A wound treatment composition containing extracellular vesicles derived from stem cells with enhanced efficacy

Abstract

The present invention relates to a wound treatment or wound healing composition containing extracellular vesicles derived from three-dimensional spheroid-shaped cell aggregates produced by a novel production method, and a wound treatment method. The extracellular vesicles produced by the novel method of the present invention can rapidly promote wound healing, and thus can be used as various pharmaceuticals, cosmetics, and foods for wound treatment, regeneration, and recovery.

Description

Technical Field

[0001] The present invention relates to a wound healing composition containing extracellular vesicles derived from three-dimensional spheroid cell aggregates produced by a novel production method.

Background Art

[0002] In various diseases, positive clinical results have been reported for treatment methods using stem cells, particularly mesenchymal stem cells (MSCs). However, stem cell therapeutics have risks of cell-related side effects such as vascular occlusion, tumor formation, and coagulation disorders as side effects, and the reality is that verification of efficacy by clinical trials is still necessary. It is known that the paracrine effect of stem cells induces the regeneration of peripheral skin cells and the enhancement of angiogenesis ability. In particular, extracellular vesicles (EVs) are known as effective factors for the main paracrine effect. Extracellular vesicles are classified into exosomes and microvesicles according to size. Exosomes have a diameter of 30 to 150 nm, and microvesicles have a size of 100 to 1,000 nm. Extracellular vesicles are released into the blood when a part of the cell membrane is released, contain both proteins and nuclear components, and are known to mediate cell-to-cell communication. Using extracellular vesicles instead of stem cells not only minimizes the side effects caused by the use of stem cells and enhances safety but is also advantageous in terms of biodistribution and production process in vivo. However, mass production and acquisition methods for using extracellular vesicles derived from stem cells have not yet been established, and there has been little research on methods that can further enhance the efficacy of extracellular vesicles while maintaining the characteristics of stem cell-derived extracellular vesicles. Therefore, there is a need for extracellular vesicles with further enhanced efficacy and novel therapeutic agents using the same.

Summary of the Invention

Problems to be Solved by the Invention

[0003] Therefore, the present inventors have studied a method that enables mass production and shortens the culture time in order to produce three-dimensional spheroid-type cell aggregates or extracellular vesicles derived therefrom. As a result, three-dimensional spheroid-type cell aggregates (3D-static-spheroids) were produced by static culture using microwells, and it was confirmed that extracellular vesicles with improved wound regeneration and wound healing ability were produced when extracellular vesicles were produced using these. Thus, the present invention has been completed.

[0004] Accordingly, an object of the present invention is to provide extracellular vesicles derived from three-dimensional spheroid-type cell aggregates with improved wound treatment ability, and a composition for wound treatment, regeneration, or recovery containing the same.

Means for Solving the Problems

[0005] To achieve the above object, the present invention provides a pharmaceutical composition for wound treatment or wound recovery, comprising extracellular vesicles derived from three-dimensional spheroid-type cell aggregates produced through: (a) three-dimensionally (3D, 3 dimension) culturing stem cells in microwells having a diameter of 200 to 800 μm and a depth of 100 to 1000 μm to produce three-dimensional spheroid-type cell aggregates; and (b) separating extracellular vesicles from the three-dimensional spheroid-type cell aggregates.

[0006] The present invention also provides a cosmetic composition for wound treatment or wound recovery, comprising extracellular vesicles derived from three-dimensional spheroid-type cell aggregates produced through: (a) three-dimensionally (3D, 3 dimension) culturing stem cells in microwells having a diameter of 200 to 800 μm and a depth of 100 to 1000 μm to produce three-dimensional spheroid-type cell aggregates; and (b) separating extracellular vesicles from the three-dimensional spheroid-type cell aggregates.

[0007] In addition, the present invention provides a pharmaceutical cosmetic composition for wound treatment or wound recovery, which contains extracellular vesicles derived from a three-dimensional spheroid-type cell aggregate produced through: (a) culturing stem cells in a three-dimensional (3D) manner in a microwell having a diameter of 200 to 800 μm and a depth of 100 to 1000 μm to produce a three-dimensional spheroid-type cell aggregate; and (b) separating extracellular vesicles from the three-dimensional spheroid-type cell aggregate.

[0008] In addition, the present invention provides a method for producing a composition for wound treatment or wound recovery, which contains extracellular vesicles derived from a three-dimensional spheroid-type cell aggregate and includes: (a) culturing stem cells in a three-dimensional (3D) manner in a microwell having a diameter of 200 to 800 μm and a depth of 100 to 1000 μm to produce a three-dimensional spheroid-type cell aggregate; and (b) separating extracellular vesicles from the three-dimensional spheroid-type cell aggregate.

[0009] In addition, the present invention provides a method for treating a wound, which includes: (a) culturing stem cells in a three-dimensional (3D) manner in a microwell having a diameter of 200 to 800 μm and a depth of 100 to 1000 μm to produce a three-dimensional spheroid-type cell aggregate; (b) producing extracellular vesicles derived from the three-dimensional spheroid-type cell aggregate through separating extracellular vesicles from the three-dimensional spheroid-type cell aggregate; and (c) treating an object in need thereof with the extracellular vesicles derived from the three-dimensional spheroid-type cell aggregate produced through the step (b).

Effects of the Invention

[0010] The extracellular vesicles produced by the novel method of the present invention can rapidly promote wound recovery, and thus can be used as various pharmaceuticals, cosmetics, and foods for wound treatment, regeneration, and recovery.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0012] The present invention provides a pharmaceutical composition for wound treatment or wound recovery, which contains extracellular vesicles derived from three-dimensional spheroid-type cell aggregates produced through: (a) culturing stem cells in a three-dimensional (3D) manner in microwells with a diameter of 200 to 800 μm and a depth of 100 to 1000 μm to produce three-dimensional spheroid-type cell aggregates; and (b) separating cells and vesicles from the three-dimensional spheroid-type cell aggregates.

[0013] In the present invention, the cells can be used without limitation as long as they are cells capable of separating extracellular vesicles, and may be cells separated from objects of natural organisms. Further, the cells may be derived from any type of animal or plant including humans and non-human mammals, and may be various types of immune cells, tumor cells, or stem cells. Preferably, the stem cells may be mesenchymal stem cells, pluripotent stem cells, induced pluripotent stem cells, or embryonic stem cells.

[0014] In the present invention, the three-dimensional culture means culturing in a three-dimensional arrangement in vitro. Different from two-dimensional culture, during three-dimensional culture, cells can grow in all directions in vitro, and an environment more similar to the in vivo cell environment can be created.

[0015] In the present invention, the three-dimensional cell culture in the 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 may be cell culture using microwell array culture, porous microparticle culture, hanging drop culture, low attachment plate culture, membrane-based cell-detachment culture, thermal lifting culture, centrifugal culture, semisolid medium culture, etc. Preferably, the three-dimensional cell culture may be dynamic culture or static culture, and more preferably, it may be static culture. In the present invention, when the three-dimensional cell culture in the step (a) is static culture, since it does not require the device necessary for shaking culture, the culture can be made easier, and large-scale culture can be enabled in a GMP (Good manufacturing practices, standards for the manufacture and quality control of excellent pharmaceuticals) manufacturing facility.

