Composition for treating wounds comprising stem cell-derived extracellular vesicles with enhanced efficacy

Abstract

The present invention relates to a composition for treating or restoring wounds, comprising extracellular vesicles derived from three-dimensional spheroidal cell aggregates prepared by a novel preparation method, and a wound treatment method. The extracellular vesicles prepared by the novel method of the present invention can rapidly promote wound recovery, and thus can be effectively used as various drugs, cosmetics, and foods for wound treatment, regeneration, and recovery.

Description

Technical Field

[0001] The present invention relates to a composition for treating wounds, which comprises extracellular vesicles derived from three-dimensional spheroid cell aggregates prepared by a new preparation method. Background Art

[0002] Treatment methods using stem cells, especially mesenchymal stem cells (MSCs), and positive clinical results have been reported for various diseases. However, stem cell therapeutic agents have the risk of producing cell-related side effects such as vascular occlusion, tumor formation, and coagulation disorders as side effects, and clinical trials are still needed to verify their efficacy. It is well known that the paracrine effect of stem cells induces the regeneration of surrounding skin cells and enhances the ability of blood vessel regeneration. In particular, it is well known that extracellular vesicles (EVs) are the main efficacy factor of the paracrine effect.

[0003] Extracellular vesicles are divided into exosomes and microvesicles according to their size. The diameter of exosomes is 30 to 150 nm, and the size of microvesicles is 100 to 1000 nm. It is well known that extracellular vesicles are part of the cell membrane released into the blood, and mediate communication between cells because they contain proteins and nuclear components. Using extracellular vesicles instead of stem cells not only improves safety by minimizing the side effects caused by the use of stem cells, but also facilitates biodistribution and production processes.

[0004] However, mass production and acquisition methods, etc., for use of extracellular vesicles derived from stem cells have not been established so far, and there are not many studies on methods that can further enhance the efficacy of extracellular vesicles while maintaining the properties of stem cell-derived extracellular vesicles.

[0005] Therefore, there is a need for extracellular vesicles with further enhanced efficacy and novel therapeutic agents utilizing the same. Summary of the invention

[0006] Technical issues

[0007] In response to this, in order to prepare three-dimensional spheroidal cell aggregates or extracellular vesicles derived from them, the present inventors have developed a method that can mass-produce and shorten the culture time. As a result, it was confirmed that three-dimensional spheroidal cell aggregates (3D-static-spheroids) can be prepared by static culture using micropores. When extracellular vesicles are prepared using them, extracellular vesicles that improve wound regeneration and wound recovery capabilities are prepared, thereby completing the present invention.

[0008] Therefore, the object of the present invention is to prepare three-dimensional spheroid cell aggregate-derived extracellular vesicles with improved wound healing ability, and to provide a composition containing the same for wound healing, regeneration or recovery.

[0009] Technical Solution

[0010] In order to achieve the above-mentioned purpose, the present invention provides a pharmaceutical composition for treating or restoring wounds, comprising extracellular vesicles derived from three-dimensional spheroid cell aggregates prepared by the following method: step (a), preparing three-dimensional spheroid cell aggregates by three-dimensionally culturing stem cells in microwells with a diameter of 200 μm to 800 μm and a depth of 100 μm to 1000 μm; and step (b), isolating extracellular vesicles from the above-mentioned three-dimensional spheroid cell aggregates.

[0011] Furthermore, the present invention provides a cosmetic composition for treating or restoring wounds, comprising extracellular vesicles derived from three-dimensional spheroid cell aggregates prepared by the following method: step (a), preparing three-dimensional spheroid cell aggregates by three-dimensionally culturing stem cells in microwells with a diameter of 200 μm to 800 μm and a depth of 100 μm to 1000 μm; and step (b), isolating extracellular vesicles from the above-mentioned three-dimensional spheroid cell aggregates.

[0012] In addition, the present invention provides a quasi-drug composition for treating or restoring wounds, comprising extracellular vesicles derived from three-dimensional spheroid cell aggregates prepared by the following method: step (a), preparing three-dimensional spheroid cell aggregates by three-dimensionally culturing stem cells in microwells with a diameter of 200 μm to 800 μm and a depth of 100 μm to 1000 μm; and step (b), isolating extracellular vesicles from the above-mentioned three-dimensional spheroid cell aggregates.

[0013] In addition, the present invention provides a method for preparing a composition for treating or restoring wounds, which comprises extracellular vesicles derived from three-dimensional spheroid cell aggregates, comprising: step (a), preparing three-dimensional spheroid cell aggregates by three-dimensionally culturing stem cells in microwells with a diameter of 200 μm to 800 μm and a depth of 100 μm to 1000 μm; and step (b), isolating extracellular vesicles from the above-mentioned three-dimensional spheroid cell aggregates.

[0014] In addition, the present invention provides a wound treatment method, comprising: step (a), preparing three-dimensional spheroidal cell aggregates by three-dimensionally culturing stem cells in microwells with a diameter of 200 μm to 800 μm and a depth of 100 μm to 1000 μm; step (b), preparing three-dimensional spheroidal cell aggregate-derived extracellular vesicles by separating extracellular vesicles from the above-mentioned three-dimensional spheroidal cell aggregates; and step (c), administering the three-dimensional spheroidal cell aggregate-derived extracellular vesicles prepared by the above-mentioned step (b) to an individual in need.

[0015] Effects of the Invention

[0016] The extracellular vesicles prepared by the novel method of the present invention can rapidly promote the recovery of wounds, and thus can be used as a variety of medicines, cosmetics, and foods for wound treatment, regeneration, and recovery. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The figure schematically shows the process of preparing three-dimensional spheroid-type cell aggregates and isolating extracellular vesicles therefrom.

[0018] Figure 2 Part A is a photograph showing the observation of a microwell filled with a culture solution of three-dimensional-dynamic-PEG spheroids.

[0019] Figure 2 Part B is a photograph showing the observation of a microwell filled with a three-dimensional static spheroid culture solution.

[0020] Figure 2 Part C is a graph showing changes in the area of ​​aggregates during the culture period using the three-dimensional-dynamic-PEG spheroid and the three-dimensional-static-spheroid prepared using microwells.

[0021] Figure 3 The graph compares the size distribution of 3D-static-spheroids and 3D-dynamic-PEG-spheroids.

[0022] Figure 4 The morphology of three-dimensional static spheroid extracellular vesicles was observed using an electron microscope.

[0023] Figure 5 The graph shows the results of nanoparticle tracking analysis (NTA) for confirming the concentration and size distribution of three-dimensional static spheroid extracellular vesicles.

[0024] Figure 6 The figure shows the results of ELISA and Western blotting for confirming the expression markers of three-dimensional static spheroid extracellular vesicles.

[0025] Figure 7 The graph compares the yields of three-dimensional static spheroid extracellular vesicles, three-dimensional dynamic PEG spheroid extracellular vesicles, and two-dimensional (2D) extracellular vesicles from each source cell.

[0026] Figure 8 A graph showing microRNAs (miRNAs) and proteins that are highly expressed in 3D-static-spheroidal extracellular vesicles compared to 2D-extracellular vesicles.

[0027] Fig. 9 The graph shows the miRNAs and proteins that are highly expressed in three-dimensional-static-spheroid extracellular vesicles compared to three-dimensional-dynamic-PEG-spheroid extracellular vesicles.

[0028] Fig.10a The graph shows the results of confirming the differences in extracellular vesicle production, extracellular vesicle size, and the amount of protein contained in the extracellular vesicles depending on the donor in two-dimensional extracellular vesicles (2D-EV) and three-dimensional static spheroid extracellular vesicles (WJ-3DEV).

[0029] Fig.10b The graph shows the results of confirming differences in miRNA expression levels associated with donor variation, angiogenesis, and neural regeneration in two-dimensional cultured WJ-MSCs, three-dimensional cultured WJ-MSCs, two-dimensional extracellular vesicles, and three-dimensional static spheroid extracellular vesicles.

[0030] Fig.11 The diagram shows changes in the expression of messenger ribonucleic acid (mRNA) after treatment of 3D-static-spheroid extracellular vesicles or 2D-extracellular vesicles to human vascular endothelial cells (HUVECs) or primitive neural stem cells (pNSCs) as neural stem cells.

[0031] Fig.12 The graph shows the results of confirming the degree of wound recovery over 14 days after treatment with three-dimensional static spheroid extracellular vesicles (3D-EV), WJ-MSC, and phosphate buffered saline (PBS).

[0032] Fig.13 This is a graph showing the recovery of wound sites by numerical calculation in the three-dimensional static spheroid extracellular vesicle and PBS (control) treatment groups.

[0033] Fig.14 A graph showing the results of staining of wound skin cross-sectional areas confirming that three-dimensional-static-spheroid extracellular vesicle treatment increased epidermal thickness.

