Method for producing a culture medium containing high-efficacy exosomes secreted from umbilical cord blood stem cells, and its applications.

Abstract

This invention provides a method for producing a culture medium in which highly effective exosomes are secreted in high concentrations from umbilical cord blood stem cells. [Solution] The manufacturing method is based on culturing umbilical cord blood stem cells in a serum-free batch to which GDF-11, EGF, FGF2, TFG-β1, and / or VEGF are added. When comparing a culture medium produced by treating umbilical cord blood stem cells with a serum-free batch to which five types of growth factors are added, with a culture medium produced when exosomes isolated from the culture medium are treated only with a serum-free batch without growth factors, the culture medium containing growth factors has a higher total protein content and extracellular matrix protein content. Furthermore, exosomes secreted by umbilical cord blood stem cells cultured in a serum-free batch containing five types of growth factors are numerous, highly concentrated, small in size, and show a uniform distribution. The umbilical cord blood stem cell culture medium produced contains a high content of highly effective exosomes and has high efficacy in skin regeneration.

Description

[Technical Field]

[0001] This invention relates to the secretion of highly effective exosomes in high content by umbilical cord blood stem cells. This relates to a method for producing culture medium and its applications.

[0002] The umbilical cord blood stem cell culture medium produced by the present invention contains a high content of highly effective exosomes, can be applied to and / or permeated into biological membranes such as skin, and can be used in cosmetic formulations. It can be usefully used as a pharmaceutical composition, such as a product or a wound treatment. [Background technology]

[0003] Mesenchymal stem cells contain epithelial growth factor. Stem cells are known to secrete a variety of growth factors and cytokines, such as fibroblast growth factor, which promote collagen production from fibroblasts and play an important role in skin regeneration (Figure 7). There is growing interest in developing cosmetics using stem cells with these characteristics, and in particular, research is being conducted on developing technologies to enhance the penetration of stem cell active ingredients into the skin, one of which is the use of exosomes.

[0004] Exosomes are small, membrane-bound vesicles secreted by various types of cells. The diameter of exosomes is reported to be approximately 30–150 nm. Electron microscopy studies suggest that exosomes do not originate directly from the plasma membrane, but rather from specific intracellular compartments called multivesicular bodies (MVBs), which are then released and secreted outside the cell. In other words, when the polyspleen and plasma membrane fuse, the vesicles are released into the extracellular environment, and these are called exosomes. The molecular mechanisms by which such exosomes are produced are not yet clear, but exosomes secreted from almost all living cells contain various components such as receptors, proteins, and miRNAs. Exosomes are known to play a crucial role in intercellular signal transduction. Furthermore, exosomes contain relatively less animal serum compared to stem cells, thus eliminating the risk of zoonosis (symptoms caused by animal serum infection). Considering these characteristics of exosomes, exosome-based therapies are expected to represent a novel mechanism that can overcome the limitations of existing stem cell therapies.

[0005] The skin is composed of the epidermis and the dermis.

[0006] The cells that make up the epidermis include keratinocytes, melanocytes, dendritic cells, and tactile cells. The cells are keratinocytes (Figure 8). The primary function of keratinocytes is to form a barrier against environmental damage caused by heat, ultraviolet radiation, water loss, pathogenic bacteria, fungi, parasites, and viruses. Numerous structural proteins (filaggrin, keratin), enzymes (proteases), lipids, and antimicrobial peptides (defensins) contribute to maintaining the skin's important barrier function. Keratin formation This is part of the physical barrier formation (keratinization) process, where keratinocytes produce even more keratin and undergo terminal differentiation. The fully keratinized keratinocytes that form the outermost layer are continuously removed and replaced with new cells.

[0007] The dermis is 15 to 40 times thicker than the epidermis (0.04 mm to 1.6 mm), makes up the majority of the skin, and is the layer that maintains skin elasticity. It acts as connective tissue between the epidermis and subcutaneous tissue. It contains skin appendages, blood vessels, lymphatic vessels, muscles, and nerves. The dermis can be broadly divided into the papillary dermis and the reticular dermis. .

[0008] The dermal papillary layer makes up a very small portion of the total thickness of the dermis and is composed of collagen, blood vessels, and fibroblasts. It supplies nutrients to the avascular basal layer of the epidermis. The dermal reticular layer makes up the majority of the dermis and consists of thick bundles of elastin and collagen fibers, giving the skin strength and flexibility, and is composed of elastic fibers and various matrix proteins. Collagen accounts for about 25% of the total connective tissue protein and plays an important role in the tensile strength of the dermal layer. On the other hand, matrix metalloprotease (MMP) is a zinc-dependent endopeptidase that hydrolyzes the extracellular matrix (ECM). MMP breaks down the ECM, increasing cell motility and circulating skin tissue, but it also promotes wrinkle formation and skin aging, promotes the metastasis of cancer cells, and its formation is promoted by ultraviolet light, stress, and antibiotics. Fibronectin is present on the cell surface, in connective tissue, and in the blood. It is a glycoprotein. Cells are not directly linked to collagen, but rather to it via fibronectin.

