Method for producing tonsil-derived mesenchymal stem cells

Abstract

The present invention relates to a method for producing tonsil-derived mesenchymal stem cells. According to the production method of the present invention, a large amount of tonsil-derived mesenchymal stem cells can be produced by isolating mononuclear cells from tonsil tissues and subculturing the mononuclear cells by an optimized normalization method, and the method can be advantageously used for developing a stem cell therapeutic agent.

Description

Method for producing tonsil-derived mesenchymal stem cells

[0001] The present invention relates to a method for producing tonsil-derived mesenchymal stem cells, and more particularly, to a method for producing tonsil-derived mesenchymal stem cells, comprising the steps of obtaining mononuclear cells from tonsil tissue; and performing primary culture and then subculturing the obtained mononuclear cells to obtain tonsil-derived mesenchymal stem cells.

[0002] Stem cells are cells that can differentiate into various cell types that make up biological tissues. They are a general term for undifferentiated cells obtained from various tissues of embryos, fetuses, and adults. Stem cells are characterized by their ability to differentiate into specific cells in response to differentiation stimuli (environment), the ability to self-renew (produce cells identical to themselves) through cell division, and the flexibility (plasticity) to differentiate into different cell types depending on differentiation stimuli.

[0003] Stem cells can be classified into totipotent stem cells, pluripotent stem cells, and multipotent stem cells according to their differentiation ability.

[0004] A pluripotent stem cell is a cell that has the pluripotent property that can develop into a complete organism. Cells up to the 8-cell stage after fertilization of an egg and sperm have this property, and if these cells are separated and transplanted into the uterus, they can develop into a complete organism. Pluripotent stem cells are cells that can develop into various cells and tissues derived from the ectoderm, mesoderm, and endoderm. They are derived from the inner cell mass located inside the blastocyst that appears 4-5 days after fertilization. These are called embryonic stem cells and can differentiate into various other tissue cells, but cannot form new living organisms. Multipotent stem cells are stem cells that can differentiate only into cells specific to the tissues and organs in which they are contained. They are involved in the growth and development of each tissue and organ in the fetal, neonatal, and adult stages, as well as in maintaining homeostasis in adult tissues and inducing regeneration when tissues are damaged. Tissue-specific multipotent cells are collectively called mesenchymal stem cells.

[0005] Stem cell therapy utilizes stem cells, which possess self-renewal and multipotentiality, to help regenerate damaged cells and improve symptoms in patients. Stem cells that can be used in cell therapy can be broadly categorized into adult stem cells, embryonic stem cells, and induced pluripotent stem cells. While each type has its own advantages and disadvantages, adult stem cells are currently the primary therapeutic choice in clinical practice.

[0006] Adult stem cells reside in various organs of the body and play a role in regenerating damaged tissue. These cells, found in nearly every organ, including the bone marrow and umbilical cord, are collectively called adult stem cells. Because their differentiation potential is relatively limited, they are also called tissue-specific stem cells. Medically, they are considered relatively safe and are therefore used in various clinical trials.

[0007] Meanwhile, mesenchymal stem cells (MSCs) are non-hematopoietic, multipotent fibroblast-like cells that exhibit the ability to differentiate into mesodermal lineages, including osteocytes, adipocytes, and chondrocytes. MSCs can be readily isolated from various tissues, including bone marrow, adipose tissue, amniotic fluid, and Wharton's jelly (substantia gelatinea funiculi umbilicalis), and possess immunomodulatory properties, enabling successful allogeneic transplantation. Furthermore, MSCs can be derived from various types of tissues. However, it is known that MSCs from various sources can exhibit heterogeneity due to differences in culturing methods under in vitro proliferation culture conditions, differences in stem cell donor sources, or differences in stem cell extraction sites.

[0008] Meanwhile, some mesenchymal stem cells (MSCs) have significant limitations in obtaining them, making their utilization difficult. For example, MSCs derived from umbilical cord blood and adipose tissue must be obtained through invasive methods. The least invasive method available is bone marrow extraction, but bone marrow extraction requires anesthesia and causes pain, limiting its utility. Alternative methods, such as those using peripheral blood to isolate patient-specific stem cells, are being sought. However, the number of MSCs that can be isolated from adults using peripheral blood alone is too small, the isolation methods are uneconomical, and even when isolated, they often do not proliferate sufficiently to meet the needs of cell therapy. Therefore, more practical alternatives are needed. Furthermore, adult stem cells from elderly patients have significantly lower proliferative capacity than those from younger patients, and their ability to secrete various factors and migrate to lesions is impaired. Therefore, it is necessary to obtain cells from tissues that can be naturally isolated from younger patients or from discarded tissues. In addition, the cells obtained in this way need to be quantitatively secured for easy experiments and have their differentiation potential well maintained during cell subculture.

[0009] The human tonsils are a type of lymphoepithelial immune tissue that exists in the oropharynx and nasopharynx of the human body. They perform their immune function until puberty and then gradually degenerate. Therefore, if problems such as abnormal enlargement of the tonsils (adenotonsillar hyperplasia) or infection (tonsillitis) occur, they are removed through tonsillectomy.

[0010] Recently, research results on extracting mesenchymal stem cells from tonsil tissue have been reported, and tonsil tissue is attracting attention as a new source of adult stem cells. Stem cells derived from tonsil tissue exhibit the same level of stem cell potential as other adult stem cells, and are capable of differentiating not only into mesodermal cells (adipogenesis, osteogenesis, chondrogenesis), but also into endodermal cells such as parathyroid hormone-secreting cells and insulin-secreting cells, and ectodermal cells such as motor neurons and Schwann cells. One advantage of tonsil tissue is that it is relatively easy to obtain as it is discarded during tonsillectomy. However, it is difficult to secure stem cells derived from tonsil tissue in therapeutically effective quantities, limiting its utilization.