[0034] Fig.15 The graph shows the results of confirming the recovery of the wound site 14 days later by staining in the three-dimensional static spheroid extracellular vesicle (EV) and PBS (Control) treatment groups.

[0035] Fig.16 This is a graph showing the effect of quantifying the thickness increase of the epidermis and the surrounding area of ​​the epidermis treated with three-dimensional static spheroid extracellular vesicles.

[0036] Fig.17 Schematic diagram showing the wound recovery experimental protocol using the chamber model.

[0037] Fig.18 A graph showing the results of confirming the wound recovery area in the three-dimensional-static-spheroid extracellular vesicle (EV) and PBS groups after the chamber was removed.

[0038] Fig.19 This is a graph showing the wound area after the chamber was removed in the three-dimensional static spheroid extracellular vesicle (EV) and PBS groups and then quantified.

[0039] Fig. 20 A graph showing the results of confirming that epidermal thickness increased with three-dimensional-static-spheroid extracellular vesicle treatment in a chamber model.

[0040] Figures 21a to 21c To show that the expression of angiogenesis-related factors vascular endothelial growth factor (VEGF), angiopoietin-1 (angiopoietin-1), and angiopoietin-2 (angiopoietin-2) was confirmed by enzyme-linked immunosorbent assay (ELISA) 7 or 14 days after the chamber was removed in the chamber model ( Fig.21a ), inflammatory factors interleukin-1b (IL-1b), tumor necrosis factor α (TNF-a) and interleukin-6 (IL-6) ( Figure 21b ), anti-inflammatory factor interleukin-10 (IL-10) ( Fig.21c )A graph expressing the results of the changes.

[0041] Fig.22a This figure shows the results of confirming the migration of fibroblasts after treating three-dimensional spheroid extracellular vesicles (EVs). Figure 22b This is a diagram showing the results of confirming the migration properties of keratinocytes.

[0042] Fig.23This is a graph showing the results of comparing the increased expression levels of VEGF, Hif-1a, and FGF associated with angiogenesis after HUVEC cells were treated with three-dimensional spheroid extracellular vesicles and two-dimensional extracellular vesicles.

[0043] Fig.24 The graph shows the results of comparing the angiogenesis effects of cells treated with three-dimensional spheroid extracellular vesicles and two-dimensional extracellular vesicles.

[0044] Fig.25 The graph shows the results of confirming the size, roundness, and solidity of three-dimensional spheroids prepared by changing the diameter and depth of microwells and the number of cells per well in the preparation of three-dimensional static spheroid extracellular vesicles.

[0045] Fig.26 The graph shows the results of comparing the expression levels of miRNA-132 and miRNA-210 expressed in three-dimensional static spheroid extracellular vesicles and two-dimensional extracellular vesicles prepared under various conditions.

[0046] Fig. 27 The graph shows the results of confirming the angiogenic ability of three-dimensional static spheroid extracellular vesicles and two-dimensional extracellular vesicles prepared under various conditions through a tube formation experiment.

[0047] Best Mode for Carrying Out the Invention

[0048] The present invention provides a pharmaceutical composition for treating or restoring wounds, comprising extracellular vesicles derived from three-dimensional spheroid cell aggregates prepared by the following method: step (a), preparing three-dimensional spheroid cell aggregates by three-dimensionally culturing stem cells in microwells with a diameter of 200 μm to 800 μm and a depth of 100 μm to 1000 μm; and step (b), isolating extracellular vesicles from the above-mentioned three-dimensional spheroid cell aggregates.

[0049] In the present invention, the above-mentioned cells can be used without limitation as long as they can be used to separate extracellular vesicles, and can be cells separated from natural biological individuals. Moreover, the above-mentioned cells can be of any type of animal or plant origin, including humans and non-human mammals, and can be various types of immune cells, tumor cells, and stem cells. Preferably, the above-mentioned stem cells can be mesenchymal stem cells, pluripotent stem cells, induced pluripotent stem cells, or embryonic stem cells.

[0050] In the present invention, the above-mentioned three-dimensional culture refers to culture in a three-dimensional configuration in a test tube. Different from two-dimensional culture, when three-dimensional culture is performed, cells can grow in all directions in vitro, which may be more similar to the cell environment in vivo.

[0051] In the present invention, the three-dimensional culture in the above step (a) can be carried out by all three-dimensional cell culture techniques known in the technical field to which the present invention belongs, for example, it can be cell culture performed by microwell array culture, porous microsphere culture, hanging drop culture, low attachment plate culture, membrane-based cell-detachment culture, thermal lifting culture, centrifugal separation culture, semisolid medium culture, etc. Preferably, the above three-dimensional culture can be a dynamic culture or a static culture, and more preferably, it can be a static culture. In the present invention, when the three-dimensional culture in step (a) is a static culture, since no device for shaking culture is required, it can be cultured more easily, and a large number of cultures can be performed in a pharmaceutical production quality management specification (Good manufacturing practices, GMP) preparation site.

[0052] In the present invention, the three-dimensional culture of the above step (a) can be cultured for 1 day to 10 days, preferably, can be cultured for 2 days to 4 days. In particular, in the present invention, when the culture time in the above step (a) is 2 days to 4 days, the cells present in the three-dimensional spheroid cell aggregates maintain a high level of survival rate, and the culture time is relatively short compared to the existing three-dimensional spheroid cell aggregate preparation process, so the three-dimensional spheroid cell aggregates and the extracellular vesicles derived therefrom can be quickly prepared.

[0053] In the present invention, the three-dimensional culture in the above step (a) can be performed by injecting mesenchymal stem cells at a density of 100 to 1000 cells / well and culturing them in microwells, preferably, by injecting and culturing them at a density of 100 to 600 cells / well, and more preferably, by injecting and culturing them at a density of 100 to 500 cells / well.

[0054] In the present invention, the step of separating extracellular vesicles in the above step (b) can be performed by physical separation or chemical separation. The above physical separation can be extrusion of a sample including cells or cell aggregates, and chemical separation can be processing a chemical substance capable of separating extracellular vesicles from cells or cell aggregates. For example, the separation step of the present invention can be performed using a method selected from the group consisting of ultrasonic decomposition, cell lysis, homogenization, freeze-thaw, electroporation, treatment with chemical substances, mechanical decomposition, and physical stimulation of applying force to cells from the outside. It can be a physical and chemical method for separation by binding ions to specific biomarkers using ion or affinity chromatography, or by immunoaffinity EV capture (immunoaffinity EV capture) of hansabiomed life sciences company, exosome purification reagent (exosome purification reagent) of creative biolabs company, ExoCAS-2 reagent or method of microgentas company, preferably, it can be separated by tangential flow filtration (TFF) method, but it is not limited thereto.

[0055] In the present invention, the naming of MicroRNA is given "mir" in the prefix and "-" and numbers in the suffix. In this case, the numbers often indicate the order of naming, for example, it is predicted that mir-123 was named before mir-156 and discovered earlier. "mir-" means microRNA precursor (pre-microRNA), and "miR-" with capital letters refers to mature microRNA (mature microRNA). MicroRNAs with almost the same sequence except for one or two sequences are named by adding lowercase letters. For example, miR-121a and miR-121b are generated from mir-121a and mir-121b, which are their respective precursors, respectively, and the sequences are also very similar. Although the mature microRNAs are the same, the microRNA precursors (pre-microRNAs) located at different sites in the genome are named by additionally adding "-" and numbers. As an example, mir-121-1 and mir-121-2, which are microRNA precursors (pre-microRNA), become the same mature microRNA (mature microRNA) (miR-121), but are located at different sites in the genome. The naming of microRNAs according to species is marked with prefixes. For example, hsa-miR-123 is a human (Homo sapiens) microRNA, and oar-miR-123 is a sheep (Ovis aries) microRNA. "v" refers to virus (viral) (miRNA encoded by a viral genome), and "d" refers to Drosophila microRNA (Drosophila microRNA). When two mature microRNAs are derived from different arms (three arms (3'arm) or five arms (5'arm)) of the same microRNA precursor (pre-microRNA), "-3p" or "-5p" is added to the suffix for naming. miR-142-3p is derived from the third arm (3'arm), and miR-142-5p is derived from the fifth arm (5'arm). The nomenclature of microRNAs generally follows the standard as described above, but there are exceptions.

