoclonal cells in the culture composition; and (4) separating the extracellular vesicles from the maintained or proliferated salivary gland epithelial cells.

In addition, the present invention provides a culture solution of monoclonal cells derived from salivary gland epithelial stem cells or progenitor cells isolated, maintained, and proliferated according to the above method; Or it provides a pharmaceutical composition for preventing or treating salivary gland inflammatory disease comprising the extracellular vesicles produced according to the above method as an active ingredient.

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, and in detail, a small amount obtained during surgery or biopsy Conditions for isolating monoclonal salivary gland epithelial cells from main salivary gland or minor salivary gland tissue were established through an optimized layered culture method, and culture medium conditions for long-term culture of salivary gland epithelial stem cells or progenitor cells in 2D were established. It was confirmed that the cultured stem cells were monoclonal salivary gland basal stem or progenitor cells, and it was confirmed that the yield and cell proliferation ability were excellent. The method for separating epithelial stem cells was verified by characterization analysis, and a method for differentiating into various cells in a 3-dimensional culture environment was presented. Finally, the process of obtaining extracellular endoplasmic reticulum and its therapeutic effect were described. These high-purity human salivary gland epithelial stem cell or progenitor cell-derived monoclonal cells can be used as candidates for cell therapy and extracellular vesicles for fundamental treatment of salivary gland dysfunction, as well as for disease modeling, pathology research, drug screening, It is expected to be widely used in salivary gland research, such as toxicity evaluation and genetic manipulation.

1 is a schematic diagram for obtaining monoclonal cells of salivary gland epithelial cells through SCM.
Figure 2 shows the appearance of salivary gland epithelial cells at the limit of growth without a combination of low molecular weight compounds in each chemically defined medium (CDM).
3 shows the results of concentration screening of low molecular weight compounds using Dermacult.
Figure 4 shows the concentration screening results in the low molecular compound complex condition.
Figure 5 shows the results of the growth limit of the salivary gland epithelial cells increased by the combination of small molecule compounds in each CDM.
Figure 6 shows the results of population doubling time shortened by the low molecular compound complex condition.
7 shows the concentration screening results in the low molecular compound complex conditions.
Figure 8 shows the result of promoting cell growth by adding WNT during cell separation by initial SCM.
9 shows the results of characterization of cultured cells through flow cytometry.
10 shows the results of confirming basal cell gene expression markers through parotid single cell transcriptome analysis.
11 shows the result of confirming salivary gland basal (progenitor) stem cells through immunofluorescence staining.
12 shows the results of cell marker verification of salivary gland epithelial stem cells through flow cytometry.
13 shows differential gene expression results from Bulk-RNA seq.
14 shows the results of character analysis of different cells for each colony.
15 shows the results of cell marker verification of salivary gland epithelial stem cells through flow cytometry.
16 shows a comparison of exosome production rates between K-SFM and Dermacult, and results of increasing exosome production by the low-molecular-weight compound.
17 shows exosome extraction and validation results from salivary gland epithelial stem cells.
18 shows the anti-inflammatory effect results of exosomes in the salivary gland ligation model.

The present inventors tested four types of chemically defined medium (CDM) as a basal medium capable of culturing epithelial cells, and tested WNT3A, a growth factor, Y-27632, a small molecule compound, or a similar ROCK I/II inhibitor, A83-01 or its Salivary gland epithelial basal stem cells or progenitor cells containing medium containing TGF-beta pathway inhibitors similar to TGF-beta pathway inhibitors and BMP pathway inhibitors such as DMH1 and LDN193189 as active ingredients are monoclonal cell groups The present invention was completed by optimizing the culture medium composition for isolation, maintenance, proliferation or differentiation in the form of (monoclonal cell population).

The present invention isolates, maintains, proliferates, or separates monoclonal cells derived from salivary gland epithelial stem cells or progenitor cells containing a medium containing Y-27632, A83-01 and a bone morphogenetic protein (BMP) inhibitor as active ingredients. A culture medium composition for differentiation is provided.

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

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.