[0011] Against this backdrop, the present inventors have endeavored to develop a method for obtaining therapeutically effective amounts of stem cells from tonsil tissue. As a result, they have confirmed that large quantities of tonsil-derived mesenchymal stem cells can be produced by isolating and subculturing mononuclear cells from tonsil tissue. This discovery, coupled with the usefulness of the method and the mesenchymal stem cells produced therefrom for the development of stem cell therapeutics, has led to the present application.

[0012] [Prior Art Literature]

[0013] [Patent Document]

[0014] Republic of Korea Patent No. 10-1508413

[0015] Republic of Korea Patent Publication No. 10-2023-0043587

[0016] [Non-patent literature]

[0017] Khatri et al., “Harvesting multipotent progenitor cells from a small sample of tonsillar biopsy for clinical applications”, Stem Cell Research & Therapy (2017) 8:174

[0018] K. E. Lee et al. "The efficacy of conditioned medium released by tonsil-derived mesenchymal stem cells in a chronic murine colitis model", PLoS ONE 14(12): e0225739

[0019] Lee et al., "Isolation and Localization of T-MSCs by W5C5", Cell Physiol Biochem 2016;38:83-93

[0020] The present invention has been devised to solve the above problems, and provides a method for producing tonsil-derived mesenchymal stem cells for obtaining a therapeutically effective amount of mesenchymal stem cells from tonsil tissue.

[0021] In one embodiment of the present invention to achieve such a task,

[0022] 1) a step of obtaining mononuclear cells from tonsil tissue isolated from a donor; and

[0023] 2) A step of obtaining tonsil-derived mesenchymal stem cells by first culturing the obtained mononuclear cells and then subculturing them up to the second passage.

[0024] A method for producing tonsil-derived mesenchymal stem cells, including:

[0025] In another embodiment of the present invention, tonsil-derived mesenchymal stem cells produced by the above production method are provided.

[0026] In another embodiment of the present invention, a composition comprising tonsil-derived mesenchymal stem cells or cells differentiated therefrom produced by the above production method is provided.

[0027] In another embodiment of the present invention, a cell therapy product is provided that includes tonsil-derived mesenchymal stem cells or cells differentiated therefrom produced by the above production method as an active ingredient.

[0028] In another embodiment of the present invention, a cosmetic composition is provided that includes tonsil-derived mesenchymal stem cells or cells differentiated therefrom produced by the above production method as an active ingredient.

[0029] The present invention relates to a method for producing tonsil-derived mesenchymal stem cells. According to the method of the present invention, a large quantity of tonsil-derived mesenchymal stem cells can be produced by isolating and subculturing mononuclear cells from tonsil tissue using an optimized standardized method, and there is an effect that this can be usefully utilized in the development of stem cell therapeutic agents.

[0030] FIG. 1 is a diagram showing a photograph of tonsil tissue extracted from a patient according to one embodiment of the present invention.

[0031] FIG. 2 is a diagram showing a photograph of a large piece of tonsil tissue that has been torn once according to one embodiment of the present invention.

[0032] FIG. 3 is a diagram showing a photograph of finely chopped tonsil tissue according to one embodiment of the present invention.

[0033] FIG. 4 is a diagram showing a process of incubating a fine tissue according to one embodiment of the present invention.

[0034] FIG. 5 is a diagram showing a process of loading a cell suspension obtained by cutting and filtering tonsil tissue on top of a lymphocyte separation medium (LSM) according to one embodiment of the present invention.

[0035] FIG. 6 is a diagram showing a process of separating and recovering mononuclear cells by subjecting a cell suspension to density gradient centrifugation using LSM according to one embodiment of the present invention.

[0036] FIG. 7 is a diagram showing a photograph of cells (P0) observed under an optical microscope on the second day of primary culture after primary culture of mononuclear cells according to one embodiment of the present invention.

[0037] FIG. 8 is a diagram showing a photograph of cells (P0) observed under an optical microscope on the fifth day of primary culture after culturing mononuclear cells according to one embodiment of the present invention.

[0038] FIG. 9 is a diagram showing a photograph of cells (P0) observed under an optical microscope on the 7th day of primary culture after culturing mononuclear cells according to one embodiment of the present invention.

[0039] FIG. 10 is a diagram showing a photograph of cells (P0) observed under an optical microscope on the 9th day of primary culture after primary culture of mononuclear cells according to one embodiment of the present invention.

[0040] FIG. 11 is a diagram showing a photograph of cells (P0) observed under an optical microscope on the 12th day of primary culture after culturing mononuclear cells according to one embodiment of the present invention.

[0041] FIG. 12 is a diagram showing a photograph of cells (P0) observed under an optical microscope on the 14th day of primary culture after culturing mononuclear cells according to one embodiment of the present invention.

[0042] FIG. 13 is a diagram showing a photograph of cells (P0) observed under an optical microscope on the 16th day of primary culture after culturing mononuclear cells according to one embodiment of the present invention.

[0043] Figure 14 is a diagram showing a photograph of cells (P1) observed under an optical microscope on the fifth day of the first subculture, after subculturing one cell according to one embodiment of the present invention.

[0044] Figure 15 is a diagram showing a photograph of cells (P2) observed with an optical microscope on the fifth day of the second subculture, after subculturing for two generations according to one embodiment of the present invention.