[0009] As shown in Figure 9, fibroblasts produce and secrete growth factors and cytokines required by keratinocytes. Keratinocytes are epidermal cells that are easily exposed to the environment. Fibroblasts can be activated through keratinocytes, and this activation reshapes the extracellular matrix, making the skin layer more uniform.

[0010] On the other hand, as a higher-level concept than skin, there is the biological membrane (covering and lining membranes). Yes, it exists. The biological membrane is the underlying layer of connective tissue. It is a continuous multicellular sheet composed of epithelial tissue (epithelium) bound to the membrane. The biological membrane is a cutaneous membrane. There are mucous membranes and serous membranes (Figure 6).

[0011] As the living standards of modern people improve and the elderly population increases, interest in and demand for functional cosmetics related to anti-aging, wrinkle reduction, skin whitening, and UV protection are on the rise. However, since basic cosmetics and functional cosmetics are mostly manufactured using chemical substances, safety concerns for the human body have been raised. As a result, interest in products containing natural and environmentally friendly ingredients is increasing, and the market size for related products is also growing.

[0012] Product development utilizing such natural raw materials is also actively taking place in the pharmaceutical field, forming the biopharmaceutical market. Biopharmaceutical raw materials are pharmaceuticals developed using cells, tissues, hormones, etc. derived from humans and other living organisms, representing a step forward from existing chemical-based pharmaceuticals. Existing chemical-based pharmaceuticals have the disadvantage of severe side effects and only showing temporary improvement, but biopharmaceuticals have the advantage of addressing the root cause of disease without side effects, and are currently being actively researched worldwide. This invention aims to utilize umbilical cord blood stem cell-derived exosomes as one of the main raw materials in the next-generation biopharmaceutical field. [Overview of the project] [Problems that the invention aims to solve]

[0013] This invention aims to provide a culture medium in which umbilical cord blood stem cells secrete a high concentration of highly effective exosomes, thereby enhancing the regenerative capacity of biological membranes such as skin.

[0014] In this invention, umbilical cord blood stem cells were grown in a serum-free batch to which a specific growth factor cocktail containing two or more growth factors was added. Analysis of the culture medium revealed that the content of extracellular matrix proteins was high. After confirming the high quantity, we isolated and analyzed exosomes from this culture medium and found that the concentration of active ingredients within the exosomes was high, resulting in improved efficacy. Furthermore, we confirmed that when human fibroblasts and keratinocytes were treated with the high-efficacy exosomes of the present invention, their proliferative and migratory abilities, as well as their wound healing abilities, were improved. The present invention is based on these findings. [Means for solving the problem]

[0015] A first aspect of the present invention involves adding one or more growth factors selected from the group consisting of GDF-11, EGF, FGF2, TGF-β1, and VEGF to umbilical cord blood stem cells at concentrations of 1 to 20 ng / ml each. culturing in the obtained serum-free medium, wherein the concentration of exosomes secreted during umbilical cord blood stem cell culture is 0.5 to 5×10 9 / ml, preferably 1 to 3×10 9 / ml, thereby producing a high-concentration culture solution There is provided a method for producing an exosome-containing culture solution secreted by umbilical cord blood stem cells, characterized by comprising the step.

[0016] For example, umbilical cord blood stem cells are cultured in a serum-free medium supplemented with GDF-11 and one or more growth factors selected from the group consisting of EGF, FGF2, TGF-β1 and VEGF each at a concentration of 1 to 20 ng / ml .

[0017] Preferably, umbilical cord blood stem cells are cultured in a serum-free medium supplemented with each of GDF-11, EGF, FGF2, TGF-β1 and VEGF at a concentration of 1 to 20 ng / ml.

[0018] A second aspect of the present invention provides a composition for application to skin or biological membranes, which contains, as an active ingredient, the umbilical cord blood stem cell exosome-containing culture solution obtained by the method of the first aspect, or high-potency exosomes isolated therefrom. The application composition includes a cosmetic composition, a quasi-drug composition, or a pharmaceutical composition. For example, the application composition is in a gelled dosage form mixed with a polymer, and undergoes solation at body temperature.

[0019] A third aspect of the present invention provides a pharmaceutical composition for treating scars or wounds, which contains, as an active ingredient, the exosome-containing culture solution of umbilical cord blood stem cells obtained by the method of the first aspect, or high-potency exosomes isolated therefrom.

[0020] The present invention is described below.

[0021] 1. Stem cell culture solution Adult stem cells are undifferentiated cells that can differentiate into cells of specific tissues as needed. Adult stem cells are mesenchymal stem cells, mesenchymal stromal cells, or pluripotent stem cells, but are not limited to these. Unlike germ stem cells extracted from human embryos, adult stem cells are extracted from already grown body tissues such as bone marrow or brain cells, which has the advantage of avoiding ethical controversies. In this invention, adult stem cells are derived from umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, or placenta, but are not limited to these.