Preferably, the medium may be applied to a subfractionation culturing method (SCM), but is not limited thereto.

In addition, the present invention comprises the steps of (1) obtaining salivary gland epithelial stem cells or progenitor cells from salivary gland tissue; (2) isolating salivary gland epithelial monoclonal cells by culturing the obtained salivary gland epithelial stem cells or progenitor cells in the culture composition; and (3) maintaining or proliferating the isolated salivary gland epithelial cells by culturing the isolated salivary gland epithelial monoclonal cells in the culture composition, isolating and maintaining the monoclonal cells derived from salivary gland epithelial stem cells or progenitor cells. and propagation methods.

Preferably, salivary gland epithelial stem cells or progenitor cells may be cultured by adding WNT3A to step (2), but it is not limited thereto.

Preferably, the salivary gland epithelial stem cell or progenitor cell culture in step (2) may be cultured according to a subfractionation culturing method (SCM), but is not limited thereto.

Preferably, the monoclonal cells derived from salivary gland epithelial stem cells or progenitor cells may be CD49f+/CD26− salivary gland basal cells, but are not limited thereto.

Preferably, the monoclonal cells derived from salivary gland epithelial stem cells or progenitor cells express salivary gland basal cell markers and salivary gland progenitor cell markers KRT14, KRT5, KRT19, SOX9 and TP63, and express salivary gland epithelial cell marker CDH1. It may, but is not limited thereto.

Preferably, the monoclonal cells derived from the salivary gland epithelial stem cells or progenitor cells may have the ability to differentiate into the salivary gland epithelial tissue.

In addition, the present invention comprises the steps of (1) obtaining salivary gland epithelial stem cells or progenitor cells from salivary gland tissue; (2) isolating salivary gland epithelial monoclonal cells by culturing the obtained salivary gland epithelial stem cells or progenitor cells in the culture composition; (3) maintaining or proliferating the salivary gland epithelial cells by culturing the isolated salivary gland epithelial monoclonal cells in the culture composition; and (4) separating the extracellular vesicles from the maintained or proliferated salivary gland epithelial cells.

Preferably, the extracellular vesicles may be salivary gland epithelial stem cell exosomes, but are not limited thereto.

The "extracellular vesicle (EV)" of the present invention refers to nano-sized vesicles derived from cells, and depending on the secretion type and size, exosomes, microvesicles, and ectosomes ), microparticles, membrane vesicles, nanovesicles, and outer membrane vesicles. Extracellular endoplasmic reticulum is a major means of communication between cells, including nucleic acids and proteins, which are the main components of cells.

In addition, the present invention provides a culture solution of monoclonal cells derived from salivary gland epithelial stem cells or progenitor cells isolated, maintained, and proliferated according to the above method; Or it provides a pharmaceutical composition for preventing or treating inflammatory diseases of the salivary gland comprising the extracellular vesicles produced according to the above method as an active ingredient.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier is one commonly used in formulation, including lactose, dextrose, sucrose, sorbitol, mannitol, and starch. , acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, stear including, but not limited to, acid magnesium and mineral oil, and the like. The composition for preventing or treating cancer metastasis of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like, in addition to the above components.

The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, direct intravascular injection, intranasal administration, intrapulmonary administration and intrarectal administration. When administered orally, since proteins or peptides are digested, oral compositions can be formulated to coat the active agent or protect it from degradation in the stomach, and the composition of the present invention can be used in any device through which the active agent can move to target cells. can be administered by

A suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration method, patient's age, weight, sex, morbid condition, food, administration time, administration route, excretion rate and reaction sensitivity, usually This allows the skilled physician to readily determine and prescribe dosages effective for the desired treatment or prophylaxis.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulation using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by those skilled in the art, or Or it can be prepared by incorporating into a multi-dose container. At this time, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, suppository, powder, granule, tablet or capsule, and may additionally contain a dispersing agent or stabilizer.

The "saliva" of the present invention is a mixed liquid secreted from the parotid, submandibular, sublingual, and mucous glands present in the oral mucosa. Saliva is a key component of the human body, produced by the salivary glands and discharged into the oral cavity. Saliva is an essential component of the human body and contains bioactive proteins, digestive enzymes, mucus, immunoglobulins, and various salts.