[0016] In the present invention, the three-dimensional cell culture in the step (a) may be cultured for 1 to 10 days, and preferably, it may be cultured for 2 to 4 days. In particular, in the present invention, when the culture in the step (a) is cultured for 2 to 4 days, the survival rate of the cells present in the three-dimensional spheroid-type cell aggregate is maintained at a high level, and the culture time is relatively short compared to the existing manufacturing process of three-dimensional spheroid-type cell aggregates, and three-dimensional spheroid-type cell aggregates and extracellular vesicles derived therefrom can be manufactured rapidly.

[0017] In the present invention, the three-dimensional cell culture in the step (a) may be to dispense mesenchymal stem cells into microwells at a density of 100 to 1000 cells / well and culture them, preferably, to dispense them at a density of 100 to 600 cells / well and culture them, and more preferably, to dispense them at a density of 100 to 500 cells / well and culture them.

[0018] In the present invention, the step of separating the extracellular vesicles in the step (b) may be by physical separation or chemical separation. The physical separation may be extrusion of a sample containing cells or cell aggregates, and the chemical separation may be treatment with a chemical substance capable of separating extracellular vesicles from cells or cell aggregates. For example, the separation step of the present invention can be carried out using a method selected from the group consisting of sonication, cell lysis, homogenization, freeze-thaw, electroporation, chemical substance treatment, mechanical disruption, and treatment with physical stimuli that externally apply force to cells, and a physicochemical method of separating through the binding of ions and specific biomarkers by ion or affinity chromatography, or immunoaffinity EV capture of hansabiomed life sciences, exosome purification reagent of creative biolabs, ExoCAS-2 reagent or method of microgentas, which destroys cells through polymers and reagents and binds and separates only extracellular vesicles. Preferably, it may be separated by a tangential flow filtration (TFF) method, but is not limited thereto.

[0019] In the present invention, MicroRNA is named by prefixing "mir" and appending "-" and a number. At this time, the number sometimes represents the order of naming. For example, mir-123 was named earlier than mir-156 and is expected to have been discovered earlier. "mir-" indicates pre-microRNA, and "miR" containing capital letters means mature microRNA. Except for one or two sequences, microRNAs with almost the same sequence are named with lowercase letters added. For example, miR-121a and miR-121b are generated from their respective precursors, mir-121a and mir-121b, and their sequences are also very similar. Although the mature microRNAs are the same, pre-microRNAs located at different sites on the genome are named by further appending "-" and a number. As an example, the pre-microRNAs mir-121-1 and mir-121-2 become the same mature microRNA (miR-121) but are located at different sites on the genome. The naming of microRNAs by species is indicated in the front. For example, hsa-miR-123 is a human (Homo sapiens) microRNA, and oar-miR-123 is a sheep (Ovis aries) microRNA. "v" means viral (miRNA encoded by a viral genome), and "d" means Drosophila microRNA. When two mature microRNAs are derived from different arms (3'arm or 5'arm) of the same pre-microRNA, "-3p" or "-5p" is appended at the back for naming. miR-142-3p is derived from the 3'arm, and miR-142-5p is derived from the 5'arm.

[0020] The nomenclature of MicroRNA generally follows the above criteria, but there are also exceptions.

[0021] In the present invention, the extracellular vesicles can be those that highly express various substances exhibiting wound healing efficacy as compared to known extracellular vesicles. For example, preferably, the extracellular vesicles of the present invention highly express one or more selected from the group consisting of miR-146a, miR-27a, miR-132, miR-184, miR-210, and miR-301b as compared to extracellular vesicles derived from spheroid-type cell aggregates obtained by three-dimensional dynamic culture of mesenchymal stem cells; or highly express one or more selected from the group consisting of miR-27a, miR-146a, and miR-146b as compared to extracellular vesicles derived from mesenchymal stem cells cultured in two dimensions, or may highly express VEGF (Vascular endothelial growth factor), Hif-1a (Hypoxia-inducible factor 1-alpha), and FGF (Fibroblast growth factor).

[0022] In addition, the extracellular vesicles can be those that are incorporated into cells and internalized when treated with cells. When incorporated into cells and internalized, clinically significant substances highly expressed in the extracellular vesicles can be effectively delivered to the cells and can be highly expressed within the cells.

[0023] In the present invention, extracellular vesicles derived from three-dimensional spheroid-type cell aggregates exhibiting wound healing, regeneration, and recovery effects can be used interchangeably with "3D-static-spheroid-EV". In addition, extracellular vesicles derived from spheroid-type cell aggregates obtained by three-dimensional dynamic culture as compared thereto can be used interchangeably with "3D-dynamic-PEG-spheroid-EV".

[0024] The "extracellular vesicles derived from spheroid-type cell aggregates obtained by three-dimensional dynamic culture of mesenchymal stem cells" in the present invention may include, without limitation, extracellular vesicles separated from spheroid-type cell aggregates obtained by three-dimensional dynamic culture of mesenchymal stem cells, and preferably, may be extracellular vesicles disclosed in Korean Registered Patent (Application No. 10-2016-0053026, Method for producing extracellular vesicles derived from stem cells).

[0025] In the present invention, the three-dimensional spheroid-type cell aggregate may have an average diameter of 74.43 ± 7.756 μm, preferably having a size range of 55 to 95.0 μm. As a result of measuring the size distribution of 155 of the three-dimensional spheroid-type cell aggregates, the average diameter may be 74.43 μm and the coefficient of variation (CV) may be 9.59%. The three-dimensional spheroid-type cell aggregate in the present invention can have a higher kurtosis of the size distribution than the "spheroid-type cell aggregate obtained by three-dimensionally culturing mesenchymal stem cells". Therefore, the size of the three-dimensional spheroid-type cell aggregate can be smaller in size compared to the "spheroid-type cell aggregate obtained by three-dimensionally culturing mesenchymal stem cells" and can have a relatively uniform size distribution.

[0026] In the present invention, the microwell may be coated with any one selected from the group consisting of TMSPMA (3-(Trimetoxysily)propylmethacrylate), HEA (Hydroxyethyl acrylate), GMA (Glycidyl methacrylate), EGDMA (diethyleneglycol dimethacrylate), THFA (Tetrahydrofurfuryl acrylate), HMAA (Hydroxymethul acrylamide), PEA (Phenyl epoxyacrylate), HOFHA (6-Hydroxy-2,2,3,3,4,4,5,5-octafluoro), EOPT (Polyethoxylated(4)pentaerythritoltetraacrylate), HPA (Hydroxypropyl acrylate), BMA (Buthylmethacrlate), PETIA (Pentaerythritol triacrylate), HDDA (Hexan diol diacrylate), EGPEA (Ethyleneglycol phenyletheracrylate), BM (Benzylmethacrylate), HPPA (Hydroxyphenoxypropyl acrylate), BHPEA (2-(4-Benzoyl-3-hydroxyphenoxy)ethylacrylate), HEMA (Hydroxyethyl methacrylate), HPMA (N-(2-Hydroxypropyl)methacrylamide), and MPC (2-Methacryloyloxyethyl Phosphorylcholine Polymer). Preferably, it may be coated with MPC (2-Methacryloyloxyethyl Phosphorylcholine Polymer), but is not limited thereto.

[0027] The microwell in the present invention may have a diameter of 200 to 800 μm, preferably 300 to 800 μm, more preferably 400 to 800 μm.

[0028] Further, the microwell may be a microwell having a flat structure without depth, or in the case of a microwell forming a depth, it may have a structure with a depth of 100 to 1000 μm, preferably 100 to 900 μm, more preferably 200 to 900 μm.