[0056] In the present invention, the extracellular vesicles can highly express a variety of substances showing wound healing efficacy compared to known extracellular vesicles. For example, preferably, compared with extracellular vesicles derived from spheroid-type cell aggregates of mesenchymal stem cells cultured in three-dimensional dynamic culture, the extracellular vesicles of the present invention can highly express one or more selected from the group consisting of miR-146a, miR-27a, miR-132, miR-184, miR-210 and miR-301b, or, compared with extracellular vesicles derived from mesenchymal stem cells cultured in two dimensions, the extracellular vesicles of the present invention can highly express one or more selected from the group consisting of miR-27a, miR-146a and miR-146b, or can highly express vascular endothelial growth factor (VEGF), hypoxia-inducible factor-1α (Hif-1a, Hypoxia-inducible factor 1-alpha) and fibroblast growth factor (FGF, Fibroblast growth factor).

[0057] Furthermore, the extracellular vesicles described above can be incorporated into cells and internalized when they are treated with cells. When incorporated into cells and internalized, clinically significant substances highly expressed in the extracellular vesicles can be effectively delivered to cells, thereby being highly expressed in cells.

[0058] In the present invention, the three-dimensional spheroid cell aggregate-derived extracellular vesicles showing wound healing, regeneration, and recovery effects can be used interchangeably with "three-dimensional-static-spheroid-extracellular vesicles". In contrast, the three-dimensionally dynamically cultured spheroid cell aggregate-derived extracellular vesicles can be used interchangeably with "three-dimensional-dynamic-PEG-spheroid-extracellular vesicles".

[0059] In the present invention, "extracellular vesicles derived from spheroid cell aggregates of mesenchymal stem cells in three-dimensional dynamic culture" can include, without limitation, extracellular vesicles isolated from spheroid cell aggregates of mesenchymal stem cells in three-dimensional dynamic culture, preferably, the extracellular vesicles disclosed in the authorized patent (application number 10-2016-0053026, method for producing extracellular vesicles derived from stem cells).

[0060] In the present invention, the average diameter of the three-dimensional spheroid cell aggregates can be 74.43±7.756 μm, preferably, the size range is 55 μm to 95.0 μm, and the average diameter of the size distribution measurement results of 155 three-dimensional spheroid cell aggregates can be 74.43 μm, and the coefficient of variation (CV) can be 9.59%. The kurtosis of the size distribution of the three-dimensional spheroid cell aggregates of the present invention can be higher than that of the "spheroid cell aggregates of mesenchymal stem cells cultured in three-dimensional dynamic culture". Therefore, the size of the three-dimensional spheroid cell aggregates can be smaller than that of the "spheroid cell aggregates of mesenchymal stem cells cultured in three-dimensional dynamic culture" and can have a relatively uniform size distribution.

[0061] In the present invention, the micropores may be coated with a material selected from 3-(Trimethoxysilane)propyl acrylate (TMSPMA), hydroxyethyl acrylate (HEA), glycidyl methacrylate (GMA), diethylene glycol dimethacrylate (EGDMA), tetrahydrofuran acrylate (THFA), hydroxymethyl acrylamide (HMAA), phenyl epoxyacrylate (PEA), 6-hydroxy-2,2,3,3,4,4,5,5-octafluoro- ...5-octafluoro, HOFHA), Polyethoxylated (4) pentaerythritoltetraacrylate (EOPT), Hydroxypropyl acrylate (HPA), Butylmethacrlate (BMA), Pentaerythritoltriacrylate (PETIA), Hexan diol diacrylate (HDDA), Ethyleneglycol phenyletheracrylate (EGPEA), Benzylmethacrylate (BM), Hydroxyphenoxypropyl acrylate (HPPA), 2-(4-Benzoyl-3-hydroxyphenoxy)ethylacrylate (BHPEA), Hydroxyethyl methacrylate (Hydroxyethyl Any one of the group consisting of N-(2-hydroxypropyl)methacrylamide (HEMA), N-(2-Hydroxypropyl)methacrylamide (HPMA) and 2-methacryloyloxyethyl phosphorylcholine (2-Methacryloyloxyethyl Phosphorylcholine Polymer, MPC), preferably, may be coated with 2-methacryloyloxyethyl phosphorylcholine, but is not limited thereto.

[0062] The diameter of the micropores of the invention may be 200 μm to 800 μm, preferably 300 μm to 800 μm, and more preferably 400 μm to 800 μm.

[0063] Furthermore, the micropores may be flat structures without depth, or when the micropores have depth, they may have a depth of 100 μm to 1000 μm, preferably 100 μm to 900 μm, more preferably 200 μm to 900 μm.

[0064] The mesenchymal stem cells cultured by the above structure can maintain a high level of survival rate even after the culture time has passed. Preferably, the production yield of the cell aggregates can be increased by preparing a microwell array comprising 1,000 to 100,000 microwells.

[0065] When extracellular vesicles are prepared by the above-mentioned "method for preparing extracellular vesicles derived from three-dimensional spheroid cell aggregates" of the present invention, not only can the advantages of static culture at the preparation level be obtained, but also extracellular vesicles with improved wound healing ability can be produced in large quantities quickly and efficiently. In particular, the method for preparing extracellular vesicles of the present invention has the characteristics of being suitable for GMP application.

[0066] In the present invention, when the three-dimensional spheroid cell aggregate-derived extracellular vesicles are treated to the wound, rapid wound recovery can be induced, the mobility of fibroblasts and / or keratinocytes at the wound site can be enhanced in the early stage of wound occurrence, and angiogenesis can be promoted, thereby achieving rapid regeneration and recovery of the wound site.

[0067] In the present invention, angiogenesis refers to angiogenesis, which refers to the process of generating new blood vessels from existing blood vessels by inducing or increasing the migration of vascular endothelial cells and promoting angiogenesis in vascular endothelial cells.

[0068] The wounds to which the three-dimensional spheroid cell aggregate-derived extracellular vesicles of the present invention are applicable may include, without limitation, all wound forms occurring on the skin, for example, burns, bedsores, traumas and other skin wounds. Burns refer to damage to skin cells caused by fire or heat, bedsores refer to chronic ulcers caused by inflammation or tissue necrosis caused by poor blood circulation, resulting in tissue death. Trauma refers to damage to skin tissue caused by external pressure, for example, may include abrasions, contusions, lacerations, cuts caused by blades, stab wounds, cuts, gunshot wounds, blast wounds, strangulations and the like.

[0069] In addition to the above-mentioned active ingredients, the pharmaceutical composition of the present invention may also contain suitable carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions. The pharmaceutical composition of the present invention may also contain other pharmaceutically active ingredients or active mixtures.

[0070] Pharmaceutical composition of the present invention can be respectively configured to the forms such as oral dosage forms such as patch, coating, powder, granule, tablet, capsule, suspension, emulsion, syrup, aerosol, external preparation, suppository and sterile injection solution and use according to conventional method.Carrier, excipient and diluent that can be included in the composition can comprise lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talcum powder, magnesium stearate and mineral oil.When preparing, diluents or excipients such as conventional fillers, extenders, adhesives, wetting agents, disintegrating agents, surfactants can be used to prepare. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., which are prepared by mixing one or more excipients in the above composition, for example, starch, calcium carbonate, sucrose or lactose. In addition, lubricants such as magnesium stearate and talc are also used in addition to simple excipients. Liquid preparations for oral administration include suspensions, internal liquids, emulsions, syrups, etc., which can contain various excipients such as wetting agents, sweeteners, aromatics, preservatives, etc. in addition to commonly used simple diluents such as water or liquid paraffin. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Vegetable oils such as propylene glycol, polyethylene glycol, olive oil, and injectable esters such as ethyl oleate can be used as non-aqueous solvents and suspensions. Semi-synthetic fatty acid ester (Witepsol), polyethylene glycol, Tween 61, cocoa butter, laurin, glyceryl gelatin, etc. can be used as the base of the suppository.

[0071] The preferred dosage of the pharmaceutical composition of the present invention varies according to the patient's state and body weight, severity of the disease, drug form, route of administration, and duration of administration, but can be appropriately selected by those skilled in the art. It can be administered once a day or divided into multiple administrations. The above dosage does not limit the scope of the present invention in any aspect.

[0072] The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, and humans in various ways. All administration methods can be predicted, for example, it can be administered orally, percutaneously, rectally, or by intravenous, intramuscular, or subcutaneous injection.

[0073] The definitions of the above-mentioned excipients, binders, disintegrants, lubricants, flavoring agents, flavor enhancers and the like related to the present invention are recorded in the literature known in the art, and include substances having the same or similar functions.

[0074] In addition, the present invention provides a wound treatment method, comprising: step (a), preparing three-dimensional spheroidal cell aggregates by three-dimensionally culturing stem cells in microwells with a diameter of 200 μm to 800 μm and a depth of 100 μm to 1000 μm; step (b), preparing three-dimensional spheroidal cell aggregate-derived extracellular vesicles by separating cells and vesicles from the above-mentioned three-dimensional spheroidal cell aggregates; and step (c), administering the three-dimensional spheroidal cell aggregate-derived extracellular vesicles prepared by the above-mentioned step (b) to an individual in need.