Saliva plays a very important role in maintaining homeostasis of the human body as well as oral health. For example, mucin and immunoglobulin, which are major components of saliva, play a primary defense role against external infections, and protect the oral mucosa and teeth by lubricating the oral cavity and teeth, maintaining moisture, and neutralizing pH. do. In addition, there are digestive enzymes such as amylase such as ptyalin in saliva, which are responsible for promoting digestion by decomposing starch to maltose units. In addition, the body's water metabolism and body temperature can be controlled by the secretion of saliva, and toxic substances (I, Hg, Pb, etc.) are excreted.

The “salivary gland” of the present invention is an organ that produces and secretes saliva, such as the parotid gland, the submaxillary gland, and the sublingual gland (sublingual gland). major salivary gland, and minor salivary glands distributed in various parts of the oral mucosa, such as mucous glands that exist in the mucous membrane of the oral cavity. .

Hereinafter, the present invention will be described in more detail through the following examples. However, the present invention is not limited by these examples.

< Experimental example >

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

1. Reagents

The following reagents were used in the present invention.

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)

2. Obtaining Human Salivary Gland Epithelial Cells from Tissues

(1) During surgical procedures such as tumor removal, normal tissue of the main salivary gland that has not been invaded by tumor is collected by a method such as biopsy, or after taking 1-2 lobes from the minor salivary gland of a patient suspected of Sjögren, they are collected using CTS™ Put in DPBS, calcium, magnesium and store in a refrigerator at 4℃ until the day or the next day.

(2) Add collagenase to CTS™ DPBS, calcium, magnesium to make 0.4 U/mL. Y27632 is added to the enzyme solution at a concentration of 10 µM. Prepare 1 mL per 10 mg of tissue. At this time, if the tissue weight is less than 10 mg, prepare a minimum volume of 1 mL.

(3) Place the tissue on a petri dish, grind it finely using a scalpel blade, and put it in the prepared enzyme solution to react at 37℃ for 1 hour. Invert once every 10 minutes to mix well the solution and tissue.

(4) Centrifuge at 300g 5 min 4℃, remove the enzyme solution, add CTS™ DPBS (1×), without calcium chloride, without magnesium chloride twice the volume of the enzyme solution, and centrifuge again at 300g 5 min 4℃ and remove the DPBS.

(5) Calculate and add CTS™ TrypLE™ Select Enzyme at 1 mL per 50 mg of original tissue weight. Y27632 is added to a concentration of 10 µM. (Add 1 mL for tissue weighing less than 50 mg.) React at 37℃ for 20 minutes and invert every 10 minutes to mix evenly.

(6) After mixing with the same amount of culture solution, pass through a 70 μm strainer, pass through a Falcon tube with strainer in succession, centrifuge at 300g 5 min 4℃, and remove the supernatant. Thereafter, the culture medium was added again and centrifuged at 300g 5 min 4°C to remove the supernatant and count the cells.

3. Epithelialization of human salivary glands using the SCM culture method monoclonal cells separation

(1) Cells are homogenized in 10 ml culture medium, spread evenly on a 100×20mm culture dish, and then stored in an incubator at 37°C and 5% CO ₂ environment. At this time, the composition of the culture medium is shown in Table 1.

(2) In order to carry out layer separation culture (SCM) for isolating epithelial monoclonal cells, after 2 hours, transfer the culture medium containing the cells to a new 100 × 20mm culture dish, and repeat this two more times.

(3) After 24 hours, culture the dish resulting from (2), remove the existing culture medium every 2-3 days, and replace it with a new culture medium.

(4) After confirming that a colony is formed, if the size of a cell cluster constituting one colony exceeds 1 cm ² , remove the culture medium, mark it with a hydrophobic pen, and remove the remaining culture medium using 1×PBS. After adding 50~100 μL of TrypLE + Y27632, store in an incubator at 37℃ and 5% CO ₂ environment for 15 minutes.