[0022] Unlike adult stem cells derived from fat or bone marrow, umbilical cord blood-derived stem cells are derived from the donor's gestational age ( Since umbilical cord blood formed during 40 weeks is used, there is no difference in efficacy depending on the donor's condition. This has the advantage.

[0023] A badge refers to a composition containing essential components necessary for cell growth and proliferation in vitro, and includes, but is not limited to, all badges commonly used in the field for stem cell culture, such as DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, DMEM / F-10 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-10), DMEM / F-12 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12), α-MEM (α-Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium), IMDM (Isocove's Modified Dulbecco's Medium), and KnockOut DMEM, which are commercially manufactured or artificially synthesized badges. The badge of the present invention generally contains a carbon source, a nitrogen source, and trace element components, and may further contain amino acids, antibiotics, and various growth factors.

[0024] In the present invention, growth factors such as GDF-11, EGF, FGF2, TGF-β1, and VEGF can be added to a batch and / or produced by umbilical cord blood stem cells during umbilical cord blood stem cell culture.

[0025] Growth factors are protein-based physiologically active substances that promote cell division, growth, and differentiation. Examples include GDF-11 (Growth and Differentiation Factor 11), brain-derived neurotropic factor (BDNF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), nerve growth factor (NGF), and vascular endothelial growth factor (VEGF). , insulin-like growth factor (IGF), transforming growth factor (TGF), platelet-derived growth factor (PDGF), bone-derived growth factor (BDF), colony stimulation factor (CSF), epidermal growth factor (EGF), keratinocytes Examples include keratinocyte growth factor (KGF).

[0026] As confirmed in Experimental Examples 1 to 5, the present invention, when GDF-11, EGF, FGF2, TGF-β1, and VEGF are added to serum-free badges, stimulates the carrier signaling pathways of each growth factor in umbilical cord blood stem cells, promoting the SMAD signaling pathway and other pathways, thereby increasing the synthesis of collagen and fibronectin proteins, and increasing the content of growth factors, collagen, and fibronectin within exosomes. The increased collagen, fibronectin, and growth factor-containing exosomes are analyzed to have affected the proliferation and migration of human fibroblasts and keratinocytes. Furthermore, the autocrine effect and paracrine effect increase the re-secretion of EGF, FGF2, TGF-β1, VEGF, and GDF-11, promoting the re-secretion of other growth factors, and thus affecting the proliferation and migration of surrounding cells.

[0027] In this invention, the culture medium refers to the supernatant of cell culture obtained by culturing umbilical cord blood stem cells. The umbilical cord blood stem cell culture medium contains various physiologically active substances secreted from cells during the culture process of umbilical cord blood stem cells. These physiologically active substances include cytokines, cell growth factors, and immunomodulatory factors that affect the functions of cells and the body. Examples of physiologically active substances include VEGF (vascular endothelial growth factor), EGF (epidermal growth factor), HGF (hepatocyte growth factor), TGF-beta (tumor growth factor-beta), and IGF (insulin growth factor). This is one example, but it is not limited to this.

[0028] 2. Stem cell secreted exosomes Exosomes are capable of delivering substances across cell membranes and are valued as ideal vesicles for drug delivery. Such lipid bilayer vesicle systems can overcome the problems of skin penetration and are therefore considered one of the most effective strategies for delivering drugs to the dermis (Saahil Arora et al., Asian Journal of Pharmaceutics, 6, 4, 237-244, 2012).

[0029] Furthermore, exosomes contain RNA, proteins, lipids, and metabolites that reflect the cell type from which they originated. Exosomes contain various molecular components (e.g., proteins and RNA) of the cell from which they originated. The protein composition varies depending on the cell and tissue from which the exosome originated, but most exosomes contain a common set of evolutionarily conserved protein molecules.

[0030] Exosomes are obtained by separating them from the culture medium after culturing cells, and the size and content of exosomes are influenced by the molecular signals received by the cells that produce them.

[0031] This invention allows the culture medium itself to be used as a raw material without separating the exosomes, but it is also possible to separate and use the exosomes. Furthermore, the exosomes can be separated to confirm their size and physical properties in the culture medium for quality assurance purposes.

[0032] Methods such as centrifugation, immunoconjugation, and filtration are used to separate exosomes from the culture medium.

[0033] Among the methods for separating exosomes, ultracentrifugation, which is commonly used, cannot separate a large amount of exosomes at once, requires expensive equipment, takes a long time to separate them, and strong centrifugation can cause physical damage to the exosomes, particularly degrading the purity of the separated exosomes. To improve these drawbacks, there is the PS affinity method, which uses a substance that specifically binds to phosphatidylserine (PS), a protein present in the exosome membrane, thereby increasing the purity of the separated exosomes. Compared to ultracentrifugation, this method can separate exosomes of higher purity, but it has the drawback of a lower yield.