[0029] The mesenchymal stem cells cultured by the above structure can maintain a high level of viability even after the passage of the culture time. Preferably, a microarray containing 1,000 to 100,000 of the microwells is prepared, and the production yield of cell aggregates can be increased.

[0030] If extracellular vesicles are produced by the "method for producing extracellular vesicles derived from three-dimensional spheroid-type cell aggregates" in the present invention, in addition to the advantages in production by static culture, extracellular vesicles with improved wound healing ability can be rapidly and efficiently mass-produced. In particular, the method for producing extracellular vesicles in the present invention is characterized by being suitable for GMP application.

[0031] In the present invention, extracellular vesicles derived from three-dimensional spheroid-type cell aggregates can induce rapid wound healing when treated on a wound, enhance the mobility of fibroblasts and / or keratinocytes at the wound site in the early stage of wound occurrence, and promote angiogenesis, thereby inducing rapid regeneration and recovery of the wound site.

[0032] In the present invention, angiogenesis means forming new blood vessels (angiogenesis), inducing or increasing the migration of vascular endothelial cells, promoting tube formation by vascular endothelial cells, and meaning the process of forming new blood vessels from existing blood vessels.

[0033] The wound to which the extracellular vesicles derived from the three-dimensional spheroid-type cell aggregates of the present invention are applied can include, without limitation, any form of wound occurring on the skin, and can be applied to various skin wounds such as burns, bedsores, and trauma. A burn means damage to skin cells caused by fire or heat, and a bedsore means a chronic ulcer that occurs when blood circulation is not smooth and tissue dies due to inflammation and tissue necrosis. Trauma means damage to skin tissue by external pressure, and can include, for example, abrasions, contusions, lacerations, incisions by a blade, puncture wounds, incised wounds, gunshot wounds, explosive wounds, bite wounds, etc.

[0034] In addition to the above active ingredient, the pharmaceutical composition in the present invention may further contain appropriate carriers, excipients, and diluents commonly used in the manufacture of pharmaceutical compositions. The pharmaceutical composition in the present invention may further contain other pharmaceutically active ingredients or active mixtures.

[0035] The pharmaceutical composition in the present invention can be formulated and used in the form of oral dosage forms such as patches, coatings, powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injection solutions, respectively, by conventional methods. Examples of carriers, excipients, and diluents that can be included in the composition are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulating, it is usually prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. Such solid preparations are prepared by mixing at least one or more excipients, such as starch, calcium carbonate, sucrose, or lactose, gelatin, etc., in the above composition. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, internal liquids, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, such as wetting agents, sweeteners, fragrances, preservatives, etc., can be included. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. As non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc., can be used. As the base of suppositories, Witepsol, macrogol, Tween 61, cocoa butter, laurin fat, glycerogelatin, etc., can be used.

[0036] The preferred dosage of the pharmaceutical composition in the present invention varies depending on the patient's condition and body weight, the degree of the disease, the drug form, the administration route, and the period, but can be appropriately selected by those skilled in the art. The administration may be carried out once a day or divided into several times. The above dosage does not limit the scope of the present invention in any way. The pharmaceutical composition in the present invention can be administered to mammals such as rats, mice, livestock, and humans by various routes. All modes of administration can be anticipated. For example, it can be administered orally, transdermally, rectally, or by intravenous, intramuscular, or subcutaneous injection. The definitions of the terms such as excipients, binders, disintegrants, lubricants, flavoring agents, and flavoring agents in the present invention are those described in the literature known in the art and include those having the same or similar functions.

[0037] The present invention also provides a wound treatment method including: (a) culturing stem cells in a three-dimensional (3D) microwell with a diameter of 200 to 800 μm and a depth of 100 to 1000 μm to produce a three-dimensional spheroid-type cell aggregate; (b) separating cells and vesicles from the three-dimensional spheroid-type cell aggregate to produce extracellular vesicles derived from the three-dimensional spheroid-type cell aggregate; and (c) treating an object in need of extracellular vesicles derived from the three-dimensional spheroid-type cell aggregate produced in step (b).

[0038] The object may be a mammal including a human, and may include a patient in need of wound treatment, a patient who has received or needs to receive treatment for treating a wound. The extracellular vesicles derived from the three-dimensional spheroid-type cell aggregate can be treated in combination with other drugs or treatment methods. When treated in combination with other drugs or treatment directions, they can be treated simultaneously or sequentially with other drugs or treatment methods.

[0039] The present invention also relates to a cosmetic composition or a quasi-drug composition for wound treatment or wound healing, which contains extracellular vesicles derived from a three-dimensional spheroid-type cell aggregate produced through the steps of: (a) three-dimensionally culturing stem cells in a microwell having a diameter of 200 to 800 μm and a depth of 100 to 1000 μm to produce a three-dimensional spheroid-type cell aggregate; and (b) separating extracellular vesicles from the three-dimensional spheroid-type cell aggregate.

[0040] As used herein, the "cosmetic composition" means a composition configured for the purpose of manufacturing cosmetics, and can be broadly interpreted to include an external preparation composition for external use. The cosmetic composition according to the present invention can be manufactured in any dosage form commonly manufactured in the art. For example, the cosmetic composition can have dosage forms such as lotions such as softening lotion or nutritive lotion, spray-type lotion, facial lotion, emulsions such as body lotion, creams such as nutritive cream, moisture cream, eye cream, stick, essence, cosmetic ointment, spray, gel, pack, sunscreen, makeup base, foundation such as liquid type or spray type, powder, cleansing lotion, cleansing oil for makeup removal, cleansing foam, soap, body wash, and other detergents, but is not limited thereto.

[0041] The cosmetic composition in the present invention can be used according to a conventional method, and the number of times of use can be varied according to the skin condition or preference of the user.

[0042] As used herein, the "quasi-drug" means an article that exhibits an effect of treating, alleviating, treating, or preventing a disease, but has a milder effect on the human body than a pharmaceutical. It includes articles according to the classification criteria separately determined by the Ministry of Health and Welfare, excluding articles used for the uses of pharmaceuticals under the Pharmaceutical Affairs Act. Specifically, it may be a topical skin preparation or a personal hygiene product, but is not limited thereto.

[0043] When adding the composition in the present invention to a quasi-drug composition for the purpose of wound healing or regeneration, the composition can be added as it is or used together with the components of other quasi-drugs, and can be appropriately used according to conventional methods. The mixing amount of the active ingredient can be appropriately determined according to the purpose of use. The topical skin preparation is not particularly limited thereto, and can be produced and used, for example, in the form of an ointment, a lotion, a spray, a patch, a cream, a powder, a suspension, a gel or a gel.

[0044] All the descriptions regarding the pharmaceutical composition can be equally cited for the cosmetic composition or the quasi-drug composition.

[0045] Also, it relates to a method for producing a composition for wound treatment or wound healing, comprising: (a) culturing stem cells in a three-dimensional (3D, 3 dimension) manner in a microwell having a diameter of 200 to 800 μm and a depth of 100 to 1000 μm to produce a three-dimensional spheroid-type cell aggregate; and (b) separating extracellular vesicles from the three-dimensional spheroid-type cell aggregate.

[0046] According to the production method, extracellular vesicles derived from a three-dimensional spheroid-type cell aggregate having an excellent wound healing effect can be rapidly mass-produced in accordance with GMP standards.