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

(6) After centrifugation at 300 g at 4℃ for 5 minutes, the supernatant is removed and 250 μL of a new culture solution is homogenized and spread in each well of a 48-well plate for each cell cluster.

[Table 1]

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

(1) After removing the culture medium at intervals of 2-3 days, replace it with a new culture medium.

(2) When the well is 80 to 90% full with cells, proceed with subculture.

(3) At this time, use Table 2 for the composition of the culture medium used during passage.

(4) After removing the culture medium, use PBS to remove the residual culture medium. Then, after adding TrypLE express (about 250 μl) enough to cover the bottom of the well, it is stored in an incubator at 37°C and 5% CO ₂ environment for 15 minutes.

(5) Put the cells separated from the culture plate by TrypLE express into separate 5 ml tubes, and neutralize by adding 1 ml of the culture medium of Table 2.

(6) After centrifugation at 300 g at 4℃ for 5 minutes, the supernatant is removed, and 1 ml of the culture solution in Table 2 is homogenized and spread in each well of a 12-well plate for each cell cluster.

(7) After removing the existing culture medium at intervals of 2-3 days, replace it with a new culture medium.

(8) The above subculture goes through the scale-up step as follows, 24 well culture plate (0.5 mL), 12 well culture plate (1 mL), 6 well culture plate (2 ml), 25cm ² cell culture flask (5 ml), 75cm ² cell culture flask (15 ml), and 175cm ² cell culture flask (35 ml). See parentheses for the volume of culture medium to be added.

(9) From 6 wells, it can be used for various experiments, and the passage proceeds at a ratio of 1:2 to 1:10.

(10) Check the population doubling, and if the growth rate is not exponential, it is judged that the cells are aged and discarded.

[Table 2]

5. Characteristic analysis of proliferated epithelial cells

(1) Initiate the following experiments when clones successfully proliferated and 70-80% confluency in culture medium conditions.

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

(3) RNA is isolated using TRIzol or an RNA extraction kit, and gene expression changes are confirmed by real time PCR and bulk-RNA seq.

(4) Using flow cytometry, check the presence or absence of stem cell-related protein expression on the cell surface.

(5) Differentiation test is performed through 3-dimensional culture using Matrigel.

(6) Harvest the extracellular vesicles by reflecting the characteristics of the cells that secreted the extracellular vesicles, and check whether CD9 or the like is highly expressed by Western blotting.

< Example 1> human salivary gland epithelial stem cells or progenitor cell origin of monoclonal cells Screening of optimal culture medium composition for isolation and long-term in vitro culture

High-purity human salivary gland epithelial stem cell or progenitor cell-based monoclonal cells were isolated using a layer separation culture (SCM) modification and various types of chemically defined medium (CDM) and small molecule mixture methods (FIG. 1). ). During the course of SCM Experiments were conducted using the culture medium of [Table 1], and after the first passage, the culture medium of [Table 2] was used.

First, the extent to which epithelial cells can be cultured was evaluated only with CDM. K-SFM, EpiLife, and CnT-PR were confirmed to stop growing and age on average after 6, 2, and 3 passages, respectively, when passages were performed at a ratio of 1:2 in the absence of small molecule compounds. Dermacult was able to be passaged 20 to 30 times without a low molecular weight compound (Fig. 2).

ROCK I/II inhibitor (Y-27632) and TGF-beta pathway inhibitor (A83-01 ), the BMP pathway inhibitor (LDN193189) was added to determine the optimum concentration for maintaining the characteristics and enabling stable long-term culture (FIG. 3).

Finally, it was confirmed that the salivary gland epithelial cell growth was maintained at the highest level under the condition of adding Y-27632 10 μM, A83-01 1 μM, and LDN193189 0.1 μM to KEM through experiments at various concentrations under the mixed conditions of the low-molecular compounds. (FIG. 4).

When the optimal combination of low-molecular compounds was added, K-SFM could be passaged 9 to 18 times, EpiLife 3 times, CnT-PR more than 30 times, and Dermacult more than 50 times (FIG. 5) .