[0034] Exosomes in umbilical cord blood mesenchymal stem cell (UCB-MSC) culture medium are derived from adipose tissue-derived mesenchymal stem cells. Alternatively, it contains a variety of growth factors such as EGF, VEGF, TGF, HGF, FGF, IGF, and PDGF at higher levels than exosomes in the culture medium of bone marrow-derived mesenchymal stem cells. Growth factors such as EGF promote the proliferation of fibroblasts, which are skin constituent cells, and promote cell migration and collagen synthesis, so the UCB-MSC-derived exosomes contribute to skin regeneration, improvement of skin elasticity, and prevention of skin wrinkles. It can produce excellent skin condition improvement effects and wound healing effects, such as stopping or improving skin aging, preventing or improving skin aging, promoting hair growth, or restoring shrunken hair follicles.

[0035] The stem cell culture medium produced by this invention not only contains a large amount of exosomes, but also provides a large amount of nano-sized exosomes that penetrate to the dermis layer of the skin due to their unique lipid bilayer structure, thereby enhancing the regenerative effect. Since the exosomes contain a large amount of various growth factors, they can exhibit skin regeneration and anti-aging effects through proliferation and activation of fibroblasts, which are constituent cells of the skin, as well as increased collagen synthesis, hair growth, restoration of shrunken hair follicles, and wound healing effects.

[0036] The exosomes of the present invention can be produced or used in a culture medium containing them, or they can be produced or used in a state where cells have been removed from the culture medium. Since the exosome-containing culture medium from which cells have been removed is a cell-free preparation, it has a low risk of carcinogenicity and can be used to refuse transplantation. Not only are there no reaction issues, but there is no risk of causing microvessel occlusion when administered systemically, and it is a non-cellular isolated substance, making it suitable for drug development as a commercial off-the-shelf product. Because it is a function, manufacturing costs can be reduced.

[0037] This invention relates to the concentration of exosomes secreted during umbilical cord blood-derived stem cell culture, which is 0.5 to 5 × 1 0 9 / ml, preferably 1-3 × 10 9 Because it produces a high culture medium at / ml, derived from umbilical cord blood. The method is characterized by culturing stem cells in serum-free batches to which EGF, FGF2, TGF-β1, VEGF, and / or GDF-11 are each added at concentrations of 1 to 20 ng / ml.

[0038] The inventors cultured umbilical cord blood mesenchymal stem cells under specific conditions in which growth factors EGF, FGF2, TGF-β1, VEGF, and GDF-11 were added at concentrations of 1 to 20 ng / ml each. The culture results showed that the concentration of exosomes in the culture medium was higher (1-3 × 10⁻¹⁰) compared to when umbilical cord blood stem cells were cultured in a serum-free batch without the aforementioned growth factor. 9 We found that the exosomes ( / ml) improve the migratory and proliferative abilities of human fibroblasts and human keratinocytes. Furthermore, we found that umbilical cord blood stem cells were treated with 1-20 ng / ml of EGF, FGF2, TGF-β1, VEGF, and GDF-11. The culture medium produced by culturing with serum-free batch added at ml concentration contains a large amount of extracellular matrix, namely fibronectin protein and / or collagen protein. Furthermore, we discovered that it produces large quantities of exosomes with a small particle size of 100 nm ± 20 nm, which are easily absorbed through the skin. This invention is based on this discovery.

[0039] According to the present invention, the size of exosomes secreted during umbilical cord blood stem cell culture is made uniform, and a stem cell culture medium containing a large amount of high-concentration growth factor-containing exosomes, or exosomes separated therefrom, can be an effective component of a composition for application to a biological membrane such as skin.

[0040] Therefore, the present invention not only mass-produces approximately 100 nm sized exosomes that permeate the skin through cell culture of umbilical cord blood stem cells, but also has a high exosome content that improves the motility and proliferation of human fibroblasts, and the permeability of the culture medium through biological membranes such as skin (covering and lining membranes) is high. It is high in fibronectin protein and / or collagen protein. Therefore, dosage forms for application to biological membranes such as skin (e.g., pharmaceutical compositions, cosmetics, It is not only desirable for therapeutic agents such as quasi-drugs and / or wound treatment agents, but it can also exhibit excellent efficacy when used.

[0041] 3. Various dosage forms The present invention provides a culture medium containing exosomes secreted by umbilical cord blood-derived stem cells, and / or culture media. Exosomes separated from the liquid can be formulated into various dosage forms depending on their intended use.

[0042] For example, exosome-containing culture medium and / or exosomes separated from the culture medium It is either gelled, or frozen and / or dried to form a powder.

[0043] It can be used in skin or biological membrane application compositions that can be mixed with components such as hydrogel and gelatin to adjust the viscosity difference based on the ratio of component content, and when mixed with components such as poloxamer, it gels at 4°C, and at body temperature The dosage form can be designed to form a sol under certain conditions.