[0045] Figures 16 and 17 are diagrams showing the results of flow cytometry analysis of tonsil-derived mesenchymal stem cells (T-MSCs) manufactured according to one embodiment of the present invention.

[0046] Figure 18 is a diagram confirming that T-MSCs manufactured according to one embodiment of the present invention are differentiated into adipocytes.

[0047] Figure 19 is a diagram confirming that T-MSCs cultured according to one embodiment of the present invention differentiate into bone cells.

[0048] Figure 20 is a diagram confirming that T-MSCs cultured according to one embodiment of the present invention differentiate into chondrocytes.

[0049] Unless otherwise defined, all terms (including technical and scientific terms) used herein may be used in their common sense to those of ordinary skill in the art to which the present invention pertains. Furthermore, terms defined in commonly used dictionaries are not to be interpreted ideally or excessively unless explicitly and specifically defined otherwise.

[0050] As used herein, the singular form may include the plural form unless the context clearly indicates otherwise.

[0051] When a part in this specification is said to "include" a component, this does not mean that it excludes other components, but rather that it may include other components, unless otherwise specifically stated.

[0052] Additionally, all numbers and expressions indicating the amounts of components, reaction conditions, etc. described in this specification should be understood to be modified by the term “about” in all cases unless otherwise specified.

[0053] Additionally, the experimental procedures specified in this specification are identical to those commonly performed in the art unless specifically described otherwise.

[0054] Hereinafter, the present invention will be described in detail.

[0055] The present invention

[0056] 1) a step of obtaining mononuclear cells from tonsil tissue isolated from a donor; and

[0057] 2) A step of obtaining tonsil-derived mesenchymal stem cells by first culturing the obtained mononuclear cells and then subculturing them up to the second passage.

[0058] A method for producing tonsil-derived mesenchymal stem cells, including:

[0059] In addition, the present invention provides tonsil-derived mesenchymal stem cells produced by the method according to the present invention.

[0060] In the present invention, the term "tonsil tissue" refers to a tissue located in the back of the throat and nose, which primarily defends the body from external invading substances such as bacteria, and at the same time functions as a lymphoepithelial immune tissue. The tonsil tissue includes the pharyngeal tonsil, the auris-pharyngeal tonsil, the palatine tonsil, the lingual tonsil, etc. For the purposes of the present invention, the tonsil tissue is used as a source tissue for providing tonsil-derived mesenchymal stem cells, but is not particularly limited thereto.

[0061] In the present invention, the term "mesenchymal stem cells (MSCs)" refers to undifferentiated stem cells that can differentiate into bone, cartilage, fat, bone marrow stroma, muscle, nerve, etc., and in adults, they generally reside in bone marrow, but also exist in umbilical cord blood, peripheral blood, and other tissues.

[0062] In the method of the present invention, mononuclear cells can be obtained from tonsil tissue in step 1) through the following steps:

[0063] i) a step of crushing and filtering the tonsil tissue to extract cells; and

[0064] ii) A step of obtaining mononuclear cells by subjecting the extracted cells to density gradient centrifugation.

[0065] A balanced salt solution may be added to the tonsil tissue separated in the above step i) and pulverized. In one specific example of the present invention, the balanced salt solution is prepared by mixing Hank's balanced salt solution (HBSS), hydroxyethylpiperazineethanesulfonic acid (HEPES), and sterile water.

[0066] Additionally, the above tonsil tissue can be pulverized to less than 5 mm.

[0067] In addition, the above-mentioned tonsil tissue can be crushed, collagenase, DNA decomposition enzyme and calcium chloride can be added, and the reaction can be carried out for 30 to 120 minutes, specifically 30 to 110 minutes, 30 to 100 minutes, 30 to 90 minutes, 30 to 80 minutes, 30 to 70 minutes, 40 to 70 minutes, 50 to 70 minutes or 55 to 65 minutes, and then filtered.

[0068] The cells extracted in the above step ii) can be subjected to density gradient centrifugation using ficoll-hypaque, histopaque, or lymphocyte separation medium (LSM), and specifically, density gradient centrifugation can be performed using lymphocyte separation medium. In addition, the intermediate cell layer generated by density gradient centrifugation can be recovered to obtain mononuclear cells.

[0069] The above lymphocyte separation medium (LSM) is a sterile iso-osmotic polysucrose and diatrizoate solution having a density of 1.077-1.080 g / mL at 20°C.

[0070] In the method of the present invention, in step 2), mononuclear cells can be inoculated into a medium and cultured for 14 to 18 days, and specifically, cultured for 15 to 17 days.

[0071] Additionally, the mononuclear cells can be inoculated into a medium and the medium can be replaced at intervals of 1 to 3 days, and specifically, the medium can be replaced at intervals of 2 to 3 days.

[0072] More specifically, the mononuclear cells can be inoculated into a medium and cultured for 14 to 18 days while replacing the medium every 1 to 3 days, and even more specifically, the primary culture can be cultured for 15 to 17 days while replacing the medium every 2 to 3 days. If the above range is exceeded, cell contamination may occur due to impurities in the medium, and cell growth may be difficult, making it difficult to obtain cells cultured for the desired number of cells.

[0073] In addition, the above mononuclear cells were 1×10 8 1×10 9 cells / cm 2 (cells / cm 2 ), specifically 1×10 6 1×10 9 cells / cm 2 (cells / cm 2 ), 1×10 7 1×10 9 cells / cm 2 (cells / cm 2 ), 1×10 8 1×10 9 cells / cm 2 (cells / cm 2 ) or 5×10 8 1×10 9 cells / cm 2 (cells / cm 2 ) can be inoculated into the badge.