[0075] The above-mentioned individual may be a mammal including a human being, and may include a patient who needs wound treatment, a patient who is receiving treatment for wound treatment, or a patient who needs to receive wound treatment. The above-mentioned three-dimensional spheroid cell aggregate-derived extracellular vesicles may be treated in combination with other drugs or treatment methods. In the case of combined treatment with other drugs or treatment methods, the other drugs or treatment methods may be treated simultaneously or sequentially.

[0076] Furthermore, the present invention relates to a cosmetic composition and a quasi-drug composition for treating or restoring wounds, comprising extracellular vesicles derived from three-dimensional spheroid cell aggregates prepared by the following steps: step (a), preparing three-dimensional spheroid cell aggregates by three-dimensionally culturing stem cells in microwells with a diameter of 200 μm to 800 μm and a depth of 100 μm to 1000 μm; and step (b), isolating extracellular vesicles from the above-mentioned three-dimensional spheroid cell aggregates.

[0077] In this specification, "cosmetic composition" refers to a composition formed for the purpose of preparing cosmetics, and can be broadly interpreted as including the meaning of the topical composition for topical preparation. The cosmetic composition of the present invention can be prepared as any dosage form commonly prepared in the technical field to which the present invention belongs. For example, the above-mentioned cosmetic composition can have a lotion such as a skin softening lotion or a nutritious lotion, a lotion of a spray type, a facial lotion, a body lotion, a nutritious cream, a moisturizing cream, an eye cream, a stick, an essence, a cosmetic ointment, a spray, a gel, a facial mask, a sunscreen, a foundation, a liquid type or aerosol type foundation cream, a powder, a cleansing milk, a cleansing oil, a makeup remover such as a cleansing foam, a soap, a shower gel, etc., but is not limited thereto.

[0078] The cosmetic composition of the present invention can be used according to a common method of use, and the frequency of use can be varied depending on the skin condition or preference of the user.

[0079] In this specification, "quasi-drugs" refer to items that have therapeutic, mitigating, treating or preventing effects on diseases, or items that have less effects on the body than drugs. Excluding items used for the purpose of drugs according to the Korean Pharmaceutical Affairs Act, including items classified according to the Korean Ministry of Health and Welfare. Specifically, it can be a skin topical agent or a personal hygiene product, but it is not limited to this.

[0080] When the composition of the present invention is added to a quasi-drug for the purpose of wound recovery or regeneration, the composition can be directly added or used together with other quasi-drug ingredients, and can be used appropriately according to the usual method. The mixing amount of the active ingredient can be appropriately determined according to the purpose of use. The above-mentioned skin external preparation can be prepared in the form of, for example, an ointment, emulsion, spray, patch, cream, powder, suspension, gel or gel for use, but is not limited thereto.

[0081] The above-mentioned cosmetic composition or quasi-drug composition also refers to all the contents of the description of the pharmaceutical composition.

[0082] Furthermore, it relates to a method for preparing a composition for treating or restoring wounds, which comprises extracellular vesicles derived from three-dimensional spheroid cell aggregates, comprising: step (a), preparing three-dimensional spheroid cell aggregates by three-dimensionally culturing stem cells in microwells with a diameter of 200 μm to 800 μm and a depth of 100 μm to 1000 μm; and step (b), isolating extracellular vesicles from the above-mentioned three-dimensional spheroid cell aggregates.

[0083] The above-mentioned preparation method can rapidly and in large quantities produce three-dimensional spheroid cell aggregate-derived extracellular vesicles with excellent wound treatment effects in accordance with GMP standards.

[0084] Considering the complexity of the specification, repeated contents will be omitted, and unless otherwise defined, the terms in this specification have the meanings commonly used in the technical field to which the present invention belongs.

[0085] The present invention will be described in detail below by way of examples, but the following examples are only used to illustrate the present invention, and the content of the present invention is not limited to the following examples. DETAILED DESCRIPTION

[0086] Western blotting

[0087] Cells and extracellular vesicles were solubilized in radioimmunoprecipitation assay (RIPA) buffer (25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.5% triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and protease inhibitors). A total of 20 μg of protein was separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). Then, the nitrocellulose membrane was incubated with primary antibodies against histone H2A.Z, histone H3, lamin A / C, raftin-1 (1:1000, Cell signaling technology, Beverly, MA, USA) or calreticulin (1:1000, ThermoFisher Scientific, Inc., Rockford, IL, USA) at 4°C overnight. After washing with Tris-buffered saline-Tween 20, the nitrocellulose membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:1000, anti-rabbit, Cell signaling technology, Beverly, MA, USA) for 2 hours. Proteins were detected using a chemiluminescent substrate from Thermo Fisher Scientific (Waltham, MA, USA). Tagged proteins were visualized by X-ray film (Agfa, Mortsel, Belgium).

[0088] Enzyme-linked immunosorbent assay (ELISA)

[0089] ELISA was performed using commercial kits according to the guidelines of each manufacturer. The following ELISA kits were used: gentamicin (5111GEN, EuroProxima, Arnhem, The Netherlands), bovine albumin (8100, Alpha Diagnostic, San Antonio, TX, USA), Hsp70 (Abcam, Cambridge, UK), CD63, CD9 and CD81 (System Biosciences, Palo Alto, CA, USA), histone H2A.Z. (Mybiosource, San Diego, CA, USA), calreticulin (Mybiosource) and cytochrome C (Thermo Fisher Scientific). All kits contain standard proteins. Therefore, the amount of protein and extracellular vesicles depends on the standard curve of each kit.

[0090] Real-time quantitative polymerase chain reaction (qPCR)

[0091] Utilize Trizol according to the manufacturer's instructions. TM RNA was extracted from extracellular vesicles and quantified by nanodrop. RNA was converted into cDNA by reverse transcription (RT), and then real-time fluorescence quantitative PCR (RT PCR) was performed using oligonucleotide probes (Taqman probes) suitable for each miRNA and mRNA according to the manufacturer's instructions.

[0092] Extracellular vesicle (EV) labeling and cell-based uptake

[0093] Purified extracellular vesicles were stained with 5-(and-6)-Carboxyfluorescein Diacetate, Succinimidyl Ester (CFSE) labeling dye (c-1157, Invitrogen) according to the manufacturer's instructions. Excess dye was removed by ultracentrifugation at 100.000 g for 1 hour. Human NSC cell lines were treated with labeled extracellular vesicles (0.4 μg / ml) After incubation for 24 hours. After treatment, the cells were washed twice with PBS and stained according to the conventional immunocytochemistry protocol. The cells were incubated with mouse anti-SMA (1:100, Sigma aldrich) antibody at 4°C overnight. Then, the cells were washed with PBS and incubated with DyLight-labeled anti-mouse IgG (1:200, 594nm, Abcam) antibody as the secondary antibody. 1.5μg / mL of 4'-6'diamidino-2-phenylindole (DAPI) (Vector Laboratories) and Vectashield TM Nuclear staining was performed and cells were imaged using a confocal microscope (LSM 700, Carl Zeiss, Germany).

[0094] Example 1. Isolation of extracellular vesicles by three-dimensional culture of mesenchymal stem cells

[0095] 1.1. Preparation of Mesenchymal Stem Cells

[0096] Human umbilical cord-derived mesenchymal stem cells (hereinafter, WJ-MSC, Samsung Medical Center, Seoul, Korea) at the 5th passage culture stage were obtained and cultured in a 37°C, 5% CO2 incubator. α-modified eagle's medium (α-MEM, GIBCO, New York (NY), USA) containing 10% fetal bovine serum (FBS) (GIBCO, New York (NY), USA) and 50 μg / mL gentamicin (GIBCO, New York, USA) was used as a growth medium. WJ-MSC at the 6th passage culture stage (passage 6) was used for the preparation of three-dimensional spheroid cell aggregates.

[0097] 1.2. Preparation of 3D Spheroid Cell Aggregate Culture Medium

[0098] The WJ-MSCs at passage 6 prepared in Example 1.1 were washed with PBS and lysed with TrypLE TMExpress, GIBCO, New York, USA) and reacted in a CO2 incubator for 5 minutes. Then, trypsin was neutralized by adding new serum-free medium to recover the cells, and cell pellets were obtained by centrifuge. Then, a cell suspension was prepared by adding new serum-free medium and the cells were counted. After the cell counting was completed, 60 ml of the cell suspension was dispensed into a microwell array at a density of 400 cells / well, wherein the microwell array contained approximately 69,000 microwells with a diameter and depth of 500 μm×200 μm, respectively, and coated with 2-methacryloyloxyethyl phosphorylcholine polymer (2-Methacryloyloxyethyl Phosphorylcholine Polymer, MPC), and then induced to spontaneously form spheroid-type cell aggregates while maintaining a static state, and cultured in a CO2 incubator at 37°C for a total of 4 days to prepare a three-dimensional spheroid-type cell aggregate culture solution (hereinafter, three-dimensional-static-spheroid culture solution).