[0044] Cosmetic compositions can be manufactured in dosage forms selected from the group consisting of solutions, topical ointments, creams, foams, nourishing lotions, softening lotions, perfumes, packs, softening waters, emulsions, makeup bases, essences, soaps, liquid cleansers, bath additives, sunscreen creams, sun oils, suspensions, emulsions, pastes, gels, lotions, powders, surfactant-containing cleansers, oils, powder foundations, emulsion foundations, wax foundations, patches, and sprays, but are not limited to these.

[0045] The cosmetic composition may further contain one or more cosmetically acceptable carriers commonly used in skin cosmetics, and may appropriately blend with typical ingredients such as oils, water, surfactants, humectants, lower alcohols, thickeners, chelating agents, pigments, preservatives, and fragrances. It is possible, but it is not limited to this.

[0046] The cosmetically acceptable carriers included in a cosmetic composition vary depending on the dosage form of the cosmetic composition.

[0047] When the dosage form is an ointment, paste, cream, or gel, animal oils, vegetable oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc., can be used as carrier components, but are not limited to these. These can be used alone or in combination of two or more.

[0048] When the dosage form is a powder or spray, lactos, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, etc. are used as carrier components, in particular In the case of sprays, additionally, chlorofluorocarbon, propane / butane, or dimethyl These can include, but are not limited to, propellants such as ether. They can be used individually or in combination of two or more.

[0049] When the dosage form is a solution or emulsion, a solvent, solubilizer, or emulsifier is used as the carrier component, for example, water, glycerin, ethanol, isopropanol, diethyl carbonate, Ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, etc., are used, and in particular, cottonseed oil, peanut oil, corn oil, olive oil, pimaja oil, and sesame oil, glycerol aliphatic esters, polyethylene glycol, or fatty acid esters of sorbitan are used, but are not limited to these. These can be used individually or in combination of two or more.

[0050] If the dosage form is a suspension, the carrier component may be a liquid diluent such as water, glycerin, ethanol, or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum methhydroxyl, bentonite, agar, or Tragacanth and other similar herbs are used, but the plant is not limited to these. These can be used individually or in combination of two or more.

[0051] When the dosage form is soap, the carrier component may include, but is not limited to, alkali metal salts of fatty acids, fatty acid hemiester salts, fatty acid protein hydrolysates, isethionic acid, lanolin derivatives, aliphatic alcohols, vegetable oils, glycerol, and sugars. These can be used individually or in combination of two or more.

[0052] In the cosmetic composition, the exosomes are present in an amount equal to 0.0001% of the total weight of the cosmetic composition. It contains up to 50% by weight, more specifically, 0.0005 to 10% by weight. Exosomes When the composition falls within the aforementioned range, it offers the advantages of excellent skin condition improvement efficacy and stable dosage form of the composition.

[0053] Generally, quasi-pharmaceuticals refer to articles used for the purpose of diagnosing, treating, improving, alleviating, managing, or preventing diseases in humans and animals, excluding articles that have a milder effect than pharmaceuticals or that are used for pharmaceutical purposes. This includes products used for treating or preventing diseases in humans and animals, and products that have a mild or no direct effect on the human body.

[0054] Quasi-pharmaceutical compositions can be manufactured in dosage forms selected from the group consisting of body cleansers, foams, soaps, masks, ointments, creams, lotions, essences, and sprays, but are not limited thereto.

[0055] If the present invention relates to a pharmaceutical composition for application to a biological membrane such as skin, or for the treatment of wounds, in addition to containing exosomes as an active ingredient, it may further contain a pharmaceutically acceptable carrier.

[0056] "Pharmacologically acceptable" means that, upon administration, it does not irritate the organism, nor does it inhibit the biological activity and properties of the administered compound, and is therefore normally usable in the pharmaceutical field.

[0057] The dosage is a pharmaceutically effective amount for improving the skin condition. The “pharmaceutically effective amount” means an amount sufficient to treat the disease with a reasonable benefit-risk ratio applicable to medical treatment. The effective dose level is determined by factors including individual species and severity, age, sex, type of disease, drug activity, sensitivity to the drug, administration time, route of administration, and excretion rate, duration of treatment, drugs used concurrently, and other factors well known in the medical field. Furthermore, the effective dose varies depending on the route of administration, the use of excipients, and the possibility of use with other drugs, as is recognized by those skilled in the art.

[0058] The type of carrier is not particularly limited and any carrier commonly used in the relevant art is acceptable. While not limited to this, specific examples include saline solution, sterile water, Ringer's solution, buffered saline, albumin injection solution, lactose, D-glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, maltodextrin, glycerol, and ethanol. These can be used individually or in combination of two or more.