[0074] In the method of the present invention, the subculture up to the second passage in step 2) can be performed through the following steps:

[0075] a) a step of inoculating the cultured cells into a medium and performing the first subculture for 4 to 7 days; and

[0076] b) A step of inoculating the subcultured cells into a medium, subculturing them for 4 to 7 days, and recovering the subcultured cells for the second time to obtain tonsil-derived mesenchymal stem cells.

[0077] 5×10 cells cultured in the above step a) 5 1×10 7 cells / cm 2 (cells / cm 2 ), specifically 7×10 5 1×10 7 cells / cm 2 (cells / cm 2 ) or 1×10 6 5×10 6 cells / cm 2 (cells / cm 2 ) can be inoculated into the medium. Alternatively, the cells cultured above can be inoculated at 1,000 to 5,000 cells / cm per flask area. 2 (cells / cm 2 ), specifically, 1,000 to 4,500 cells / cm2, 1,000 to 4,000 cells / cm2, 1,000 to 3,500 cells / cm2, 1,500 to 3,500 cells / cm2, 2,000 to 3,500 cells / cm2, 2,500 to 3,500 cells / cm2 or 2900 to 3100 cells / cm2 can be inoculated into the medium.

[0078] Additionally, the cells cultured above can be inoculated into a medium and subcultured once without changing the medium for 4 to 7 days, specifically 4 to 6 days, and more specifically 5 days.

[0079] 1×10 cells cultured once in step b) above 61×10 8 cells / cm 2 (cells / cm 2 ), specifically 5×10 6 5×10 7 cells / cm 2 (cells / cm 2 ) or 1×10 7 5×10 7 cells / cm 2 (cells / cm 2 ) can be inoculated into the medium. Alternatively, the cells cultured for the first passage can be inoculated at 1,000 to 5,000 cells / cm per flask area. 2 (cells / cm 2 ), specifically, 1,000 to 4,500 cells / cm2, 1,000 to 4,000 cells / cm2, 1,000 to 3,500 cells / cm2, 1,500 to 3,500 cells / cm2, 2,000 to 3,500 cells / cm2, 2,500 to 3,500 cells / cm2 or 2900 to 3100 cells / cm2 can be inoculated into the medium.

[0080] In addition, the cells cultured in the first subculture can be inoculated into a medium and cultured in the second subculture without changing the medium for 4 to 7 days, specifically 4 to 6 days, more specifically 5 days.

[0081] In the method of the present invention, the tonsil-derived mesenchymal stem cells have an immunophenotype characteristic that is negative for CD11b, CD19, CD34, CD45 and HLA-DR, and an immunophenotype characteristic that is positive for CD73, CD90 and CD105.

[0082] Additionally, the above tonsil-derived mesenchymal stem cells have a phenotypic characteristic that is positive for SUSD2.

[0083] In addition, the tonsil-derived mesenchymal stem cells can be differentiated into mesodermal tissue cells, such as adipocytes, osteocytes, or chondrocytes, into endodermal tissue cells, such as parathyroid hormone-secreting cells, hepatocytes, or insulin-secreting cells, or into ectodermal tissue cells, such as motor neurons or Schwann cells, but are not limited thereto.

[0084] The term "differentiation" of the present invention generally refers to a phenomenon in which a relatively simple system is separated into two or more qualitatively different subsystems. Specifically, it refers to a phenomenon in which cells become specialized in structure or function while they divide and proliferate and grow, that is, a phenomenon in which cells, tissues, etc. of an organism change in form or function to perform their respective assigned tasks. For example, during ontogeny, qualitative differences arise between parts of an egg that were initially homogeneous, such as the head or torso, or differences arise among cells, such as muscle cells or nerve cells, or a phenomenon in which qualitative differences arise between parts of a certain organism that were initially almost homogeneous, or a phenomenon in which they are divided into qualitatively distinguishable subregions or subsystems as a result. Relatively speaking, "undifferentiated" refers to a state in which the aforementioned differentiation has not occurred and still contains the characteristics of stem cells.

[0085] In addition, the present invention provides a composition comprising tonsil-derived mesenchymal stem cells or cells differentiated therefrom, manufactured by a manufacturing method according to the present invention.

[0086] In addition, the present invention provides a pharmaceutical composition comprising tonsil-derived mesenchymal stem cells or cells differentiated therefrom, manufactured by a manufacturing method according to the present invention.

[0087] The pharmaceutical composition of the present invention may additionally contain pharmaceutical adjuvants such as preservatives, stabilizers, wetting agents or emulsifying agents, salts for osmotic pressure control, and / or buffers, and other therapeutically useful substances, and may be formulated into various oral or parenteral dosage forms according to conventional methods.

[0088] The oral dosage forms include, for example, tablets, pills, hard and soft capsules, liquids, suspensions, emulsions, syrups, powders, granules, granules, pellets, etc., and these dosage forms may contain, in addition to the active ingredient, a surfactant, a diluent (e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and glycine), and a lubricant (e.g., silica, talc, stearic acid and its magnesium or calcium salts, and polyethylene glycol). The tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, and polyvinylpyrrolidine, and optionally pharmaceutical additives such as disintegrating agents such as starch, agar, alginic acid or its sodium salt, absorbents, coloring agents, flavoring agents, and sweetening agents. The tablets may be manufactured by conventional mixing, granulating, or coating methods.

[0089] In addition, the above-mentioned non-oral administration form may be a transdermal administration formulation, and examples thereof include, but are not limited to, injections, drops, ointments, lotions, gels, creams, sprays, suspensions, emulsions, suppositories, patches, etc.