[0047] Duplicate content is omitted in consideration of the complexity of this specification, and terms not specifically stated in this specification have the meanings usually used in the technical field to which the present invention pertains.

Examples

[0048] Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following examples.

[0049] Western blot Cells and extracellular vesicles were lysed in radioimmunoprecipitation assay (RIPA) buffer (25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.5% Triton X-100, 1% Na-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 a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). Next, the nitrocellulose membrane was incubated overnight at 4 °C with primary antibodies against histone H2A.Z, histone H3, lamin A / C, flotillin-1 (1:1,000, Cell signaling technology, Beverly, MA, USA), or calreticulin (1:1,000, ThermoFisher Scientific, Inc., Rockford, IL, USA). After washing with Tris-buffered saline-Tween 20, the nitrocellulose membrane was incubated for 2 h with a horseradish peroxidase (HRP)-conjugated secondary antibody (1:1,000, anti-rabbit, Cell Signaling Technology, Beverly, MA, USA). Proteins were detected using a chemiluminescent substrate from ThermoFisher Scientific, Inc. (Waltham, MA, USA). The labeled proteins were visualized through X-ray film (Agfa, Mortsel, Belgium).

[0050] ELISA ELISA was performed using commercial kits according to the manuals of individual manufacturers. The following ELISA kits were used: gentamicin (5111GEN, EuroProxima, Arnhem, Nederland), 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 (ThermoFisher Scientific, Inc.). All kits contain standard proteins; therefore, the amounts of proteins and extracellular vesicles are determined based on the standard curves of each kit.

[0051] qPCR RNA was extracted from EVs using Trizol (trademark application) according to the manufacturer's instructions and quantified with a nanodrop. RNA was made into cDNA through the reverse transcription (RT) process and Real Time PCR was carried out according to the manufacturer's manual using Taqman probes suitable for each miRNA and mRNA.

[0052] EV Labeling and Uptake by Cells Purified EVs were stained with CFSE (5-(and-6)-Carboxyfluorescein Diacetate, Succinimidyl Ester) labeling dye (c-1157, Invitrogen) according to the manufacturer's instructions. Ultracentrifugation was performed at 100,000 g for 1 hour to remove excess dye. Labeled EVs (0.4 μg / ml) were treated with human NSC cell line (ReNcells™) and cultured for 24 hours. After treatment, the cells were washed twice with PBS and stained using a normal immunocytochemistry protocol. The cells were incubated overnight at 4 °C with mouse anti-SMA (1:100, Sigma aldrich) antibody. Then, the cells were washed with PBS and incubated with a secondary antibody, DyLight-labeled anti-mouse IgG (1:200, 594 nm, Abcam) antibody. Nuclear staining was performed using Vectashield® with 1.5 μg / mL 4’-6’diamidino-2-phenylindole (DAPI) (Vector Laboratories), and the cells were imaged using a confocal microscope (LSM 700, Carl Zeiss, Germany).

[0053] Example 1. Isolation of extracellular vesicles through three-dimensional culture of mesenchymal stem cells 1.1 Preparation of mesenchymal stem cells Human umbilical cord-derived mesenchymal stem cells at passage 5 (hereinafter, WJ-MSC, Samsung medical center, Seoul, Korea) were obtained through assignment and cultured in an incubator at 37 °C with 5% CO 2 2. The growth medium used was α-modified eagle’s medium (α-MEM, GIBCO, NY, USA) containing 10% fetal bovine serum (FBS) (GIBCO, NY, USA) and 50 μg / mL gentamicin (GIBCO, NY, USA). WJ-MSC at passage 6 was used for the preparation of 3D spheroid-type cell aggregates.

[0054] 1.2 Preparation of 3D spheroid-type cell aggregate culture solution The WJ-MSCs prepared in Example 1.1 were washed with PBS, treated with trypsin (TrypLE (trademark application) Express, GIBCO, NY, USA), and reacted in a CO 2 incubator for 5 minutes. Then, fresh serum-free medium was added to neutralize the trypsin, the cells were collected, and a cell pellet was obtained using a centrifuge. Next, fresh serum-free medium was added to prepare a cell suspension, and the cells were counted. After cell counting, 60 ml of the cell suspension was uniformly dispensed onto a microarray containing microwells with a diameter and depth of 500 μm × 200 μm, respectively, coated with MPC (2-Methacryloyloxyethyl Phosphorylcholine Polymer), at a density of 400 cells / well, maintaining a static state to induce the formation of spontaneous spheroid-type cell aggregates, and cultured in a CO 2 incubator at 37°C for a total of 4 days to produce a 3D spheroid-type cell aggregate culture solution (hereinafter, 3D-static-spheroid culture solution).

[0055] 1.3 Separation of extracellular vesicles derived from 3D spheroid-type cell aggregates The 3D-static-spheroid culture solution prepared in Example 1.2 was collected, centrifuged at 2,500 g for 10 minutes to remove cell debris, and filtered through a 0.22 μm syringe filter. Then, the 3D-static-spheroid culture solution was passed through a 300 kDa hollow fiber membrane (Pall, NY, USA) using a tangential flow filtration (TFF) system to remove proteins and perform a primary separation of extracellular vesicles, and then purified once again with physiological saline to obtain highly pure extracellular vesicles derived from the 3D-static-spheroid of the present invention (hereinafter, 3D-static-spheroid EV). The processes of Examples 1.1 to 1.3 were schematically shown in FIG. 1.

[0056] Example 2. Analysis of the characteristics of 3D spheroid-type cell aggregates The characteristics of 3D spheroid-type cell aggregates (hereinafter referred to as 3D-static-spheroids) present in the 3D-static-spheroid culture solution produced in Example 1.2 were analyzed. As the experimental group, the 3D-static-spheroid culture solution produced in Example 1.2 was used. As the control group, according to the dynamic 3D cell culture method disclosed in the Korean Registered Patent (Application No. 10-2016-0053026, Method for Producing Extracellular Vesicles Derived from Stem Cells), mesenchymal stem cells were cultured for 5 days, and the produced 3D mesenchymal stem cell spheroid-type cell aggregate (hereinafter referred to as 3D-dynamic-PEG spheroid) culture solution was used. The microwells containing each culture solution were observed with an optical microscope, which are shown in FIGS. 2A and 2B, and the area change of the spheroid-type cell aggregates after culture compared to the initial stage of culture is shown in FIG. 2C. As shown in FIG. 2, as a result of comparing the areas of 3D-static-spheroids, which are the experimental group, and 3D-dynamic-PEG spheroids, which are the control group, the 3D-static-spheroids showed a statistically significantly decreased spheroid-to-area at the initial stage of culture compared to the 3D-dynamic-PEG spheroids according to the characteristics of cells being densely condensed and forming spheroids (p = 0.0011).

[0057] Example 3. Analysis of the Size of 3D Spheroid-Type Cell Aggregates The size distribution of 3D-static-spheroids produced in Example 1.2 and 3D-dynamic-PEG spheroids produced in Example 3 was measured, and based on the measured size distribution data, the coefficient of variation, skewness, and kurtosis were analyzed. Skewness is a parameter that can know the direction and degree of the distribution inclination from the transition of the median. The closer it is to 1, the more it shows a distribution with a long tail on the right side, and the closer it is to -1, the more it shows a distribution with a long tail on the left side. The skewness in the case of a normal distribution is 0. Kurtosis is a parameter that indicates the sharpness of the data distribution. When the value is a positive number, it means that a relatively large number of data points are concentrated in the central part, and when it is a negative number, it means that a relatively small number of data points are concentrated in the central part. In the case of a normal distribution, the kurtosis is 0. The measured size distribution data are shown in Figure 3, and the results of the analysis are shown in Table 1.