[0059] Furthermore, if necessary, it can be used with the addition of excipients, diluents, antioxidants, buffers, bacteriostatic agents, and other pharmaceutically acceptable additives, as well as fillers, bulking agents, wetting agents, decomposing agents, dispersants, surfactants, binders, or lubricants. [Effects of the Invention]

[0060] The umbilical cord blood mesenchymal stem cell culture medium produced by the present invention contains a high content of highly effective exosomes with a uniform size distribution, and can be usefully used in cosmetic compositions that require skin penetration and pharmaceutical compositions for wound and wound treatment. [Brief explanation of the drawing]

[0061] [Figure 1] Figure 1 shows the results of a comparative analysis of the total protein concentration and extracellular matrix protein content present in umbilical cord blood stem cell culture media prepared under different conditions according to Example 1. [Figure 2]Figure 2 shows the results of a comparative analysis of the distribution, size, and concentration of exosomes present in umbilical cord blood-derived stem cell culture media prepared under different conditions according to Example 1. [Figure 3] Figure 3 shows the results of a comparative analysis of the effects of exosomes in umbilical cord blood-derived stem cell culture media prepared under different conditions according to Example 1 on the motility of human fibroblasts and keratinocytes. [Figure 4] Figure 4 shows the results of a comparative analysis of the effects of exosomes in umbilical cord blood-derived stem cell culture media prepared under different conditions according to Example 1 on the proliferation ability of human fibroblasts and keratinocytes. [Figure 5] Figure 5 shows the results of an analysis of the effects of umbilical cord blood stem cell culture medium on skin cell death and secretion of inflammation-inducing substances in an inflammatory environment, as demonstrated in Experiment Example 5. [Figure 6] Figure 6 shows the results of a comparative analysis of the effects of treating an animal wound model with exosomes from umbilical cord blood-derived stem cell culture medium, prepared under different conditions according to Example 1, on skin cell regeneration. [Figure 7] Figure 7 is a conceptual diagram showing the various active ingredients produced by fibroblasts. [Figure 8] Figure 8 is a conceptual diagram showing the main structural features of the skin epidermis. [Figure 9] Figure 9 is a conceptual diagram illustrating the interaction between fibroblasts and keratinocytes.

[0062] [Specific details for carrying out the invention] The present invention will be described in detail below with reference to examples. However, the following examples merely illustrate the technical features of the present invention and do not limit the scope of protection of the present invention.

[0063] Example 1. Preparation of umbilical cord blood stem cell culture medium and extraction of exosomes secreted by umbilical cord blood stem cells. Human umbilical cord blood stem cells are mixed with 10% fetal bovine serum. The cultures were incubated in KSB-3 (Basal Culture Medium) containing bovine serum at 37°C in a 5% CO2 incubator for 1 to 4 days. After removing the culture badge, the cultures were washed with PBS.

[0064] Washed cells were placed in DMEM badges containing GDF-11 (1-20 ng / ml), EGF (1-20 ng / ml), FGF2 (1-20 ng / ml), TGF-β1 (1-20 ng / ml), and VEGF (1-20 ng / ml), and in DMEM badges without growth factors. These were cultured in a 37°C, 5% CO2 incubator for 1-4 days, then centrifuged at 500 g for 5 minutes. The upper layer was separated and filtered through a 0.2 μm-0.8 μm filter.

[0065] The culture medium was produced under each condition, filtered through a 0.2 μm to 0.8 μm filter, and then processed using Capturem. TM Exosomes were extracted using the Exosome Isolation Kit (Takara Bio). The extracted exosomes were then buffered in PBS using the Amicon Ultra Centrifugal Filter (Merck Millipore) and used in each test.

[0066] Experimental Example 1. Analysis of extracellular matrix protein content in umbilical cord blood stem cell culture medium.

[0067] The extracellular matrix (Extracellular Molecule) within connective tissue, which is responsible for structural support of cells, typically includes components such as fibronectin, collagen, and elastin, and plays a role in regenerating damaged tissue.

[0068] To analyze the total protein content in umbilical cord blood stem cell culture medium, a BCA Assay was performed, and the umbilical cord blood To analyze the extracellular matrix protein content in stem cell culture media, enzyme-linked immunosorbent assay (ELISA) was performed. The culture media prepared under each condition in Example 1 were analyzed using the Human Fibronectin Quantikine ELISA Kit (R&D System, MN, USC) and Procollagen Type I C-peptide (PIP)EIA. A comparative analysis was performed using Kit (Takara bio inc, Japan).

[0069] Specifically, a culture medium (DMEM) using a batch that does not contain growth factors, and GDF-11, EGF, FGF2 Using a batch containing TGF-β1 and VEGF at concentrations of 1-20 ng / ml each, The total protein content of the nutrient solution and the content of extracellular matrix protein components were compared and analyzed.

[0070] As a result, Figure 1(a) shows the total amount of protein present in the culture medium using batches to which GDF-11, EGF, FGF2, TGF-β1, and VEGF were added at concentrations of 1-20 ng / ml, and the amount of phi We confirmed that the bronnectin (fibronectin) protein content was higher compared to the control group (DMEM). Furthermore, Figure 1(b) shows that the collagen protein (PIP) content was higher in the group compared to the control group (DMEM). It was confirmed that it contained it at a high level.