[0099] 1.3. Isolation of extracellular vesicles from three-dimensional spheroidal cell aggregates

[0100] The three-dimensional static-spheroid culture medium prepared in Example 1.2 was recovered, centrifuged at 2500 g for 10 minutes, and then filtered with a 0.22 μm syringe filter to remove cell impurities. Then, the protein was removed by a tangential flow filtration (TFF) system through a 300 kDa hollow fiber membrane (Pall, New York, USA) to separate the extracellular vesicles from the three-dimensional static-spheroid culture medium for the first time, and then purified again with physiological saline to obtain the three-dimensional static-spheroid-derived extracellular vesicles of the present invention with high purity (hereinafter, three-dimensional static-spheroid extracellular vesicles).

[0101] The processes of the above-mentioned embodiments 1.1 to 1.3 are shown in a graphical manner. Figure 1 middle.

[0102] Example 2. Analysis of the properties of three-dimensional spheroid-type cell aggregates

[0103] The characteristics of the three-dimensional spheroid-type cell aggregates (hereinafter referred to as three-dimensional-static-spheroids) present in the three-dimensional-static-spheroid culture solution prepared in the above-mentioned Example 1.2 were analyzed. The three-dimensional-static-spheroid culture solution prepared in Example 1.2 was used as the experimental group, and the three-dimensional mesenchymal stem cell spheroid-type cell aggregates (hereinafter referred to as three-dimensional-dynamic-PEG spheroids) culture solution prepared by culturing mesenchymal stem cells for 5 days according to the dynamic three-dimensional cell culture method disclosed in the authorized patent (application number 10-2016-0053026, method for producing extracellular vesicles derived from stem cells) was used as the control group. The microwells filled with each culture solution were observed with an optical microscope and are shown in FIG. Figure 2 Part A of Figure 2 Part B shows the change in the area of ​​spheroid cell aggregates after culture compared to the initial culture stage. Figure 2 Part C of

[0104] like Figure 2 As shown, the results of comparing the areas of the three-dimensional-static-spheroids as the experimental group and the three-dimensional-dynamic-PEG spheroids as the control group showed that based on the characteristic of cells tightly aggregating to form spheroids, the area of ​​the three-dimensional-static-spheroids was statistically significantly reduced compared with the spheroids in the initial stage of culture (p=0.0011) compared with the three-dimensional-dynamic-PEG spheroids.

[0105] Example 3. Analysis of the size of three-dimensional spheroid-type cell aggregates

[0106] The size distribution of the three-dimensional static spheroid prepared in Example 1.2 and the three-dimensional dynamic PEG spheroid prepared in Example 3 was determined, and the coefficient of variation, skewness and kurtosis were analyzed based on the determined size distribution data.

[0107] Skewness is a parameter that understands the direction and degree of the distribution tilt based on the median trend. As it approaches 1, it shows a distribution with the tail of the curve extending to the right, and as it approaches -1, it shows a distribution with the tail of the curve extending to the left. In the case of a standard distribution, the skewness is 0.

[0108] Kurtosis is a parameter that indicates the sharpness of data distribution. In the case of a positive value, it means that the central part accumulates relatively more data points, and in the case of a negative value, it means that the central part accumulates relatively fewer data points. In the case of a standard distribution, the kurtosis is 0.

[0109] The size distribution data are shown in Figure 3 The analysis results are shown in Table 1.

[0110] Table 1

[0111]

[0112] like Figure 3 As shown in Table 1, the average size of the three-dimensional static spheroid was confirmed to be 74.43um, and the coefficient of variation (CV) was 9.59%. In contrast, the average size of the three-dimensional dynamic PEG spheroid was confirmed to be 148.66um, and the coefficient of variation (CV) was 14.1%. In order to compare the coefficients of variation measured by the test, the results of the Feltz and Miller's (1996) asymptotic test were performed, and the p value was calculated to be 0.01765604, and the results of the Krishnamoorthy and Lee's (2014) modified signed-likelihood ratio test were performed, and the p value was calculated to be 0.01853969. Therefore, it was confirmed that the size distributions of 3D-static-spheroids and 3D-dynamic-PEG-spheroids showed statistically significant differences in both tests.

[0113] The results of comparing the size distributions of the three-dimensional-static-spheroid and the three-dimensional-dynamic-PEG spheroid confirmed that the kurtosis value of the size distribution of the three-dimensional-static-spheroid was relatively large, and thus it was confirmed that the size distribution of the three-dimensional-static-spheroid of the present invention showed that most of it was concentrated in the median. This result confirmed that when a three-dimensional spheroid is prepared by the three-dimensional-static-spheroid preparation method of the present invention, a three-dimensional spheroid of a relatively specified size can be prepared.

[0114] Example 4. Analysis of the morphology of extracellular vesicles derived from three-dimensional spheroid cell aggregates

[0115] In order to observe the morphology of the three-dimensional-static-spheroid extracellular vesicles isolated in Example 1.3, transmission electron microscopy (TEM) was used for photography. Specifically, the three-dimensional-static-spheroid extracellular vesicles were fixed with 1% OsO4 dissolved in 0.1M phosphate buffer (PB) for 2 hours. The EM grid was adsorbed for 1 minute in such a way that the polymethylvinyl acetate (formvar) side was facing downward on the extracellular vesicle droplet. Then, the liquid was blotted with filter paper and reacted with 2% uranyl acetate for 15 seconds. After removing the excess uranyl acetate, the EM grid was observed using TEM (JEM-1011, JEOL, Japan), and the observed image is shown in Figure 4 middle.

[0116] like Figure 4 As shown, the three-dimensional static spheroidal extracellular vesicles were confirmed to be round, which is a typical morphology of extracellular vesicles.

[0117] Example 5. Analysis of the concentration and size of extracellular vesicles derived from three-dimensional spheroid cell aggregates

[0118] Nanoparticle tracking analysis (NTA) was performed using NanoSight NS300 (Malvern, Worcestershire, UK) to analyze the concentration and size distribution of the three-dimensional-static-spheroid extracellular vesicles isolated in Example 1.3. For optimal analysis, the three-dimensional-static-spheroid extracellular vesicles were pre-diluted in vesicle-free phosphate buffered saline (PBS). The average size and concentration (particle / mL) were calculated from the three records and the results are shown in Figure 5 middle.

[0119] like Figure 5 As shown, it was confirmed that the average particle diameter of the three-dimensional-static-spheroid extracellular vesicles was 182.5 nm and the mode diameter was 106.1 nm.

[0120] Example 6. Analysis of expression markers of extracellular vesicles derived from three-dimensional spheroid cell aggregates

[0121] Experiments were conducted to confirm the expression markers of the three-dimensional-static-spheroid extracellular vesicles isolated in Example 1.3. Cell lysates and secretomes were used as control groups. Cell lysates were prepared by washing the three-dimensional-static-spheroids with PBS, recovering the cells after treatment with trypsin, and then centrifuging the cells to obtain cell pellets. Secretomes were prepared by the following method, that is, after obtaining the above-mentioned cell pellets, extracellular vesicles were separated in the culture medium as the supernatant by the process of Example 1.3 to obtain the remaining culture secretomes. For marker analysis, ELISA was used to quantify the extracellular vesicle-specific positive markers CD9, CD63, CD81 and HSP70, and calreticulin, histone H2A.Z, cytochrome C, albumin, and antibiotics as specific contaminating protein markers were quantified. In addition, histone H2A.Z, histone H3, lamin A / C, and calreticulin, which are specific contaminating protein markers of extracellular vesicles, were quantified by Western blotting, and raftin-1, which is a positive marker for extracellular vesicles, was quantified. The results of ELISA analysis and Western blotting are shown in Figure 6 middle.

[0122] like Figure 6 As shown, it was confirmed that CD9, CD63, CD81 and HSP70, which are specific positive markers for extracellular vesicles, were expressed in all three-dimensional static-spheroid extracellular vesicles, and calreticulin, histone H2A.Z, histone H3, cytochrome C, albumin (BSA, bovine serum albumin), lamin A / C and antibiotics, which are highly expressed contaminating protein markers in cell lysates and secretomes, were hardly expressed. In particular, it was confirmed that Flotilin-1, which is a positive marker for extracellular vesicles and is expressed at a very low level in cell lysates, was relatively highly expressed in three-dimensional static-spheroid extracellular vesicles.