[0058]

Table 1

[0059] As shown in Figure 3 and Table 1, it was confirmed that the average size of the 3D-static-spheroid was 74.43 μm and the coefficient of variation (CV) was 9.59%. In contrast, for the 3D-dynamic-PEG spheroid, it was confirmed that the average size was 148.66 μm and the coefficient of variation (CV) was 14.1%. To compare and test the measured coefficients of variation, as a result of performing the Feltz and Miller’s (1996) asymptotic test, the p value was calculated as 0.01765604, and as a result of performing the Krishnamoorthy and Lee’s (2014) modified signed-likelihood ratio test, the p value was calculated as 0.01853969. Therefore, it was confirmed that in both tests, the size distributions of the 3D-static-spheroid and the 3D-dynamic-PEG spheroid showed a statistically significant difference. As a result of comparing the size distributions of the 3D-static-spheroid and the 3D-dynamic-PEG spheroid, it was confirmed that the kurtosis value of the size distribution of the 3D-static-spheroid was relatively large, and it was confirmed that the size distribution of the 3D-static-spheroid of the present invention showed a tendency to concentrate more on the median. From such results, it was confirmed that when manufacturing a 3D spheroid by the method for manufacturing a 3D-static-spheroid of the present invention, it is possible to manufacture a 3D spheroid with a relatively constant size.

[0060] Example 4. Analysis of the Morphology of Extracellular Vesicles Derived from 3D Spheroid-Type Cell Aggregates To observe the morphology of 3D-static-spheroid EVs isolated in Example 1.3, images were taken using a transmission electron microscope (TEM). Specifically, the 3D-static-spheroid EVs were fixed with 1% OsO 4 dissolved in 0.1 M phosphate buffer (PB) for 2 hours. An EM grid was adsorbed onto the droplet of extracellular vesicles with the formvar side facing down for 1 minute. Then, blotting was performed with filter paper, and the grid was reacted with 2% uranyl acetate for 15 seconds. Excess uranyl acetate was removed, and the EM grid was observed using a TEM (JEM-1011, JEOL, Japan), and the observation images are shown in Fig. 4. As shown in Fig. 4, it was confirmed that the 3D-static-spheroid EVs have a round shape, which is a typical extracellular vesicle morphology.

[0061] Example 5. Analysis of the concentration and size of extracellular vesicles derived from 3D spheroid-type cell aggregates To confirm the concentration and size distribution of 3D-static-spheroid EVs isolated in Example 1.3, nanoparticle tracking analysis (NTA) was performed using a NanoSight NS300 (Malvern, Worcestershire, UK). For optimal analysis, the 3D-static-spheroid EVs were pre-diluted in phosphate-buffered saline (PBS) without vesicles. The average size and concentration (particles / mL) were calculated by integrating three recordings, and the results are shown in Fig. 5. As shown in Fig. 5, it was confirmed that the average particle diameter of the 3D-static-spheroid EVs is 182.5 nm and the mode diameter is 106.1 nm.

[0062] Example 6. Analysis of the expression markers of extracellular vesicles derived from 3D spheroid-type cell aggregates An experiment was conducted to confirm the expression markers of 3D-static-spheroid EVs isolated in Example 1.3. Cell lysate and secretome were used as the control groups. The cell lysate was prepared by washing 3D-static-spheroids with PBS, treating them with trypsin, recovering the cells, and obtaining cell pellets from the recovered cells using a centrifuge. The secretome was prepared by separating EVs through the steps of Example 1.3 using the culture medium, which is the supernatant, after obtaining the cell pellets to obtain the remaining culture secretome. Marker analysis was performed to quantify the specific positive markers CD9, CD63, CD81, and HSP70 of extracellular vesicles using ELISA, and to quantify the specific contaminating protein markers calreticulin, histone H2A.Z, cytochrome C, albumin, and antibiotics. Western blot was also used to quantify the specific contaminating protein markers histone H2A.Z, histone H3, lamin A / C, and calreticulin of extracellular vesicles, and to quantify flotillin-1, which is a positive marker of extracellular vesicles. The results of the ELISA analysis and the Western blot results are shown in Figure 6. As shown in Figure 6, it was confirmed that 3D-static-spheroid EVs expressed all of the specific positive markers CD9, CD63, CD81, and HSP70 of extracellular vesicles, and that the contaminating protein markers calreticulin, histone H2A.Z, histone H3, cytochrome C, albumin (BSA, bovine serum albumin), lamin A / C, and antibiotics, which are highly expressed in cell lysate and secretome, were hardly expressed. In particular, it was confirmed that flotillin-1, a positive marker of extracellular vesicles that is very lowly expressed in cell lysate, was relatively highly expressed in 3D-static-spheroid EVs.

[0063] Example 7. Analysis of the production amount of extracellular vesicles derived from 3D spheroid-type cell aggregates An experiment was conducted to compare the production yields of extracellular vesicles (EVs) obtained from 3D-static-spheroid EVs, which are extracellular vesicles produced by the manufacturing method of Example 1, 3D-dynamic-PEG spheroid-derived extracellular vesicles (3D-dynamic-PEG spheroid EVs), and extracellular vesicles (2D-EVs) isolated from stem cells cultured by a normal 2D culture method. The 2D-EVs were prepared by the following steps. Stem cells cultured for 3 days in a cell stack were washed with PBS, replaced with a serum-free medium, and further cultured for 2 days. The culture medium was collected, and cell debris was continuously removed using centrifugation and a 0.2-μm filter. Thereafter, the culture medium was passed through a hollow fiber membrane using a TFF system to remove proteins, and the EVs were separated at the primary stage. They were purified once again with physiological saline to obtain highly pure 2D-EVs. The production yields per cell derived from the prepared 3D-static-spheroid EVs, 3D-dynamic-PEG spheroid EVs, and 2D-EVs were compared and are shown in Table 2 and Figure 7.

[0064]

Table 2

[0065] Example 8. Analysis of microRNA (miRNA) expressed in extracellular vesicles derived from 3D spheroid-type cell aggregates 8.1 Confirmation of miRNAs with increased expression in 3D-static-spheroid EVs To confirm the differences in the characteristics of 3D-static-spheroid EVs, which are extracellular vesicles produced by the manufacturing method of Example 1, 3D-dynamic-PEG spheroid-derived extracellular vesicles (3D-dynamic-PEG spheroid EVs), and 2D-EVs produced in Example 7, the expression of miRNAs and protein expression were measured through qPCR. Figure 8 shows the miRNAs and proteins that are highly expressed in 3D-static-spheroid EVs compared with 2D-EVs, and Figure 9 shows the miRNAs and proteins that are highly expressed in 3D-static-spheroid EVs compared with 3D-dynamic-PEG spheroid EVs. As shown in Fig. 8, compared with 2D-EV, 3D-static-spheroid EV highly expresses miR-27a, miR-146b, and miR-146a, which are miRNAs showing efficacy in angiogenesis / neurogenesis and immunomodulation, and integrin 1 / 2 and VEGF / R2 (Vascular endothelial growth factor / R2), which are proteins showing efficacy in angiogenesis / neurogenesis. As shown in Fig. 9, compared with 3D-dynamic-PEG spheroid EV, 3D-static-spheroid EV, which is an extracellular vesicle produced by the production method of Example 1, highly expresses miR-146a, which is a miRNA showing efficacy in angiogenesis / neurogenesis, miR-27a, miR-132, miR-184, and miR-210, which are miRNAs showing efficacy in angiogenesis / neurogenesis, and miR-301b showing an antitumor effect, and integrin 1 / 2 and VEGF / R2, which are proteins showing efficacy in angiogenesis / neurogenesis. From such results, 3D-static-spheroid EV, which is an extracellular vesicle produced by the production method of Example 1, is a new extracellular vesicle with different miRNA and protein expressions compared with 2D-EV or 3D-dynamic-PEG spheroid EV. In particular, it highly expresses miRNAs related to angiogenesis / neurogenesis, immunomodulation, rejuvenation, or antitumor, and it was confirmed that it is an extracellular vesicle that can be usefully used clinically.