[0090] The pharmaceutical composition of the present invention can be manufactured in the form of a unit dose or can be manufactured by placing it in a multi-dose container by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains, and the like. 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 dispersing agent or stabilizer.

[0091] Pharmaceutically acceptable carriers that may be included in the pharmaceutical composition of the present invention are those commonly used in formulations, and include, but are 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 pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.

[0092] The pharmaceutical composition of the present invention can be administered orally and parenterally, and can be administered by, for example, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, topical administration, intranasal administration, intrapulmonary administration, rectal administration, intrathecal administration, ocular administration, skin administration, and transdermal administration.

[0093] The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity, and a generally skilled physician can easily determine and prescribe a dosage effective for the desired treatment or prevention.

[0094] The determination of the dosage of the above-mentioned effective ingredient is within the level of a person skilled in the art, and the daily administration dose of the drug varies depending on various factors such as the degree of progression, onset time, age, health condition, and complications of the subject to be administered, but for adults, in one aspect, 1 ㎍ / kg to 200 mg / kg of the composition, and in another aspect, 50 ㎍ / kg to 50 mg / kg may be administered once to three times a day in divided doses, and the above dosage does not limit the scope of the present invention in any way.

[0095] In addition, the present invention provides a cell therapy product comprising, as an active ingredient, tonsil-derived mesenchymal stem cells or cells differentiated therefrom, manufactured by a manufacturing method according to the present invention.

[0096] The differentiated cells are not particularly limited thereto, but may be any cells that can be used for cell therapy, more specifically, may be mesodermal cells or endoderm cells, and even more specifically, may be adipocytes, chondrocytes, osteocytes, etc.

[0097] The term "cellular therapeutic agent" of the present invention refers to a medicine (US FDA regulation) used for the purposes of treatment, diagnosis, and prevention by separating, culturing, and manufacturing cells and tissues from an individual through special manipulation, and means a medicine used for the purposes of treatment, diagnosis, and prevention through a series of actions such as proliferating and selecting living autologous, allogeneic, or xenogeneic cells in vitro or changing the biological characteristics of cells by other methods to restore the function of cells or tissues.

[0098] In addition, the present invention provides a cosmetic composition comprising, as an active ingredient, tonsil-derived mesenchymal stem cells or cells differentiated therefrom, manufactured by a manufacturing method according to the present invention.

[0099] The cosmetic composition of the present invention may be formulated as a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, leave-on type, mist, spray, etc., but is not limited thereto. More specifically, it may be formulated as a cleansing agent such as shampoo, rinse, body cleanser, etc., a hair styling agent such as hair tonic, gel, or mousse, a hair nourishing toner, hair essence, hair serum scalp treatment, hair treatment, hair conditioner, hair shampoo, hair lotion, hair tonic, or hair dye, etc., and as a base cosmetic such as an oil-in-water (O / W) type, a water-in-oil (O / W) type, etc.

[0100] In addition, the composition, in addition to the essential ingredients mentioned above, can be appropriately selected and mixed by those skilled in the art without difficulty depending on the type of external preparation or intended use, etc., for each formulation. For example, the composition may further include a sunscreen, a hair conditioner, a fragrance, etc.

[0101] The cosmetic composition may contain a cosmetically acceptable medium or base. This may be provided in any formulation suitable for topical application, for example, in the form of a solution, gel, solid or paste anhydrous product, an emulsion obtained by dispersing an oil phase in an aqueous phase, a suspension, a microemulsion, a microcapsule, a microgranule, or an ionic (liposome) and / or non-ionic vesicular dispersion, or in the form of a cream, skin lotion, lotion, powder, ointment, spray, or concealer stick. These compositions may be prepared according to conventional methods in the art.

[0102] When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

[0103] When the formulation of the present invention is a suspension, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth may be used as a carrier component.

[0104] When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component.

[0105] When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and particularly in the case of a spray, a propellant such as chlorofluorohydrocarbon, propane / butane or dimethyl ether may be additionally included.

[0106] In one embodiment of the present invention, the cosmetic composition may contain various appropriate bases and additives as needed, the types and amounts of which can be readily selected by the inventor. The composition may contain acceptable additives as needed, and for example, may additionally include preservatives, colorants, additives, and other ingredients commonly used in the art.

[0107] The above preservative may be specifically phenoxyethanol or 1,2-hexanediol, and the fragrance may be an artificial fragrance.

[0108] And, in one embodiment of the present invention, the cosmetic composition may include a composition selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, high molecular peptides, high molecular polysaccharides, sphingolipids, and seaweed extracts. In addition, examples of compounding ingredients that may be added include fat components, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, bactericides, antioxidants, plant extracts, pH adjusters, alcohols, pigments, fragrances, blood circulation promoters, cooling agents, antiperspirants, purified water, and the like.

[0109] In addition, the compounding ingredients that may be added are not limited to these, and any of the above ingredients may be compounded within a range that does not impair the purpose and effect of the present invention.

[0110] Hereinafter, the present invention will be described in detail by examples.

[0111] However, the following examples are only illustrative of the present invention, and the content of the present invention is not limited to the following examples.

[0112] <Example 1> Sample preparation

[0113] The following samples were prepared and manufactured for the production of tonsil-derived stem cells.

[0114] A total of 500 ml of cell culture medium was prepared by mixing 10% FBS (hyclone) and 50 μg / ml of gentamicin (Shin Poong Pharmaceutical) in MEM-α medium (gibco).