[0123] Example 7. Analysis of the yield of extracellular vesicles from three-dimensional spheroid cell aggregates

[0124] An experiment was conducted to compare the yield of three-dimensional-static-spheroid extracellular vesicles as extracellular vesicles prepared by the preparation method of Example 1, extracellular vesicles isolated from the three-dimensional-dynamic-PEG spheroids of Example 2 (three-dimensional-dynamic-PEG spheroid extracellular vesicles), and extracellular vesicles isolated from stem cells cultured by a conventional two-dimensional culture method (two-dimensional-extracellular vesicles). Two-dimensional-extracellular vesicles were prepared by the following process. Stem cells cultured in a cell culture chamber (Cellstack) for 3 days were washed with PBS, then replaced with serum-free medium and further cultured for 2 days. After recovering the culture medium, cell impurities were continuously removed by centrifugation and using a 0.2μm filter. Then, the protein was removed by a TFF system through a hollow fiber membrane to separate the extracellular vesicles from the culture medium for the first time, and then purified again with physiological saline to obtain a high-purity two-dimensional-extracellular vesicle. The yields of the three-dimensional static spheroid extracellular vesicles-derived cells, the three-dimensional dynamic PEG spheroid extracellular vesicles-derived cells, and the two-dimensional extracellular vesicles-derived cells were compared and are shown in Table 2 and Figure 7 middle.

[0125] Table 2

[0126]

[0127] Example 8. Analysis of the expression of microRNA (miRNA) in extracellular vesicles derived from three-dimensional spheroid cell aggregates

[0128] 8.1. Identification of miRNAs with increased expression in 3D-static-spheroid EVs

[0129] In order to confirm the differences in characteristics between the three-dimensional-static-spheroid extracellular vesicles as extracellular vesicles prepared by the preparation method of Example 1, the three-dimensional-dynamic-PEG spheroid extracellular vesicles as extracellular vesicles separated from the three-dimensional-dynamic-PEG spheroid of Example 2, and the two-dimensional-extracellular vesicles prepared in Example 7, the expression of miRNAs and protein expression were measured by qPCR, and the miRNAs and proteins highly expressed in the three-dimensional-static-spheroid extracellular vesicles compared with the two-dimensional-extracellular vesicles are shown in Figure 8 In the figure, the miRNAs and proteins highly expressed in the three-dimensional-static-spheroidal extracellular vesicles compared with the three-dimensional-dynamic-PEG-spheroidal extracellular vesicles are shown. Fig. 9 middle.

[0130] like Figure 8 As shown, it was confirmed that compared with two-dimensional extracellular vesicles, three-dimensional static-spheroid extracellular vesicles highly expressed miR-27a, miR-146b, and miR-146a as miRNAs that exhibit effects on angiogenesis / neurogenesis and immunomodulation, and highly expressed integrin 1 / 2 and vascular endothelial growth factor receptor (VEGF / R2) as proteins that exhibit effects on angiogenesis / neurogenesis.

[0131] like Fig. 9 As shown, it was confirmed that compared with the three-dimensional-dynamic-PEG spheroid extracellular vesicles, the three-dimensional-static-spheroid extracellular vesicles highly expressed miR-146a, which is a miRNA showing angiogenesis / neurogenesis and immunomodulatory effects, miR-27a, miR-132, miR-184 and miR-210, which are miRNAs showing angiogenesis / neurogenesis effects, and miR-301b showing anti-tumor effects, and highly expressed integrin 1 / 2 and vascular endothelial growth factor receptor, which are proteins showing angiogenesis / neurogenesis effects.

[0132] This result confirmed that the three-dimensional-static-spheroid extracellular vesicles, which are extracellular vesicles prepared by the preparation method of Example 1, are a new type of extracellular vesicles that have different expressions of miRNA and protein compared to two-dimensional extracellular vesicles or three-dimensional-dynamic-PEG spheroid extracellular vesicles, and in particular, highly express miRNAs related to angiogenesis / neurogenesis, immunoregulation, rejuvenation or anti-tumor, and are therefore an extracellular vesicle that is useful for clinical use.

[0133] 8.2. Analysis of miRNA expression differences between different donors

[0134] It is well known that stem cell therapeutics have the problem of donor variation in which the components vary depending on the donor. Experiments were conducted to confirm whether the three-dimensional-static spheroid extracellular vesicles of the present invention show a more consistent miRNA profile without the problem of donor variation. Samples of each donor were obtained and used from Samsung Medical Center (Seoul, South Korea). The two-dimensionally cultured WJ-MSCs of Example 1.1 were compared with the three-dimensionally cultured WJ-MSCs and the three-dimensionally cultured spheroid extracellular vesicles as the extracellular vesicles prepared by the preparation method of Example 1 in terms of the number of extracellular vesicles produced according to different donors, the amount of extracellular vesicle protein, and the miRNA profile. The miRNA profile was analyzed by the small RNA sequencing method, and the results are shown in Figure 10a to Figure 10b middle.

[0135] like Fig.10a As shown, the two-dimensional cultured WJ-MSC-derived two-dimensional-extracellular vesicles showed deviations in the amount, size, and amount of protein produced by the extracellular vesicles according to different donors, but the three-dimensional-static-spheroid extracellular vesicles showed no significant deviation between donors and had consistent results.

[0136] And, if Fig.10b As shown, it was confirmed that the miRNA composition produced by two-dimensional cultured WJ-MSC and two-dimensional extracellular vesicles varies greatly depending on the donor, while on the contrary, the difference between three-dimensional cultured WJ-MSC and three-dimensional static-spheroid extracellular vesicles is reduced depending on the donor. In particular, it was confirmed that three-dimensional static-spheroid extracellular vesicles showed a consistent miRNA profile without differences depending on the donor, and at the same time, compared with two-dimensional extracellular vesicles, they uniformly contained more miR-27a-3p (1.5 times), miR-146a-5p (2.3 times), miR-210 (2.6 times), and miR-132 (2.6 times), which are miRNAs that can show angiogenic effects. Furthermore, it was confirmed that miR-199a, miR-125b, miR-26a, let-7, miR-125a, miR-181b, and miR-92a, which are miRNAs related to angiogenesis, were also expressed at a uniform level among the donors.

[0137] This result indicates that three-dimensional-static-spheroid extracellular vesicles can reduce the differences in therapeutic active ingredients according to different donors and show better therapeutic effects.

[0138] Example 9. Analysis of mRNAs expressed when cells are treated with extracellular vesicles derived from three-dimensional spheroid-type cell aggregates

[0139] After treating 3D-static-spheroid extracellular vesicles as extracellular vesicles prepared by the preparation method of Example 1 and 2D-extracellular vesicles as extracellular vesicles isolated from stem cells cultured by a conventional 2D culture method to HUVECs or primitive neural stem cells as neural stem cells, the expression changes of mRNA were confirmed by qPCR. The results of qPCR are as follows: Fig.11 shown.

[0140] like Fig.11 As shown, it was confirmed that the vascular endothelial cells treated with three-dimensional static-spheroid extracellular vesicles increased the expression of vascular endothelial growth factor, hypoxia-inducible factor-1α and fibroblast growth factor compared with the cells treated with two-dimensional extracellular vesicles, and the pNSCs treated with three-dimensional static-spheroid extracellular vesicles increased the expression of VEGF, BDNF (brain-derived neurotrophic factor), FGF, NGF (nerve growth factor), and HGF (hepatocyte growth factor). In particular, compared with the control group, the pNSCs treated with two-dimensional extracellular vesicles showed a decrease in the expression of NGF and HGF, but the pNSCs treated with three-dimensional static-spheroid extracellular vesicles showed an effect of increasing the expression of NGF and HGF, thus confirming that both three-dimensional static-spheroid extracellular vesicles and two-dimensional extracellular vesicles normally showed other effects.

[0141] VEGF, BDNF, NGF, HGF, and Hif-1a are proteins related to angiogenesis / neurogenesis, and FGF is a factor equivalent to a protein that regulates biological functions related to cell proliferation, survival, and differentiation. Therefore, it was confirmed that three-dimensional-static-spheroid extracellular vesicles with relatively high effects of increasing the expression of VEGF, BDNF, NGF, HGF, Hif-1a, and FGF are excellent extracellular vesicles suitable for clinical use.

[0142] Example 10. Confirmation of the wound treatment effect of extracellular vesicles derived from three-dimensional spheroid cell aggregates on animal models

[0143] It was confirmed through Example 8 that the three-dimensional static-spheroid extracellular vesicles, which are extracellular vesicles prepared by the preparation method of Example 1, are new extracellular vesicles whose expression patterns of miRNA and protein are different from those of existing two-dimensional extracellular vesicles or three-dimensional dynamic-PEG spheroid extracellular vesicles.

[0144] In order to confirm whether the three-dimensional-static-spheroid extracellular vesicles, which are newly prepared extracellular vesicles, show a wound healing effect, a wound animal model was prepared and an experiment for confirming the wound healing effect was performed in the following manner.