[0066] 8.2. Analysis of miRNA expression differences by donor Stem cell therapies are known to have a problem of donor variation, in which components vary depending on the donor. An experiment was conducted to confirm whether the 3D-static-spheroid EVs of the present invention exhibit a more consistent miRNA profile without the problem of donor variation. Samples from each donor were obtained under license from Samsung medical center (Seoul, Korea). The miRNA profiles of donors of 2D-cultured WJ-MSCs and 3D-cultured WJ-MSCs of Example 1.1 and 3D-static-spheroid EVs, which are extracellular vesicles produced by the production method of Example 1, were compared. miRNA was profiled by Small RNA sequencing, and the results were compared with the number of EVs produced, the amount of EV protein, and the miRNA profile for each donor of 2D-cultured WJ-MSCs and 3D-cultured WJ-MSCs of Example 1.1 and 3D-static-spheroid EVs, which are extracellular vesicles produced by the production method of Example 1. miRNA was profiled by Small RNA sequencing, and the results are shown in FIGS. 10a to 10b. As shown in FIG. 10a, for 2D-EVs derived from 2D-cultured WJ-MSCs, the amount, size, and amount of EV-producing protein of the produced EVs varied among donors, whereas 3D-static-spheroid EVs showed consistent results without significant variation among donors. Also, as shown in Fig. 10b, there are significant differences in the miRNA composition produced by donors between 2D-cultured WJ-MSCs and 2D-EVs, while it was confirmed that the differences among donors decreased in 3D-cultured WJ-MSCs and 3D-static-spheroid EVs. In particular, 3D-static-spheroid EVs showed a consistent miRNA profile with no donor differences, and miR-27a-3p (1.5-fold), miR-146a-5p (2.3-fold), miR-210 (2.6-fold), and miR-132 (2.6-fold), which are miRNAs capable of showing an angiogenesis effect compared to 2D-EVs, were confirmed to be more uniformly contained. In addition, it was confirmed that other angiogenesis-related miRNAs, such as miR-199a, miR-125b, miR-26a, let-7, miR-125a, miR-181b, and miR-92a, also showed uniform expression levels among donors. Such results indicate that 3D-static-spheroid EVs can reduce the differences in therapeutic active ingredients among donors and show a better therapeutic effect.

[0067] Example 9. Analysis of mRNA expression upon treatment of cells with extracellular vesicles derived from 3D spheroid-type cell aggregates After treating human umbilical vein endothelial cells (HUVECs) or primitive neural stem cells (pNSCs), which are neural stem cells, with 3D-static-spheroid EVs, which are extracellular vesicles produced by the production method of Example 1, and 2D-EVs, which are extracellular vesicles isolated from stem cells cultured by a normal 2D culture method, the changes in mRNA expression were confirmed by qPCR, and the qPCR results are shown in Fig. 11. As shown in Fig. 11, compared with the cells treated with 2D-EV, the vascular endothelial cells (HUVECs) treated with 3D-static-spheroid EV showed increased expression of VEGF (Vascular endothelial growth factor), Hif-1a (Hypoxia-inducible factor 1-alpha), and FGF (Fibroblast growth factor). It was confirmed that the pNSCs treated with 3D-static-spheroid EV had increased expression of VEGF, BDNF (Brain-derived neurotrophic factor), FGF, NGF (Nerve growth factor), and HGF (Hepatocyte growth factor). In particular, in the case of pNSCs treated with 2D-EV compared with the control group, the expression of NGF and HGF decreased, but pNSCs treated with 3D-static-spheroid EV showed an effect of increasing the expression of NGF and HGF. Thus, it was confirmed that 3D-static-spheroid EV and 2D-EV show qualitatively different effects. VEGF, BDNF, NGF, HGF, and Hif-1a are angiogenesis / neurogenesis-related proteins, and FGF is a factor corresponding to a protein that regulates biological functions related to cell proliferation, survival, and differentiation. It was confirmed that 3D-static-spheroid EV, which has a relatively high effect of increasing the expression of VEGF, BDNF, NGF, HGF, Hif-1a, and FGF, is an excellent extracellular vesicle for clinical application.

[0068] Example 10. Confirmation of the wound healing effect of an animal model of extracellular vesicles derived from 3D spheroid-type cell aggregates Through Example 8, it was confirmed that 3D-static-spheroid EV, an extracellular vesicle produced by the production method of Example 1, is a new extracellular vesicle with different miRNA and protein expression patterns from normal 2D-EV or 3D-dynamic-PEG spheroid EV. In order to confirm whether 3D-static-spheroid EV, the newly produced extracellular vesicle, shows a wound healing effect, a wound animal model was produced, and an experiment to confirm the wound recovery effect was conducted as follows. After anesthetizing 8-week-old SD rats by inhalation, the hair on their backs was removed, the skin on the back was folded around the midline of the back, and a wound model was created by piercing the skin with an 8-mm skin biopsy punch. At a total of 4 points around the wound, 6×10 8 / 100 μl of 3D-static-spheroid EVs were subcutaneously injected, and the control group was injected with 100 μl of the same amount of PBS. Furthermore, to compare the wound healing effects of 3D-static-spheroid EVs and WJ-MSCs, a WJ-MSC administration group was set as a comparative experimental group. After creating a skin wound on the back of the rats, 2×10 6 Cells / 100 μl were intradermally injected at 4 locations around the wound site, and then observed for 2 weeks. The injection was performed once a day for 3 days. To prevent wound contraction, a silicon pad with a 10-mm diameter hole was sutured to the wound with nylon 6.0 for fixation. The results of observing the wound healing effect after injection are shown in Fig. 12. As shown in Fig. 12, the 3D-static-spheroid EV treatment group showed a faster wound healing effect compared to the control group and the WJ-MSC treatment group. The difference in the wound healing effects between 3D-static-spheroid EVs and the control group was quantified and shown in Fig. 13. It was confirmed that in the 3D-static-spheroid EV treatment group, a significantly faster wound healing effect was shown compared to the control group from the 4th day after the start of treatment. The wound healing effect of 3D-static-spheroid EVs was confirmed by hematoxylin and eosin staining (H&E staining) experiments of the skin cross-sectional area. After the previously created rat wound modeling, the cross-sectional area of the damaged skin was stained on the 3rd, 7th, and 14th days of 3D-static-spheroid EV treatment, and the thickness of the epidermal part in the skin 14 days after treatment was measured. As shown in Fig. 14, it was confirmed that the skin was generated quickly in the 3D-static-spheroid EV treatment group. As shown in Fig. 15, 14 days after treatment, it was confirmed that the thickness of the epidermis increased significantly in the 3D-static-spheroid EV treatment group. More specifically, the thickness of the epidermis and the surrounding area of the epidermis was measured, quantified, and shown in Fig. 16. As shown in Fig. 16, in the epidermis and the peripheral part of the epidermis, it was confirmed that 3D-static-spheroid EV showed a fast skin thickness increasing effect and could rapidly recover wounds.