[0115] A total of 50 ml of HBSS solution was prepared by mixing 1× HBSS (Hank's Balanced Salt Solution, gibco) and 1 mM HEPES (gibco) in sterile distilled water.

[0116] 10 mg of collagenase NB6 (Nordmark) was added to 10 ml of the above HBSS solution, mixed well, and dispensed in 1 ml (0.16 PZ U / ml) portions. The dispensed collagenase was frozen at -20°C and then taken out and thawed on ice when used.

[0117] DNase I (NEB) was used as a DNA degrading enzyme mixed with reaction buffer (Cat No. B0303, NEB) and nuclease-free water (Cat No. R0581, Thermo).

[0118] In addition, lymphocyte separation medium (LSM: Lymphocyte Separation Medium, Cat No. 25-072-CI, Corning), calcium chloride solution (Sigma Aldrich), 1X CTS TM DPBS (gibco) was prepared.

[0119] <Example 2> Production of tonsil-derived mesenchymal stem cells

[0120] <2-1> Tonsil tissue removal and dissection

[0121] A 14-year-old male patient underwent tonsillectomy, and tonsil tissue was donated (Fig. 1). The obtained tonsil tissue was placed in a 50-mL tube containing 70% ethanol (25 mL) for 1 minute, then transferred to a 50-mL tube containing HBSS solution (25 mL) and placed for 1 minute to remove any residual ethanol.

[0122] As shown in Fig. 2, the tonsil tissue was transferred to a cell culture dish with a diameter of 100 mm, 5 ml of HBSS solution was poured, and the tissue was torn into large pieces using tweezers. The solution containing the cells that initially flowed out during this process was removed by suction.

[0123] Next, as shown in Fig. 3, the previously torn tonsil tissue was transferred to a new 100 mm diameter cell culture dish, 10 ml of HBSS solution was added, and the tissue was minced into pieces of less than 5 mm using surgical scissors and tweezers. The minced tissue was recovered, along with the HBSS, and transferred to a new 50 ml conical tube, and 15 ml of HBSS was added to completely recover any remaining tissue remaining in the dish.

[0124] As shown in Fig. 4, the finely chopped tissue including the HBSS solution was transferred to a 50 ml conical tube, and then the HBSS solution was adjusted to a total volume of 19.73 ml, and 31.25 μl of the collagenase prepared in <Example 1>, 200 μl of the DNA degrading enzyme, and 40 μl of 1 M calcium chloride were added. Afterwards, the tube was cultured for 1 hour in a 37°C CO2 incubator. During the culture, the tube was shaken periodically every 15 minutes.

[0125] <2-2> Separation of mononuclear cells using density-gradient centrifugation

[0126] In the above Example <2-1>, the cell tissue fluid that had been cultured for 1 hour was slowly poured into a new 50 ml conical tube equipped with a cell strainer, and the tissue was filtered to obtain a cell suspension. If it was clogged and not filtered, carefully scrape it with a tip or use a new cell strainer to filter. In addition, HBSS solution (20 ml) was additionally added to the tube containing the cell tissue fluid and then poured into the cell strainer where the tissue remained to obtain an additional cell suspension.

[0127] The cell suspension obtained above was centrifuged at 300 ×g for 5 minutes, and the supernatant was removed to obtain a pellet. The obtained pellet was suspended with 20 ml of HBSS solution using a pipette, centrifuged at 300 ×g for 5 minutes, the supernatant was removed again, and 10 ml of cell culture medium was added and suspended with a pipette to obtain a cell suspension.

[0128] Next, LSM (15 ml) was added to a new 50 ml tube as shown in Fig. 5, and the cell suspension suspended in the cell culture medium was carefully loaded so that layers were formed, and centrifuged at 900 ×g for 20 minutes.

[0129] As shown in Fig. 6, the intermediate cell layer generated by centrifugation was recovered, HBSS solution (30 ml) was added, and centrifuged at 300 ×g for 5 minutes. After centrifugation, the supernatant was removed and suspended in 30 ml of cell culture medium using a pipette to obtain a cell suspension containing mononuclear cells.

[0130] <2-3> Production of tonsil-derived mesenchymal stem cells through cell subculture of mononuclear cells

[0131] The mononuclear cells obtained in the above Example <2-3> were cultured for primary (P0), 1 passage (P1), and 2 passages (P2) under the conditions of [Table 1] below to obtain tonsil-derived mesenchymal stem cells (T-MSC).

[0132] Major categorySubcategory unitP0P1P2Culture conditionCultureDaily1655Medium change cycleDay2--Culture vesselCulture vesselN / AT-75 flask(T-75 flask, SPL)T-175 flask(T-175 flask, SPL)Hyper flask(Hyper flask, Corning)Culture vessel area㎠751751750Venture quantityea432Total area of ​​culture vesselcells2255253500Number of cells inoculated per areacells / ㎠N / A30003000Total number of cells inoculatedcells / ㎠8.2.E+081.5.E+061.0.E+07

[0133] Primary culture (P0) Count the number of monocytes in the cell suspension containing the monocytes obtained in the above Example <2-3> and inoculate 5x10 into 20 ml of medium per T-75 flask. 8 Each cell was inoculated into four T-75 flasks. Two days after inoculation, the cells were observed under an optical microscope.

[0134] As a result, as shown in Fig. 7, it was confirmed that the culture medium contained a large number of impurities on the second day of initial culture, indicating that the cell culture medium needs to be replaced at two-day intervals.