[0145] After respiratory anesthesia, 8-week-old SD rats were depilated of the back hair, and the back skin was folded with the midline of the back as the center. The skin was pierced with an 8-mm skin biopsy punch to prepare a wound model. A total of 6 × 10 8 / 100μl of 3D-static-spheroid extracellular vesicles, and the control group was injected with an equal amount of 100ul PBS. In addition, in order to compare the wound treatment effects of 3D-static-spheroid extracellular vesicles and WJ-MSC, the WJ-MSC-administered group was set as a comparative experimental group. After skin wounds were prepared on the backs of rats, 2×10 6 Cells / 100μl of WJ-MSC were injected and observed for 2 weeks. The injection was performed once a day for 3 days. In order to prevent the wound from shrinking, a 6.0 nylon thread was used to seal and fix a 10mm diameter silicone sheet on the wound. The results of wound recovery after injection are shown in the figure. Fig.12 shown.

[0146] like Fig.12 As shown in Figure 2, compared with the control group and the WJ-MSC treatment group, the three-dimensional static-spheroid extracellular vesicle treatment group still showed a more rapid wound recovery effect. The results of quantifying the difference in wound recovery effect between the three-dimensional static-spheroid extracellular vesicle and the control group are shown in Figure 2. Fig.13 As shown, it was confirmed that in the three-dimensional static-spheroid extracellular vesicle treatment group, a significantly faster wound recovery effect was shown than in the control group starting from the fourth day after the start of treatment.

[0147] The wound healing effect of 3D-static-spheroid extracellular vesicles was confirmed by hematoxylin and eosin staining (H&E staining) of the skin cross-sectional area. After preparing the aforementioned rat wound model, the cross-sectional area of ​​the skin where the wound occurred was stained on the third, seventh, and fourteenth days of 3D-static-spheroid extracellular vesicle treatment, and the thickness of the epidermis in the skin was measured after 14 days of treatment.

[0148] like Fig.14 As shown, it was confirmed that skin was rapidly generated in the three-dimensional-static-spheroid extracellular vesicle treatment group. Fig.15 As shown, after 14 days of treatment, a significant increase in epidermal thickness was confirmed in the 3D-static-spheroid extracellular vesicles-treated group.

[0149] More specifically, the thickness of the epidermis and the surrounding area of ​​the epidermis is measured and the result is digitized as follows: Fig.16 shown.

[0150] like Fig.16 As shown, it was confirmed that three-dimensional static spheroid extracellular vesicles can quickly heal wounds by rapidly increasing skin thickness in the epidermis and the peri-epidermal area.

[0151] Example 11. Wound treatment effect of the chamber model of extracellular vesicles derived from three-dimensional spheroid cell aggregates

[0152] The wound healing effect of extracellular vesicles derived from three-dimensional spheroid cell aggregates was confirmed in a chamber model. A rat skin wound model was prepared. The experimental method was as follows Fig.17 shown.

[0153] After the chamber was removed, the wound recovery effect was photographed every day. Fig.18 As shown in the figure, the result of quantifying the size change of the wound site is as follows Fig.19 shown.

[0154] like Fig.18 and Fig.19 As shown, significant differences in the wound recovery area were confirmed on the third and tenth days after the removal of the chamber, and it was confirmed that the wound recovery speed after the removal of the chamber was faster in the group treated with extracellular vesicles derived from three-dimensional spheroid cell aggregates.

[0155] The thickness of the epidermis was confirmed by hematoxylin and eosin staining in the chamber model. Fig. 20 shown.

[0156] like Fig. 20 As shown, on the seventh day after the chamber was removed, an increase in the thickness of the epidermis layer was confirmed in the three-dimensional spheroid-type cell aggregate-derived extracellular vesicles-treated group compared with PBS, and on the fourteenth day thereafter, the epidermis thickness remained at a similar level.

[0157] Additionally, in the chamber model, the expression changes of VEGF, Angpt-2, IL-1b and IL-10 were confirmed by ELISA on the seventh or fourteenth day after the chamber was removed. Figures 21a to 21c shown.

[0158] like Figures 21a to 21cAs shown, in the three-dimensional spheroid cell aggregate-derived extracellular vesicles treatment group, it was confirmed that on the seventh and fourteenth days after the removal of the chamber, the expression of VEGF, Angpt-1, and Angpt-2 related to angiogenesis increased significantly, the expression of IL-10 related to anti-inflammation increased, and the expression of IL-1b, TNF-a, and IL-6 related to verification decreased.

[0159] Example 11. Confirmation of cell migration in the group treated with extracellular vesicles derived from three-dimensional spheroid cell aggregates

[0160] After preparing the scratch model, the migration of fibroblasts and keratinocytes treated with the three-dimensional spheroid cell aggregate-derived extracellular vesicles of the present invention was confirmed. The results are as follows: Fig.22a and Figure 22b shown.

[0161] like Fig.22a and Figure 22b As shown, the migration of fibroblasts and keratinocytes in the group treated with three-dimensional spheroid-type cell aggregates was significantly increased, and it was confirmed that rapid wound recovery can be achieved by this.

[0162] Example 12: Confirmation of the angiogenesis-promoting effect of extracellular vesicles derived from three-dimensional spheroid cell aggregates

[0163] After treating vascular endothelial cells with three-dimensional static spheroid extracellular vesicles, which are extracellular vesicles prepared by the preparation method of Example 1, and two-dimensional extracellular vesicles, which are extracellular vesicles isolated from stem cells cultured by a conventional two-dimensional culture method, the expression changes of VEGF, Hif-1a, and FGF, which are angiogenesis-related factors, were confirmed. The results were as follows: Fig.23 shown.

[0164] Furthermore, in order to confirm the effect of extracellular vesicles obtained by three-dimensional culture of stem cells on the proliferation of new blood vessels, the angiogenic effect was confirmed by treating HUVECs adhered to Matrigel with WJ-two-dimensional extracellular vesicles and the three-dimensional static-spheroid extracellular vesicles of the present invention. Specifically, HUVECs were cultured in M199 medium (Gibco) supplemented with 20% FBS, 5 U / mL heparin, and 3 ng / mL bFGF. The cells were cultured at 1.7×10 4The cells were seeded at a density of 10 cells / mL on growth factor-reduced Matrigel Matrix (BD Bioscience, Massachusetts, USA) on angiogenesis slides (μ-Slides Angiogenesis) (ibidi, Graefelfing, Germany) and then allowed to form blood vessels for 4 hours in a humidified chamber at 37°C and 5% CO2. Images were taken under a phase contrast microscope (Olympus), and the number of tubular structures under the microscope field (4x magnification) was quantified using ImageJ software. The results are shown in Table 1. Fig.24 shown.

[0165] like Fig.23 As shown, it was confirmed that the expression of VEGF, Hif-1a, and FGF in vascular endothelial cells treated with three-dimensional static-spheroid extracellular vesicles increased compared with cells treated with two-dimensional extracellular vesicles. VEGF and Hif-1a are proteins related to angiogenesis, and FGF is a factor equivalent to a protein that regulates biological functions related to cell proliferation, survival, and differentiation, thus confirming that three-dimensional static-spheroid extracellular vesicles are excellent extracellular vesicles suitable for clinical angiogenesis.

[0166] like Fig.24 As shown, it was confirmed that the angiogenesis was significantly increased in the experimental group treated with the three-dimensional-static-spheroid extracellular vesicles of the present invention compared with the angiogenesis-related protein VEGF-treated group, and still showed an excellent angiogenesis effect compared with the WJ-two-dimensional-extracellular vesicle-treated group.

[0167] Example 13. Examination of the effect of three-dimensional-static-spheroid extracellular vesicles according to preparation conditions

[0168] 13.1. Experimental methods and conditions

[0169] The above examples confirm the excellent effects of the three-dimensional-static-spheroid extracellular vesicles of the present invention. In order to confirm whether the same effect is exhibited in the extracellular vesicles prepared by the same preparation method as in Example 1 but with changed micropore specifications and cell number conditions, the cell number specification of each micropore is changed from 400 cells / well to 200 cells / well, or the diameter and depth specifications of the micropore are changed from 500μm×200μm to 500μm×600μm, or changed to a flat pore with a diameter of 800μm and no depth, and then the cells are cultured.

[0170] The experimental conditions changed in the preparation method of Example 1 are shown in Table 3 below.

[0171] Table 3

[0172]

[0173] The WJ-MSCs at passage 6 prepared in Example 1.1 were washed with PBS and then lysed with TrypLE TM Express, GIBCO, New York, USA) and reacted in a CO2 incubator for 5 minutes. Then, trypsin was neutralized by adding new serum-free medium to recover cells, and then cell pellets were obtained by centrifugation. Then, a cell suspension was prepared by adding new serum-free medium and then the cells were counted. After the cell counting was completed, the cells were evenly dispensed into the microwells under the same conditions as in Table 4 above, and spontaneous formation of spheroid-type cell aggregates was induced while maintaining a static state, and cultured in a CO2 incubator at 37°C for a total of 4 days to prepare a three-dimensional spheroid-type cell aggregate culture solution.

