Composition for promoting angiogenesis comprising extracellular vesicles derived from 3 d spheroid-type cell aggregate

Abstract

[Disclosure] [Technical Problem] The present invention has been made in an effort to provide an extracellular vesicle derived from a three dimensional spheroid-type cell aggregate having improved angiogenesis-promoting ability, and a composition for promoting angiogenesis including the same.SOLUTION: Disclosed is a pharmaceutical composition for promoting angiogenesis, which comprises an extracellular vesicle derived from a three dimensional spheroid cell aggregate produced by (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 and (b) separating the extracellular vesicle from the three dimensional spheroid cell aggregate.SELECTED DRAWING: Figure 13

Description

[Technical Field]

[0001] The present invention relates to a composition for promoting angiogenesis, which contains extracellular vesicles derived from three-dimensional spheroid cell aggregates produced by a novel production method, and a method for promoting angiogenesis. [Background technology]

[0002] Positive clinical results have been reported for the treatment of various diseases using stem cells, particularly mesenchymal stem cells (MSCs). However, stem cell therapy agents carry the risk of cell-related side effects such as vascular occlusion, tumor formation, and coagulation disorders, and efficacy verification through clinical trials is still required. The paracrine effect of stem cells is known to induce the regeneration of surrounding skin cells and promote vascular regeneration, and extracellular vesicles (EVs) in particular are known to be the main effective factor in this paracrine effect.

[0003] Extracellular vesicles are classified into exosomes and microvesicles based on their size. Exosomes range in diameter from 30 to 150 nm, while microvesicles range in size from 100 to 1,000 nm. Extracellular vesicles are portions of the cell membrane released into the bloodstream. They contain both proteins and nuclear components and are known to mediate intercellular communication. Using extracellular vesicles instead of stem cells not only minimizes side effects associated with stem cell use and increases safety, but also offers advantages in terms of biodistribution and production processes.

[0004] However, methods for mass production and acquisition of stem cell-derived extracellular vesicles have not yet been established, and little research has been done on methods for further enhancing the efficacy of stem cell-derived extracellular vesicles while maintaining their properties.

[0005] Therefore, there is a need for extracellular vesicles with improved efficacy and novel therapeutic agents using the same. Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, the present inventors researched a method for producing three-dimensional spheroid cell aggregates or extracellular vesicles derived therefrom that allows for mass production and shortens the culture time. As a result, they produced three-dimensional spheroid cell aggregates (3D-static-spheroids) by static culture using microwells, and confirmed that when these are used to produce extracellular vesicles, extracellular vesicles with improved angiogenesis-promoting ability are produced, thereby completing the present invention.

[0007] Therefore, an object of the present invention is to produce extracellular vesicles derived from three-dimensional spheroid cell aggregates with improved angiogenesis-promoting ability, and to provide a composition for promoting angiogenesis containing the same. [Means for solving the problem]

[0008] To achieve the above object, the present invention provides a pharmaceutical composition for promoting angiogenesis, comprising extracellular vesicles derived from three-dimensional spheroid cell aggregates prepared through the steps of: (a) three-dimensionally (3D, 3-dimensionally) culturing stem cells in microwells having a diameter of 200 to 800 μm and a depth of 100 to 1000 μm to prepare three-dimensional spheroid cell aggregates; and (b) isolating extracellular vesicles from the three-dimensional spheroid cell aggregates.

[0009] The present invention also provides a food composition for promoting angiogenesis, comprising extracellular vesicles derived from three-dimensional spheroid cell aggregates produced through the steps of: (a) three-dimensionally 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 cell aggregates; and (b) isolating extracellular vesicles from the three-dimensional spheroid cell aggregates.

[0010] The present invention also provides an in vitro composition for promoting angiogenesis, comprising extracellular vesicles derived from three-dimensional spheroid cell aggregates prepared through the steps of: (a) three-dimensionally (3D, 3-dimensionally) culturing stem cells in microwells having a diameter of 200 to 800 μm and a depth of 100 to 1000 μm to prepare three-dimensional spheroid cell aggregates; and (b) isolating extracellular vesicles from the three-dimensional spheroid cell aggregates.

[0011] The present invention also provides a method for producing a composition for promoting angiogenesis, which comprises extracellular vesicles derived from three-dimensional spheroid cell aggregates, the method comprising: (a) three-dimensionally (3D, 3-dimensionally) 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 cell aggregates; and (b) isolating extracellular vesicles from the three-dimensional spheroid cell aggregates.