In particular, when Dermacult was added, it was confirmed that cells proliferated faster and could be cultured for a long time in the experimental group in which the cells were cultured with the addition compared to the control group in which the cells were cultured without the addition of the low-molecular compound, and the population doubling time of the cells was also significantly reduced. (FIG. 6).

In the case of K-SFM in CDM, when various types of low-molecular compound combinations were confirmed, Y-27632 and A83-01 conditions generally gave good results, but Chir99021 or PD0325901 showed poor results. Isoproterenol had no effect. In addition, when LDN193189 was replaced with another type of BMP pathway inhibitor, DMH-1, similar effects to Dermacult's YAL were obtained, but a higher concentration of 1 uM was required (FIG. 7).

In the case of using K-SFM in CDM, rapid cell growth was observed when WNT was initially added, but it was not essential in the low molecular weight compound culture condition, and it was confirmed that the cell shape changed when added for a long time. If WNT3A is treated, it can be said that it is preferable to use it only in the SCM separation process and exclude it from subsequent passaging (FIG. 8).

< Example 2> Flow cytometry department immunofluorescence staining through human salivary gland epithelial stem cells or progenitor cell origin of monoclonal cells phylogenetic analysis

In order to identify which cells the isolated and cultured cells were among the basal, luminal, myoepithelial, and acinar cells constituting the epithelial cells of the salivary gland, the cells of the parotid tissue were sorted by flow cytometry using CD49f and CD26 antibodies, and then cultured. As a result of flow cytometry analysis of the cultured cells once again, they were composed of CD49f+/CD26- basal cells (FIG. 9).

In order to further verify that only salivary gland basal progenitors and stem cells can selectively grow under the conditions of the culture medium established as above, markers specifically expressed in salivary gland basal cells were selected as candidates by single cell transcriptome analysis (FIG. 10). ).

Thereafter, basal cell expression markers were confirmed through immunofluorescence staining in cultured cells. KRT14, KRT5, KRT19, SOX9, and TP63, which are salivary gland basal cell markers and are also used as precursor cell markers, were identified. KRT7, a marker for differentiated tubular epithelial cells, and SOX2, a precursor marker for acinar cells, were not identified. In addition, it was confirmed by expression of CDH1 and lack of VIM that epithelial cell characteristics were not lost even in long-term culture (FIG. 11).

Next, in order to verify that they were epithelial stem cells, flow cytometry analysis was performed to compare with mesenchymal stem cell markers, and CD34 and CD90, known to be positive in mesenchymal stem cells, were found to be negative (FIG. 12).

< Example 3> Analysis of the characteristics of monoclonal cells derived from human salivary gland epithelial stem cells or progenitor cells by bulk-RNA sequencing

The characteristics of these cells were identically expressed in bulk RNA-seq, which analyzed each of the five colonies. SOX2 and VIM were not expressed, and KRT14, KRT5, KRT19, SOX9, TP63, and CDH1 were highly expressed, and KRT7 was highly expressed. It was confirmed that the expression was low (FIG. 13).

In addition, it was reconfirmed that monoclonal epithelial stem cell isolation is possible through the SCM method through each colony having slightly different characteristics (FIG. 14).

< Example 4> Analysis of differentiation capacity of monoclonal cells derived from human salivary gland epithelial stem cells or progenitor cells through immunofluorescence staining

When organoids were prepared by embedding the corresponding cells in Matrigel, it was confirmed whether luminal cells were formed in cells mainly composed of basal cells. After embedding, the size of the organoids could be confirmed to grow in the bright field, and at the same time, immunofluorescence staining and real-time PCR confirmed that KRT7 was expressed and increased (FIG. 15).

< Example 5> extracellular endoplasmic reticulum Isolation and characterization and potency confirmation

As a result of comparing the production of exosomes in K-SFM and Dermacult, even if the low-molecular compound combination was added to K-SFM, fewer exosomes were produced than in Dermacult, and more exosomes were produced when the low-molecular compound combination was added to Dermacult. It was confirmed (FIG. 16).