[0071] Experimental Example 2. Analysis of the size and concentration of exosomes present in umbilical cord blood stem cell culture medium. In Example 1, exosomes were separated from umbilical cord blood stem cell culture media prepared under different conditions. The size and concentration of exosomes present in the culture media were then compared and analyzed using Nanosight. Nanosight can observe and analyze individual particles undergoing nanoparticle diffusion motion of 10 nm to 2000 nm size in solution, thereby analyzing the distribution and concentration of nanoparticle sizes.

[0072] As a result, as shown in Figure 2, compared to the control group (DMEM) using a badge without growth factors, GDF-11, EGF, FGF2, TGF-β1, and VEGF were added at concentrations of 1-20 ng / ml, respectively. When using a badge, we confirmed that the number of exosomes was large and that the size distribution appeared uniformly at around 100 nm.

[0073] Experimental Example 3. Effects of exosomes in umbilical cord blood stem cell culture medium on the migratory ability of human fibroblasts and keratinocytes. In wound healing, epidermal cell migration is a major factor. To investigate the effects of exosomes in umbilical cord blood stem cell culture on the migration of human dermal fibroblasts (HDFs) and human keratinocytes (HKCs), a trans-well migration assay was performed. .

[0074] Human fibroblasts and keratinocytes are placed in a Trans-well Insert, 2x10. 4 Cells / well were divided and cultured for 1 day at 37°C under 5% CO2 conditions. After removing the culture batch, the cells were washed with PBS. Serum-free (blood) containing the exosomes prepared and extracted under different conditions in Example 1. DMEM (without water) was treated at 100 ul / well, and after 2 hours, the bottom plate... Add 400 µl of DMEM containing 10% serum, and leave at 37°C and 5% CO2 for 1 day. The cells were cultured. To stain the migrated cells, they were stained with Crystal Violet Solution and then comparatively analyzed under a light microscope.

[0075] Specifically, a culture medium using a batch (DMEM) that does not contain growth factors, and GDF-11, EGF, FGF2 Exosomes 2×10¹ were extracted from a culture medium using batches to which TGF-β1 and VEGF were each added at concentrations of 1-20 ng / ml. 8Cells were treated to human fibroblasts, and basal DMEM without culturing cord blood stem cells was used as the negative control group (non treat).

[0076] As a result, from FIG. 3, exosomes isolated from cord blood stem cell culture medium using a medium supplemented with GDF-11, EGF, FGF2, TGF-β1, and VEGF at a concentration of 1 to 20 ng / ml, respectively, were It was confirmed that the migration ability of treated human fibroblasts and keratinocytes was increased compared with the control group.

[0077] Experimental Example 4. Effect of exosomes in cord blood stem cell culture medium on the proliferation ability of human fibroblasts and keratinocytes In wound healing, the proliferation of fibroblasts and keratinocytes is also an important part in the regeneration process. Therefore, to confirm the effect of culture medium on the proliferation of human fibroblasts and keratinocytes, a proliferation assay was performed. In a 6-well plate, human fibroblasts and keratinocytes were each plated at 1 ×10 5 cells / well, and cultured under conditions of 37°C and 5% CO₂. The number of cells was measured at 24-hour intervals.

[0078] Specifically, each 2×10 exosomes extracted from a culture medium using a growth factor-free medium (DMEM) and a medium supplemented with GDF-11, EGF, FGF2, TGF-β1, and VEGF at a concentration of 1 to 20 ng / ml, respectively, were used to treat 8 human fibroblasts and keratinocytes, and basal DMEM without culturing cord blood stem cells was used as the negative control group (non treat) .

[0079] As a result, from FIG. 4, exosomes isolated from cord blood stem cell culture medium using a medium supplemented with growth factors GDF-11, EGF, FGF2, TGF-β1, and VEGF at a concentration of 1 to 20 ng / ml, respectively We confirmed that the proliferative capacity of human fibroblasts and keratinocytes treated with somatosomals was increased compared to the control group.

[0080] Experimental Example 5. Exosomes in umbilical cord blood stem cell culture medium are involved in skin tissue regeneration in animal wound models. influence

[0081] To confirm whether umbilical cord blood stem cell secretory exosomes, which were identified in vitro, improve the proliferation and migration of human skin cells in vivo, we will investigate whether they do so in vivo. A physical wound model test was conducted. A 15 mm wound was created on the back of a 6-week-old SD rat, and then 2 × 10⁻¹⁶ wounds were applied. 8 Individual exosomes were treated. After 14 days, the effect of skin tissue regeneration was confirmed.

[0082] Specifically, in Example 1, 2x exosomes were extracted from a culture medium using a batch to which GDF-11, EGF, FGF2, TGF-β1, and VEGF were each added at concentrations of 1-20 ng / ml. 10 8 Each individual was treated in an animal wound model, and an untreated animal wound model was used as a negative control group (NC).