[0135] Afterwards, the primary culture was performed by replacing the entire medium every two days until the 16th day. In addition, the cells were observed under an optical microscope on the 5th day of culture (Fig. 8), the 7th day of culture (Fig. 9), the 9th day of culture (Fig. 10), the 12th day of culture (Fig. 11), the 14th day of culture (Fig. 12), and the 16th day of culture (Fig. 13).

[0136] As a result, as shown in Figures 8 to 13, it was confirmed that the cells reached a cell density (confluency) of 80% by culturing them for up to 16 days while replacing the medium every two days.

[0137] To recover cells on the 16th day of primary culture, 5 ml of Tryple select CTS was added to each flask and incubated for 5 minutes. The flasks were gently tapped to detach the cells, 8 ml of medium was added to neutralize, and the entire cell was collected using a pipette in a 50 ml conical tube, which was then washed twice with PBS.

[0138] 1 subculture (P1)

[0139] On the 16th day of the initial culture, the cells were collected, washed, counted, and inoculated into three T-75 flasks at a density of 3,000 cells per area in 50 ml of medium per T-175 flask. In addition, the cells were observed under an optical microscope during the culture process.

[0140] As a result, as shown in Fig. 14, it was confirmed that cell culture was possible for up to 5 days without replacing the medium and that the cell density reached 80%.

[0141] Accordingly, on the 5th day of the first subculture, cells were harvested and washed in the same manner as above.

[0142] 2nd subculture (P2)

[0143] On the fifth day of the first subculture, the cells were collected and washed, counted, and inoculated into two hyperflasks at a density of 3,000 cells per area in 500 ml of medium per hyperflask. In addition, the cells were observed under an optical microscope during the culture process.

[0144] As a result, as shown in Fig. 15, it was confirmed that cell culture was possible for up to 5 days without replacing the medium and that a cell density of 80% or more was reached.

[0145] Here, on the 5th day of the second subculture, cells were harvested and washed in the same manner as above to obtain T-MSCs. The T-MSCs were suspended in a cryopreservation solution (cryomedium, 50% FBS, 40% MEM-α, and 10% DMSO), and the suspension was dispensed so that each container contained 2 × 10 cells. The containers into which the suspension was dispensed were frozen at -70°C for 24 hours, stored in liquid nitrogen at -200°C, and thawed and used for experiments using a known method.

[0146] <Example 3> Cell phenotype analysis of tonsil-derived mesenchymal stem cells

[0147] The cell phenotype of T-MSCs obtained in the above example <2-3> was confirmed by flow cytometry.

[0148] Specifically, T-MSCs obtained in the above Example <2-3> and each antibody (2 μg / ml) capable of reacting with the surface antigen were stained at 4°C for 30 minutes and analyzed using a FACSCaliur system.

[0149] As a result, as shown in Fig. 16, it was confirmed that the T-MSCs obtained in the above Example <2-3> exhibited an immunophenotype that was negative for CD11b, CD19, CD34, CD45, and HLA-DR, and a immunophenotype that was positive for CD73, CD90, and CD105.

[0150] In addition, SUSD2 (sushi containing domain 2) is well known as a cell surface marker indicating pluripotency in human stem cells and has been reported as a marker of tonsil-derived mesenchymal stem cells (Lee et al., Isolation and Localization of T-MSCs by W5C5, Cell Physiol Biochem 2016;38:83-93). Therefore, SUSD2 expression was analyzed in the T-MSCs obtained in Example <2-3>.

[0151] Specifically, flow cytometry analysis was performed using the T-MSCs obtained in the above Example <2-3> using the same method described in Lee et al. In addition, as a control group, T-MSCs were isolated and cultured using the same method described in Lee et al., and flow cytometry analysis was performed.

[0152] As a result, as shown in Fig. 17, it was confirmed that the expression of SUSD2 in the T-MSC of the comparison group was 1.5%, while the expression of SUSD2 in the T-MSC obtained in Example <2-3> was high, at 8 to 16%.

[0153] The above results confirm that the cells obtained through <Example 1> and <Example 2> of the present invention are T-MSCs. Furthermore, it can be seen that a large quantity of high-purity T-MSCs can be produced through <Example 1> and <Example 2> of the present invention.

[0154] <Example 4> Confirmation of cell differentiation potential of tonsil-derived mesenchymal stem cells

[0155] In order to determine whether T-MSCs obtained through <Example 1> and <Example 2> of the present invention can differentiate into mesodermal cells, differentiation into mesodermal cells such as adipocytes, osteocytes, and chondrocytes was induced using T-MSCs obtained through <Example 1> and <Example 2> of the present invention.

[0156] <4-1> Confirmation of differentiation ability into adipocytes

[0157] T-MSCs obtained in Example <2-3> were seeded in a 12-well plate containing adipocyte differentiation medium (Invtorgen) containing 3-isobutyl-1-methyl-gentin, dexamethasone, indomethacin, and insulin, at a density of 10 per well. 4 The cells were seeded in 10 μg ml medium and cultured for 3 weeks, replacing the medium every 3-4 days. After the culture was completed, the medium was removed, the cells attached to the bottom of each well were washed with PBS, fixed with 4% paraformaldehyde for 5 minutes, dried, and stained with Oil red O (Sigma-Aldrich) for 10 minutes at room temperature. After the staining was completed, Oil red O was removed, and the cells were immediately washed 4 times with distilled water, and then observed under a phase contrast microscope.

[0158] As a result, as shown in Figure 18, it was confirmed that T-MSCs differentiated into adipocytes.