[0174] 13.2. Confirmation of three-dimensional spheroid-type cell aggregate morphology

[0175] It was confirmed that, similar to Example 1, spheroids were uniformly formed under the conditions of Experimental Examples 1 to 3, and the culture medium of three-dimensional spheroid-type cell aggregates was recovered and spheroid-derived extracellular vesicles were obtained by the method of Example 1.3. The size distribution of the obtained extracellular vesicles was measured, and the roundness and firmness of the spheroids were additionally confirmed. The results were as follows: Fig.25 shown.

[0176] like Fig.25 As shown, it was confirmed that the sizes of the three-dimensional spherical cell aggregates prepared in Experimental Examples 1 to 3 were all in the range of 55 μm to 131 μm, which was the size range of the cell aggregates prepared in Example 1, and the average value of the size distribution was 70.34 μm to 99.51 μm. Furthermore, the results of confirming the roundness and firmness showed that, similar to the average roundness value of 0.8697 (CV 2.64%) of the three-dimensional spheroidal cell aggregates in Example 1, the spheroidal cell aggregates of Experimental Examples 1 to 3 also showed average roundness values ​​of 0.8751, 0.8669, and 0.8601, respectively. Similar to the average firmness value of 0.9488 (CV 2.64%) of the three-dimensional spheroidal cell aggregates in Example 1, the spheroidal cell aggregates of Experimental Examples 1 to 3 also showed average firmness values ​​of 0.9744, 1, and 0.9752, respectively.

[0177] This result indicates that even if the number of cells per microwell is changed to 200, 400, and the diameter of the microwell is changed to 400 to 800, three-dimensional spheroid-type cell aggregates having similar morphology can be effectively formed by the method of Example 1.

[0178] 13.3. Comparison of miRNA expression patterns in 3D spheroid-derived extracellular vesicles

[0179] Since it was confirmed that spheroid cell aggregates with a similar morphology to Example 1 can be prepared under the conditions of Experimental Examples 1 to 3, extracellular vesicles were isolated and obtained from them by the method of Example 1.3, and it was further confirmed whether the isolated extracellular vesicles also showed the same miRNA expression pattern. The comparison of miRNA expression patterns was confirmed using extracellular vesicles derived from spheroid cell aggregates prepared by the methods of Experimental Examples 1 and 2 with changed micropore conditions. Expression miRNA analysis was confirmed by the same qPCR method as Example 8. The results are shown in Fig.26 The two-dimensional extracellular vesicles prepared in Example 7 and the miRNA expression patterns were compared.

[0180] like Fig.26 As shown, it was confirmed that, compared with the existing two-dimensional extracellular vesicles, even if the conditions of the micropores were changed to 500 μm and 800 μm in diameter, the expression of miR-132 and miR-210, which are miRNAs showing efficacy in angiogenesis / neurogenesis and immunomodulation, was significantly increased, similar to the extracellular vesicles prepared in Example 1. This result shows that the three-dimensional spheroid-type cell aggregate-derived extracellular vesicles obtained by the method of the present invention using micropores with a diameter of 200 μm to 800 μm can collectively express miRNA marker expression characteristics. Therefore, they can all be called three-dimensional-static-spheroid extracellular vesicles.

[0181] 13.4. Confirmation of the angiogenic effect of 3D spheroid-derived extracellular vesicles

[0182] An experiment was conducted to confirm the angiogenic effect of three-dimensional static spheroid extracellular vesicles obtained by the method of the present invention using micropores with a diameter of 200 μm to 800 μm. Three-dimensional static spheroid extracellular vesicles prepared under the conditions of Experimental Examples 1 and 2 and Example 1 and two-dimensional extracellular vesicles prepared in Example 7 were used as experimental groups, and the angiogenic effect was confirmed by the same method as Example 11. The results are shown in Fig. 27 middle.

[0183] like Fig. 27As shown, compared with the control group and the two-dimensional extracellular vesicles, the three-dimensional static spheroid extracellular vesicles prepared under the conditions of Experimental Examples 1, 2 and Example 1 all showed significantly excellent angiogenesis effects.

[0184] Based on the above results, it was confirmed that three-dimensional-static-spheroid extracellular vesicles have an outstanding effect in promoting angiogenesis, and through this, even compared with other extracellular vesicles such as WJ-two-dimensional-extracellular vesicles, excellent wound recovery can be achieved.

[0185] Above, specific parts of the present invention are described in detail, but it is obvious to those skilled in the art that such specific description is only a preferred embodiment, and the scope of the present invention is not limited thereto. Therefore, the essential scope of the present invention should be defined by the attached invention claims and their equivalents.

Claims

1. A pharmaceutical composition for treating or restoring wounds, characterized in that: The invention comprises three-dimensional spheroid cell aggregate-derived extracellular vesicles prepared by the following method: Step (a), three-dimensionally culturing stem cells in microwells with a diameter of 200 μm to 800 μm and a depth of 100 μm to 1000 μm to prepare three-dimensional spheroid cell aggregates; as well as Step (b), isolating extracellular vesicles from the three-dimensional spheroid cell aggregates.

2. The pharmaceutical composition for treating or restoring wounds according to claim 1, characterized in that: The stem cells are at least one selected from the group consisting of mesenchymal stem cells, pluripotent stem cells, induced pluripotent stem cells, and embryonic stem cells.

3. The pharmaceutical composition for treating or restoring wounds according to claim 1, characterized in that: In the above step (a), the three-dimensional culture is a static culture.

4. The pharmaceutical composition for treating or restoring wounds according to claim 1, characterized in that: In the above step (a), mesenchymal cells are dispensed into microwells at a density of 100 to 1000 cells / well to perform three-dimensional culture.

5. The pharmaceutical composition for treating or restoring wounds according to claim 1, characterized in that: Compared with the extracellular vesicles derived from spheroid-type cell aggregates of three-dimensional dynamic cultured mesenchymal stem cells, the extracellular vesicles highly express one or more selected from the group consisting of miR-146a, miR-27a, miR-132, miR-184, miR-210 and miR-301b; or Compared with extracellular vesicles derived from mesenchymal stem cells in two-dimensional culture, the extracellular vesicles highly express one or more selected from the group consisting of miR-27a, miR-146a, and miR-146b.

6. The pharmaceutical composition for treating or restoring wounds according to claim 1, characterized in that: The extracellular vesicles described above enhance the migration of fibroblasts or keratinocytes at the wound site.

7. The pharmaceutical composition for treating or restoring wounds according to claim 1, characterized in that: These extracellular vesicles promote angiogenesis at the wound site.

8. A cosmetic composition for treating or restoring wounds, characterized in that: The invention comprises three-dimensional spheroid cell aggregate-derived extracellular vesicles prepared by the following method: Step (a), three-dimensionally culturing stem cells in microwells with a diameter of 200 μm to 800 μm and a depth of 100 μm to 1000 μm to prepare three-dimensional spheroid cell aggregates; as well as Step (b), isolating extracellular vesicles from the three-dimensional spheroid cell aggregates.

9. A quasi-drug composition for treating or restoring a wound, characterized in that: The invention comprises three-dimensional spheroid cell aggregate-derived extracellular vesicles prepared by the following method: Step (a), three-dimensionally culturing stem cells in microwells with a diameter of 200 μm to 800 μm and a depth of 100 μm to 1000 μm to prepare three-dimensional spheroid cell aggregates; as well as Step (b), isolating extracellular vesicles from the three-dimensional spheroid cell aggregates.

10. A method for preparing a composition for treating or restoring wounds comprising extracellular vesicles derived from three-dimensional spheroid cell aggregates, comprising: Step (a), three-dimensionally culturing stem cells in microwells with a diameter of 200 μm to 800 μm and a depth of 100 μm to 1000 μm to prepare three-dimensional spheroid cell aggregates; as well as Step (b), isolating extracellular vesicles from the three-dimensional spheroid cell aggregates.

11. A method for treating wounds, characterized in that: include: Step (a), three-dimensionally culturing stem cells in microwells with a diameter of 200 μm to 800 μm and a depth of 100 μm to 1000 μm to prepare three-dimensional spheroid cell aggregates; Step (b), preparing three-dimensional spheroid cell aggregate-derived extracellular vesicles by separating extracellular vesicles from the three-dimensional spheroid cell aggregates; as well as Step (c), administering the three-dimensional spheroid cell aggregate-derived extracellular vesicles prepared in step (b) above to an individual in need thereof.

Images