[0069] Example 11. Effect of extracellular vesicles derived from 3D spheroid-type cell aggregates on wound healing in a chamber model The wound healing effect of extracellular vesicles derived from 3D spheroid-type cell aggregates was confirmed from a chamber model, and rat skin wound modeling was advanced. The experimental method is shown in Fig. 17. After chamber removal, the results of photographing the wound healing effect every day are shown in Fig. 18, and the change in the size of the wound site was quantified and shown in Fig. 19. As shown in Figs. 18 and 19, after chamber removal, a significant difference was confirmed in the wound healing area on the 3rd and 10th days. After chamber removal, it was confirmed that the wound healing rate was faster in the extracellular vesicle-treated group derived from 3D spheroid-type cell aggregates. The increase in epidermal thickness in the chamber model was confirmed by hematoxylin and eosin staining (H&E staining) experiment, and the results are shown in Fig. 20. As shown in Fig. 20, after chamber removal, on the 7th day, it was confirmed that the thickness of the epidermal layer significantly increased in the extracellular vesicle-treated group derived from 3D spheroid-type cell aggregates compared with PBS, and on the 14th day, the epidermal thickness was maintained at the same level. Furthermore, in the chamber model, 7 or 14 days after chamber removal, the changes in the expressions of VEGF, angiopoietin-2 (Angpt-2), IL-1b and IL-10 were confirmed by ELISA, and the results are shown in Fig. 21. As shown in Fig. 21, in the extracellular vesicle-treated group derived from 3D spheroid-type cell aggregates, the expressions of VEGF, angiopoietin-1 (Angpt-1), angiopoietin-2 (Angpt-2) involved in angiogenesis increased 7 and 14 days after chamber removal, the expression of IL-10 involved in anti-inflammation increased, and the expressions of IL-1b, TNF-a and IL-6 involved in inflammation decreased.

[0070] Example 11. Confirmation of cell motility by the extracellular vesicle treatment group derived from 3D spheroid - type cell aggregates A scratch model was prepared, and the motility of fibroblasts and keratinocytes after treatment with extracellular vesicles derived from the 3D spheroid - type cell aggregates of the present invention was confirmed, and the results are shown in Fig. 22. As shown in Figs. 22a and 22b, in the group treated with 3D spheroid - type cell aggregates, the motility of fibroblasts and keratinocytes was significantly increased, and it was confirmed that rapid wound healing could be achieved.

[0071] Example 12. Confirmation of the effect of promoting angiogenesis by treatment with extracellular vesicles derived from 3D spheroid - type cell aggregates After treating vascular endothelial cells (HUVECs) with 3D - static - spheroid EV, which is an extracellular vesicle produced by the production method of Example 1, and 2D - EV, which is an extracellular vesicle isolated from stem cells cultured by a conventional 2D culture method, the expression changes of angiogenesis - related factors VEGF (Vascular endothelial growth factor), Hif - 1a (Hypoxia - inducible factor 1 - alpha), and FGF (Fibroblast growth factor) were confirmed, and the results are shown in Fig. 23. In addition, to show the effect of promoting angiogenesis by extracellular vesicles obtained by three - dimensional stem cell culture, HUVECs attached to Matrigel were treated with WJ - 2D - EV and 3D - static - spheroid EV of the present invention, and the tube - forming effect was confirmed. Specifically, HUVECs were cultured in M199 medium (Gibco) supplemented with 20% FBS, 5 U / mL heparin, and 3 ng / mL bFGF. The cells were inoculated into growth factor - reduced Matrigel Matrix (BD Bioscience, MA, USA) in μ - Slides Angiogenesis (ibidi, Graefelfing, Germany) at a cell density of 1.7×10 4 cells per well and cultured at 37°C in 5% CO 2The tube was formed in an environmental humidification chamber for 4 hours. Images were taken with a phase contrast microscope (Olympus), and the number of tube-like structures was quantified in the microscope field (4x magnification) using ImageJ software. The results are shown in Fig. 24. As shown in Fig. 23, it was confirmed that the expression of VEGF (Vascular endothelial growth factor), Hif-1a (Hypoxia-inducible factor 1-alpha), and FGF (Fibroblast growth factor) increased in vascular endothelial cells (HUVECs) treated with 3D-static-spheroid EVs compared to cells treated with 2D-EVs. VEGF and Hif-1a are angiogenesis-related proteins, and FGF is a factor corresponding to a protein that regulates biological functions related to cell proliferation, survival, and differentiation. It was confirmed that 3D-static-spheroid EVs are excellent for clinical application in angiogenesis. As confirmed from Fig. 24, in the experimental group treated with the 3D-static-spheroid EVs of the present invention, tube formation was significantly increased compared to the VEGF treatment group, which is an angiogenesis-related protein, and it was also confirmed that the tube formation effect was excellent compared to the WJ-2D-EV treatment group.

[0072] Example 13. Verification of the effect of 3D-static-spheroid EVs according to production conditions 13.1 Experimental methods and conditions Based on the above examples, the excellent effects of the 3D-static-spheroid EVs of the present invention were confirmed. The production method of Example 1 was carried out in the same manner, but in order to confirm whether the same effects were shown in EVs produced with different micro-well specifications and cell number conditions, the cell number specification per micro-well was changed from 400 cells / well to 200 cells / well, or the micro-well specifications were changed to 500 μm × 600 μm or a flat well with a diameter of 800 μm and no depth under the conditions of a diameter and depth of 500 μm × 200 μm, respectively, and the cells were cultured. The experimental conditions changed by the production method of Example 1 are shown in Table 3 below.

[0073]

Table 3

[0074] The passage 6 WJ-MSCs prepared in Example 1.1 were washed with PBS, treated with trypsin (TrypLETM Express, GIBCO, NY, USA), and reacted in a CO 2 incubator for 5 minutes. Then, fresh serum-free medium was added to neutralize the trypsin, and the cells were collected to obtain a cell pellet using a centrifuge. Next, fresh serum-free medium was added to prepare a cell suspension, and the cells were counted. After cell counting, they were uniformly dispensed into microwells under the conditions shown in Table 3 above, maintained in a static state, and induced to form spontaneous spheroid-type cell aggregates, and cultured in a CO 2 incubator at 37°C for 4 days to produce a 3D spheroid-type cell aggregate culture solution.