[0159] <4-2> Confirmation of differentiation ability into bone cells

[0160] T-MSCs obtained in Example <2-3> were seeded at 10 per well in a 12-well plate containing osteocyte differentiation medium (Invtorgen) containing ascorbic acid 2-phosphate, dexamethasone, and β-glycerophosphate. 4 The cells were seeded in the same number as the number of dogs and cultured for 3 weeks, replacing the medium every 3-4 days. After the culture was completed, the medium was removed, the cells attached to the bottom of each well were washed with PBS, fixed for 5 minutes using 60% isopropyl alcohol, washed twice with distilled water, and stained with 2% Alizarin red O solution (pH 4.2) for 3 minutes. After the staining was completed, the Alizarin red O solution was removed, immediately washed three times with distilled water, and the cells were observed under a phase contrast microscope.

[0161] As a result, as shown in Figure 19, it was confirmed that T-MSCs differentiated into bone cells.

[0162] <4-3> Confirmation of differentiation ability into chondrocytes

[0163] 2 × 10 cells were placed in a 14 ml conical tube containing chondrocyte culture medium (Invtorgen) containing dexamethasone, ascorbic acid, L-proline, TGF-3, BMP-6, and ITS. 6 T-MSCs from dogs were seeded and cultured for 3 weeks, changing the culture medium every 3-4 days. After the culture was completed, the cultured cells were harvested, fixed with 4% paraformaldehyde, and embedded in paraffin to obtain paraffin blocks. The obtained paraffin blocks were cut into 5 μm thick sections, placed on slide glasses, and the paraffin was removed to fix the cell sections on the slide glasses. The sulfated preteoglycan contained in the cell sections fixed on the slide glasses was stained with Alcian blue (pH 2.3), and the cells were observed under a phase contrast microscope.

[0164] As a result, as shown in Figure 20, it was confirmed that T-MSCs differentiated into chondrocytes.

[0165] According to the manufacturing method of the present invention, a large quantity of tonsil-derived mesenchymal stem cells can be manufactured by isolating and subculturing mononuclear cells from tonsil tissue using an optimized standardized method, and this has the effect of being useful for developing stem cell therapeutic agents.

Claims

1. 1) A step of obtaining mononuclear cells from tonsil tissue isolated from a donor; and 2) A step of obtaining tonsil-derived mesenchymal stem cells by first culturing the obtained mononuclear cells and then subculturing them up to the second passage. A method for producing tonsil-derived mesenchymal stem cells, comprising:

2. A manufacturing method in the first paragraph, wherein in step 1), mononuclear cells are obtained from tonsil tissue through the following steps. i) a step of crushing and filtering the tonsil tissue to extract cells; and ii) A step of obtaining mononuclear cells by subjecting the extracted cells to density gradient centrifugation.

3. A manufacturing method in the second paragraph, wherein in step ii), density gradient centrifugation is performed using ficoll-hypaque, histopaque or lymphocyte separation medium (LSM).

4. A manufacturing method in the first paragraph, wherein in step 2), mononuclear cells are inoculated into a medium and cultured for 14 to 18 days.

5. A manufacturing method in claim 4, wherein the mononuclear cells are inoculated into a medium and the medium is replaced at intervals of 1 to 3 days.

6. In paragraph 4, the mononuclear cells are 1×10 8 Inside 1×10 9 cells / cm 2 (cells / cm 2 ) is inoculated into a medium.

7. A manufacturing method in which, in the first paragraph, the subculture up to the second generation in step 2) is performed in the following steps. a) a step of inoculating the cultured cells into a medium and performing the first subculture for 4 to 7 days; and b) A step of inoculating the subcultured cells into a medium, subculturing them for 4 to 7 days, and recovering the subcultured cells for the second passage to obtain tonsil-derived mesenchymal stem cells.

8. A manufacturing method according to claim 7, wherein the first subculture and the second subculture are performed without changing the medium.

9. In paragraph 7, the cells cultured above are 5×10 5 Inside 1×10 7 cells / cm 2 (cells / cm 2 ) is inoculated into a medium.

10. In the 9th paragraph, the cells cultured above are 1,000 to 5,000 cells / cm per flask area. 2 (cells / cm 2 ) is inoculated with a manufacturing method.

11. In the 7th paragraph, the cells cultured for the first passage are 1×10 6 Inside 1×10 8 cells / cm 2 (cells / cm 2 ) is inoculated into a medium.

12. In the 11th paragraph, the cells cultured for the first passage are 1,000 to 5,000 cells / cm per flask area. 2 (cells / cm 2 ) is inoculated, a manufacturing method.

13. A method for producing a method according to claim 1, wherein the tonsil-derived mesenchymal stem cells have an immunophenotypic characteristic that is negative for CD11b, CD19, CD34, CD45 and HLA-DR, and a immunophenotypic characteristic that is positive for CD73, CD90 and CD105.

14. A manufacturing method according to claim 1, wherein the tonsil-derived mesenchymal stem cells have a phenotypic characteristic that is positive for SUSD2.

15. A manufacturing method in claim 1, wherein the tonsil-derived mesenchymal stem cells are differentiated into at least one selected from the group consisting of mesodermal tissue cells, endoderm tissue cells, and ectoderm tissue cells.

16. A manufacturing method in claim 15, wherein the mesodermal tissue cells are at least one selected from the group consisting of adipocytes, bone cells, and cartilage cells.

17. A tonsil-derived mesenchymal stem cell produced by any one of the production methods of claims 1 to 16.

18. A cell therapy product containing the tonsil-derived mesenchymal stem cells of Article 17 or cells differentiated therefrom as an active ingredient.

19. A cosmetic composition comprising the tonsil-derived mesenchymal stem cells of Article 17 or cells differentiated therefrom as an effective ingredient.

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