It was confirmed that the extracellular vesicles generated from epithelial stem cells have characteristics of epithelial stem cell exosomes through Western blotting, NTA, and transmission scanning microscopy (FIG. 17).

After ligating the submandibular conduit of the rat for 2 weeks, the released model showed excellent anti-inflammatory, anti-fibrotic and tissue protective effects compared to the control group injected with PBS (FIG. 18).

According to the present invention, it is possible to culture high-purity human salivary gland epithelial stem cells with a combination of low-molecular weight compounds under serum-free and BPE-free conditions (Dermacult). In addition, it shows the potential to be successful even under the conditions achieved up to animal free (CnT-PR). In addition, it was confirmed that it can be used as a treatment for inflammatory diseases of the salivary gland by increasing the secretion of extracellular vesicles under the conditions.

Having described specific parts of the present invention in detail above, it is clear to those skilled in the art that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (14)

A culture medium for isolation, maintenance, proliferation or differentiation of monoclonal cells derived from salivary gland epithelial stem cells or progenitor cells containing a medium containing Y-27632, A83-01 and a bone morphogenetic protein (BMP) inhibitor as an active ingredient composition. The culture medium composition according to claim 1, wherein the BMP inhibitor is DMH1 or LDN193189. The culture medium composition according to claim 1, wherein 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. The culture medium composition according to claim 2, wherein 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. The culture medium composition according to any one of claims 1 to 4, wherein the medium is applied to a subfractionation culturing method (SCM). (1) obtaining salivary gland epithelial stem cells or progenitor cells from salivary gland tissue;
(2) isolating salivary gland epithelial monoclonal cells by culturing the obtained salivary gland epithelial stem cells or progenitor cells in the culture medium composition according to any one of claims 1 to 4; and
(3) maintaining or proliferating salivary gland epithelial cells by culturing the isolated salivary gland epithelial monoclonal cells in the culture composition according to any one of claims 1 to 4; salivary gland epithelial stem cells Or a method for isolating, maintaining, and proliferating monoclonal cells derived from progenitor cells. The method according to claim 6, wherein the salivary gland epithelial stem cells or progenitor cells are cultured by adding WNT3A in step (2). The method according to claim 6, wherein the salivary gland epithelial stem cells or progenitor cells in step (2) are cultured according to the Subfractionation Culturing Method (SCM). The method according to claim 6, wherein the monoclonal cells derived from salivary gland epithelial stem cells or progenitor cells are CD49f+/CD26− salivary gland basal cells. The method of claim 6, wherein the salivary gland epithelial stem cell or progenitor cell-derived monoclonal cells express salivary gland basal cell markers and salivary gland progenitor cell markers KRT14, KRT5, KRT19, SOX9 and TP63, and the salivary gland epithelial cell marker CDH1 A method characterized by being expressed. 7. The method according to claim 6, wherein the monoclonal cells derived from salivary gland epithelial stem cells or progenitor cells have the ability to differentiate into salivary gland epithelial tissue. (1) obtaining salivary gland epithelial stem cells or progenitor cells from salivary gland tissue;
(2) isolating salivary gland epithelial monoclonal cells by culturing the obtained salivary gland epithelial stem cells or progenitor cells in the culture medium composition according to any one of claims 1 to 4;
(3) maintaining or proliferating the salivary gland epithelial cells by culturing the isolated salivary gland epithelial monoclonal cells in the culture medium composition according to any one of claims 1 to 4; and
(4) A method for producing extracellular vesicles from monoclonal cells derived from salivary gland epithelial stem cells or progenitor cells, comprising the step of separating extracellular vesicles from the maintained or proliferated salivary gland epithelial cells. The method of claim 12, wherein the extracellular vesicles are salivary gland epithelial stem cell exosomes. a culture medium of monoclonal cells derived from salivary gland epithelial stem cells or progenitor cells isolated, maintained, and proliferated according to the method of claim 6; Or a pharmaceutical composition for preventing or treating inflammatory diseases of the salivary gland comprising the extracellular vesicles produced according to the method of claim 12 as an active ingredient.

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