[0075] 13.2 Confirmation of the morphology of 3D spheroid-type cell aggregates Under the conditions of Experimental Examples 1 to 3 above, it was confirmed that spheroids were uniformly formed in the same manner as in Example 1. The 3D spheroid-type cell aggregate culture solution was collected, and extracellular vesicles derived from the spheroids were obtained by the method of Example 1.3. The size distribution of the obtained extracellular vesicles was measured, and the Rundness and Solidity of the spheroids were further confirmed, and the results are shown in Fig. 25. As shown in Fig. 25, it was confirmed that in all of Experimental Examples 1 to 3, the size of the manufactured 3D spheroid-type cell aggregates was within the range of 55 to 131 μm, which is the range of the size of the cell aggregates manufactured in Example 1, and it was confirmed that the average value of the size distribution was shown to be 70.34 to 99.51 μm. Further, as a result of confirming Roundness and Solidity, the spheroid-type cell aggregates of Experimental Examples 1 to 3 also showed Roundness values with an average of 0.8751, 0.8669, and 0.8601, respectively, similar to the average Roundness value of 0.8697 (CV 2.64%) of the 3D spheroid-type cell aggregates of Example 1, and the spheroid-type cell aggregates of Experimental Examples 1 to 3 also showed average values of 0.9744, 1, and 0.972, respectively, similar to the average value of Solidity of 0.9488 (CV 2.64%) of the 3D spheroid-type cell aggregates of Example 1. Such results indicate that 3D spheroid-type cell aggregates having a similar morphology can be effectively formed by the method of Example 1 even when the number of cells per microwell is changed to 200 or 400 and the diameter of the microwell is changed to 400 to 800.

[0076] 13.3 Comparison of miRNA Expression Patterns of EVs Derived from 3D Spheroid Type Since it was confirmed that spheroid-type cell aggregates having a similar morphology to that of Example 1 could be produced even under the conditions of Experimental Examples 1 to 3, extracellular vesicles were further separated and obtained from these by the method of Example 1.3, and it was confirmed whether the separated and obtained EVs also showed a similar miRNA expression pattern. The comparison of the miRNA expression patterns was confirmed using extracellular vesicles derived from spheroid-type cell aggregates produced by the methods of Experimental Examples 1 and 2 with different microwell conditions. The analysis of the expressed miRNAs was confirmed by the same qPCR method as in Example 8. The results are shown in Fig. 26. The miRNA expression pattern was compared with the 2D-EV produced in Example 7. As shown in Fig. 26, even when the conditions of the microwells were changed to diameters of 500 and 800 μm, miR-132 and miR-210, which are miRNAs showing efficacy in angiogenesis / neurogenesis and immunomodulation similar to the EVs produced in Example 1, were confirmed to show a significant increase in expression compared to existing 2D-EVs. Such results indicate that EVs derived from three-dimensional spheroid-type cell aggregates obtained by the method of the present invention using microwells with diameters of 200 to 800 μm can commonly exhibit miRNA marker expression characteristics. Therefore, these can all be referred to as 3D-static-spheroid EVs.

[0077] 13.4 Confirmation of the angiogenesis effect of EVs derived from the 3D spheroid type An experiment was conducted to confirm the angiogenesis effect of 3D-static-spheroid EVs obtained by the method of the present invention using microwells with diameters of 200 to 800 μm. As experimental groups, 3D-static-spheroid EVs produced under the conditions of Experimental Examples 1 and 2 and Example 1, and 2D-EVs produced in Example 7 were used, and the tube formation effect was confirmed in the same manner as in Example 11. The results are shown in Fig. 27. As shown in Fig. 27, all of the 3D-static-spheroid EVs produced under the conditions of Experimental Examples 1 and 2 and Example 1 showed a significantly superior tube formation effect compared to the control group and 2D-EVs. Based on the above results, it was confirmed that 3D-static-spheroid EVs have an excellent effect of promoting angiogenesis, and through this, excellent wound healing can be achieved even when compared to other EVs such as WJ-2D-EV.

[0078] As described in detail the specific parts of the content of the present invention above, for those with ordinary knowledge in the art, such specific descriptions are merely preferred embodiments and it is obvious that the scope of the present invention is not limited thereby. Therefore, it can be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. (a) culturing stem cells in a three-dimensional (3D) manner in a microwell having a diameter of 200 to 800 μm and a depth of 100 to 1000 μm to produce a three-dimensional spheroid-type cell aggregate; and (b) separating cells and vesicles from the three-dimensional spheroid-type cell aggregate; A pharmaceutical composition for wound treatment or wound recovery, comprising extracellular vesicles derived from the three-dimensional spheroid-type cell aggregate produced through the above steps.

2. The pharmaceutical composition for wound treatment or wound recovery according to claim 1, wherein the stem cells are any one or more 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 wound treatment or wound recovery according to claim 1, wherein the three-dimensional culture in step (a) is a static culture.

4. The pharmaceutical composition for wound treatment or wound recovery according to claim 1, wherein in step (a), mesenchymal stem cells are dispensed into the microwell at a density of 100 to 1000 cells / well and cultured.

5. 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 iR-301b as compared with extracellular vesicles derived from spheroid-type cell aggregates obtained by three-dimensional dynamic culture of mesenchymal stem cells; or highly express one or more selected from the group consisting of miR-27a, miR-146a, and miR-146b as compared with extracellular vesicles derived from mesenchymal stem cells cultured in two dimensions. The pharmaceutical composition for wound treatment or wound recovery according to claim 1.

6. The pharmaceutical composition for wound treatment or wound recovery according to claim 1, wherein the extracellular vesicles enhance the motility of fibroblasts or keratinocytes at the wound site.

7. The pharmaceutical composition for wound treatment or wound recovery according to claim 1, wherein the extracellular vesicles promote angiogenesis at the wound site.

8. (a) culturing stem cells in a three-dimensional (3D) manner in a microwell having a diameter of 200 to 800 μm and a depth of 100 to 1000 μm to produce a three-dimensional spheroid-type cell aggregate; and (b) separating extracellular vesicles from the three-dimensional spheroid-type cell aggregate; A cosmetic composition for wound treatment or wound recovery, comprising extracellular vesicles derived from three-dimensional spheroid-shaped cell aggregates produced through [specific process].

9. (a) A step of culturing stem cells three-dimensionally (3D, 3 dimension) in a microwell having a diameter of 200 to 800 μm and a depth of 100 to 1000 μm to produce three-dimensional spheroid-shaped cell aggregates; and (b) A step of separating extracellular vesicles from the three-dimensional spheroid-shaped cell aggregates; A quasi-drug composition for wound treatment or wound recovery, comprising extracellular vesicles derived from three-dimensional spheroid-shaped cell aggregates produced through [specific process].

10. (a) A step of culturing stem cells three-dimensionally (3D, 3 dimension) in a microwell having a diameter of 200 to 800 μm and a depth of 100 to 1000 μm to produce three-dimensional spheroid-shaped cell aggregates; and (b) A step of separating extracellular vesicles from the three-dimensional spheroid-shaped cell aggregates; A method for producing a composition for wound treatment or wound recovery, comprising extracellular vesicles derived from three-dimensional spheroid-shaped cell aggregates, the method including

11. (a) A step of culturing stem cells three-dimensionally (3D, 3 dimension) in a microwell having a diameter of 200 to 800 μm and a depth of 100 to 1000 μm to produce three-dimensional spheroid-shaped cell aggregates; (b) A step of producing extracellular vesicles (extracellular vesicle) derived from three-dimensional spheroid-shaped cell aggregates through a step of separating extracellular vesicles from the three-dimensional spheroid-shaped cell aggregates; and (c) A step of treating an object in need thereof with the extracellular vesicles (extracellular vesicle) derived from the three-dimensional spheroid-shaped cell aggregates produced through the step (b); A wound treatment method including

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