[0012] The present invention also provides a method for promoting angiogenesis, including: (a) three-dimensionally (3D) culturing stem cells in microwells having a diameter of 200 to 800 μm and a depth of 100 to 1000 μm to prepare three-dimensional spheroid cell aggregates; (b) preparing extracellular vesicles derived from the three-dimensional spheroid cell aggregates by isolating extracellular vesicles from the three-dimensional spheroid cell aggregates; and (c) processing the three-dimensional spheroid cell aggregate-derived extracellular vesicles prepared through step (b) into a required object. [Effects of the Invention]

[0013] The extracellular vesicles produced by the novel method of the present invention can highly express various factors that can promote angiogenesis and induce vascular regeneration, and therefore can be useful in the treatment of various diseases that require vascular regeneration. [Brief explanation of the drawings]

[0014] [Figure 1] This figure shows a schematic diagram of the process of producing 3D spheroid cell aggregates and isolating extracellular vesicles from them. [Figure 2] A is an image of a microwell containing 3D-dynamic-PEG spheroid culture medium. B is an image of a microwell containing 3D-static-spheroid culture medium. C is a graph showing the change in aggregate area over the culture period for 3D-static-spheroids produced using 3D-dynamic-PEG spheroids and microwells. [Figure 3] FIG. 10 compares the size distribution of 3D-static and 3D-dynamic-PEG spheroids. [Figure 4] Electron microscopic observation of the morphology of 3D static spheroid EVs. [Figure 5] FIG. 1 shows the results of nanoparticle tracking analysis (NTA) to confirm the concentration and size distribution of 3D-static-spheroid EVs. [Figure 6] Figure 1 shows the results of ELISA and Western blot to confirm the expression markers of 3D-static-spheroid EVs. [Figure 7] Figure 1 shows a comparison of the production amounts of 3D-static spheroid EVs, 3D-dynamic-PEG spheroid EVs, and 2D-EVs per cell of origin. [Figure 8] Figure 1 shows miRNAs and proteins that are highly expressed in 3D-static-spheroid EVs compared to 2D-EVs. [Figure 9] FIG. 1 shows angiogenesis-related miRNAs that are highly expressed in 3D-static spheroid EVs compared to 3D-dynamic PEG spheroid EVs. [Figure 10]This figure shows the results of comparing the expression of miR-210, an angiogenesis-related miRNA, in 2D-EVs and 3D-static-spheroid EVs, confirming GAPDH in HUVEC cells treated with 2D-EVs and 3D-static-spheroid EVs, and confirming the effect on the suppression of Ephrin A3 expression. [Figure 11a] This figure shows the results of confirming the differences in EV production by donor, EV size, and the amount of protein contained in EVs between 2D-EVs and 3D-static-spheroid EVs (WJ-3D EVs). [Figure 11b] This figure shows the results of confirming donor variation and differences in angiogenesis-related miRNA expression levels among 2D-cultured WJ-MSCs, 3D-cultured WJ-MSCs, 2D-EVs, and 3D-static-spheroid EVs. [Figure 12] This figure shows the results of examining changes in the expression of angiogenesis-related factors VEGF, Hif-1a, and FGF after treating vascular endothelial cells (HUVECs) with 3D static spheroid EVs. [Figure 13] This figure shows the results of comparing the tube formation effects of vascular endothelial cells (HUVECs) treated with PBS (con), VEGF, 2D-EVs, and 3D-static-spheroid EVs. [Figure 14] This figure shows the results of examining the extent of changes in cerebral infarction lesions and ventricle volume after treating an animal model of cerebral infarction with PBS as a control group or 3D-static-spheroid EVs as an experimental group. [Figure 15] This figure shows the results of examining the effect of neurovascularization after treating an animal model of cerebral infarction with PBS as a control group or 3D-static-spheroid EVs as an experimental group. [Figure 16] This figure shows the results of confirming the effect of injecting 3D static spheroid EVs into an animal model of cerebral infarction on recovery from motor function loss. [Figure 17]This figure shows the results of examining the size, roundness, and solidity of 3D spheroids produced after varying the diameter and depth of microwells and the number of cells per well during 3D static spheroid EV production. [Figure 18] This figure shows the results of comparing the expression levels of miRNA-132 and miRNA-210 in 3D-static spheroid EVs and 2D-EVs produced under various conditions. [Figure 19] This figure shows the results of a tube formation experiment confirming the angiogenic potential of 3D-static spheroid EVs and 2D-EVs produced under various conditions. BEST MODE FOR CARRYING OUT THE INVENTION