[0083] As a result, Figure 6 shows that exosomes isolated from umbilical cord blood stem cell culture medium using batches to which GDF-11, EGF, FGF2, TGF-β1, and VEGF were added at concentrations of 1-20 ng / ml each were obtained. We confirmed that the skin tissue regeneration ability of the treated animal wound models was far superior to that of the control group.

Claims

1. Umbilical cord blood stem cells are cultured in serum-free batches supplemented with one or more growth factors selected from the group consisting of GDF-11, EGF, FGF2, TGF-β1, and VEGF, each at a concentration of 1 to 20 ng / ml. When nourished, the concentration of exosomes secreted during umbilical cord blood stem cell culture is 0.5 to 5 × 10 ９ A method for producing an exosome-containing culture medium secreted by umbilical cord blood stem cells, characterized by including a step of producing a culture medium with a high concentration of / ml.

2. Umbilical cord blood stem cells were cultured in serum-free batches supplemented with GDF-11 and one or more growth factors selected from the group consisting of EGF, FGF2, TGF-β1, and VEGF, each at a concentration of 1–20 ng / ml. A method for producing a culture medium containing exosomes secreted by umbilical cord blood stem cells as described in claim 1.

3. The method according to claim 1, characterized in that umbilical cord blood stem cells are cultured in a serum-free batch to which GDF-11, EGF, FGF2, TGF-β1, and VEGF are each added at concentrations of 1 to 20 ng / ml. A method for producing a culture medium containing exosomes secreted by umbilical cord blood stem cells.

4. The method for producing an exosome-containing culture medium secreted by umbilical cord blood stem cells according to claim 1, characterized in that the exosome-containing culture medium secreted by umbilical cord blood stem cells in a serum-free batch to which the growth factor has been added has an even higher level of fibronectin and / or collagen (PIP), which are extracellular matrix proteins, compared to a culture medium produced by culturing umbilical cord blood stem cells in a serum-free batch to which the growth factor has not been added.

5. The method for producing an exosome-containing culture medium secreted by umbilical cord blood stem cells according to claim 1, characterized in that, compared to a culture medium produced by culturing umbilical cord blood stem cells in a serum-free badge to which the growth factor has been added, the exosome-containing culture medium secreted contains a greater number of exosomes, contains an even higher concentration of active ingredients within the exosomes, and has an even higher skin regeneration capacity.

6. The method for producing a culture medium containing exosomes secreted by umbilical cord blood stem cells, as described in claim 1, characterized by the mass production of small-particle exosomes with a diameter of 100 nm ± 20 nm that are easily absorbed through the skin.

7. The badges are DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, DMEM / F-10 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-10), DMEM / F-12 (Dulbecco's Modified Eagle's Medium: A method for producing an exosome-containing culture medium secreted by umbilical cord blood stem cells according to claim 1, characterized in that it is one or more badges selected from the group consisting of Nutrient Mixture F-12), α-MEM (α-Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium), IMDM (Isocove's Modified Dulbecco's Medium), and KnockOut DMEM.

8. Furthermore, the method for producing an exosome-containing culture medium secreted by umbilical cord blood stem cells according to claim 1 is characterized by including the step of forming a dosage form according to the intended use of the exosome-containing culture medium secreted by umbilical cord blood stem cells, or exosomes separated from the culture medium.

9. The method for producing an exosome-containing culture medium secreted by umbilical cord blood stem cells according to claim 8, characterized in that the exosome-containing culture medium, or exosomes separated from the culture medium, are gelled, frozen, or dried to form a powder.

10. Exosomes secreted during umbilical cord blood stem cell culture in a serum-free badge to which the growth factor is added are the same as exosomes secreted during umbilical cord blood stem cell culture in a serum-free badge to which the growth factor is not added. A method for producing a culture medium containing exosomes secreted by umbilical cord blood stem cells according to claim 1, characterized in that it improves the migratory and / or proliferative capacity of human fibroblasts and / or human keratinocytes compared to sosomes.

11. A composition for topical application to skin or a biological membrane, characterized by containing, as an active ingredient, a culture medium containing umbilical cord blood stem cells exosomes obtained by the method described in any one of claims 1 to 10, or exosomes separated therefrom.

12. The composition for application to skin or biological membrane according to claim 11, characterized in that it is a cosmetic composition, a quasi-pharmaceutical composition, or a pharmaceutical composition.

13. The skin or biological membrane application composition according to claim 11, characterized in that the small exosomes of umbilical cord blood stem cells are used as a transdermal carrier.

14. The composition for application to skin or biological membrane according to claim 11, which is a dosage form that is mixed with a polymer and gelled, and which becomes a sol under body temperature conditions.

15. A pharmaceutical composition for treating wounds or wounds, characterized by containing, as an active ingredient, an exosome-containing culture medium of umbilical cord blood stem cells obtained by the method described in any one of claims 1 to 10, or exosomes isolated therefrom.

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