[0015] The present invention provides a pharmaceutical composition for promoting angiogenesis, comprising extracellular vesicles derived from three-dimensional spheroid cell aggregates prepared through the steps of: (a) three-dimensionally (3D, 3-dimensionally) culturing stem cells in microwells having a diameter of 200 to 800 μm and a depth of 100 to 1000 μm to prepare three-dimensional spheroid cell aggregates; and (b) isolating extracellular vesicles from the three-dimensional spheroid cell aggregates.

[0016] The cells of the present invention may be any cells capable of isolating extracellular vesicles, and may be cells isolated from natural biological objects. The cells may be derived from any type of animal, including humans and non-human mammals, or plants, 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.

[0017] The three-dimensional culture in the present invention means culturing in a three-dimensional configuration in a test tube. Unlike two-dimensional culture, three-dimensional culture allows cells to grow in all directions in vitro, which can be more similar to the cellular environment in vivo.

[0018] In the present invention, the three-dimensional culture in step (a) can be performed using any three-dimensional cell culture technique known in the art, such as microwell array culture, porous microparticle culture, hanging drop culture, low attachment plate culture, membrane-based cell-detachment culture, thermal lifting culture, centrifugation culture, and semisolid medium culture. Preferably, the three-dimensional culture can be dynamic or static culture, more preferably static culture. In the present invention, static culture in step (a) can be performed without the need for equipment required for shaking culture, making the culture easier and enabling large-scale culture in GMP (Good Manufacturing Practices) manufacturing facilities.

[0019] In the present invention, the three-dimensional (3-dimensional) culture in step (a) may be performed for 1 to 10 days, preferably 2 to 4 days. When the culture in step (a) is performed for 2 to 4 days in the present invention, the viability of cells present in the three-dimensional spheroid cell aggregates is maintained at a high level, and the culture time is relatively short compared to existing processes for producing three-dimensional spheroid cell aggregates, allowing three-dimensional spheroid cell aggregates and extracellular vesicles derived therefrom to be rapidly produced.

[0020] In the present invention, the three-dimensional culture in step (a) may involve dispensing mesenchymal stem cells into microwells at a density of 100 to 1,000 cells / well and culturing them, preferably at a density of 100 to 600 cells / well and culturing them, and more preferably at a density of 100 to 500 cells / well and culturing them.

[0021] In the present invention, the step (b) of isolating extracellular vesicles can be performed using a method selected from the group consisting of extrusion of a sample containing cells or cell aggregates, sonication, cell lysis, homogenization, freeze-thaw, electroporation, chemical treatment, mechanical disintegration, and physical stimulation treatment by externally applying force to the cells. Preferably, the separation can be performed using a tangential flow filtration (TFF) method, but is not limited to these.

[0022] In the present invention, microRNAs are named by prefixing them with "mir" followed by a "-" and a number. The numbers often indicate the order in which they were named; for example, mir-123 was named before mir-156 and is expected to have been discovered earlier. "mir-" indicates a pre-microRNA, while "miR" with a capital letter indicates a mature microRNA. MicroRNAs with nearly identical sequences, except for one or two sequences, are named with a lowercase letter. For example, miR-121a and miR-121b are generated from their respective precursors, mir-121a and mir-121b, and are very similar in sequence. Pre-microRNAs that are identical in mature microRNA but located at different sites on the genome are named with an additional "-" and a number. For example, the pre-microRNAs mir-121-1 and mir-121-2 are the same mature microRNA (miR-121) but are located at different sites on the genome. MicroRNAs are named according to species, starting with the prefix. For example, hsa-miR-123 is a human (Homo sapiens) microRNA, and oar-miR-123 is a sheep (Ovis aries) microRNA. The "v" stands for viral (miRNA encoded by a viral genome), and the "d" stands for Drosophila microRNA. When two mature microRNAs are derived from different arms (3' or 5' arms) of the same pre-microRNA, the suffix "-3p" or "-5p" is added to the end of the name. miR-142-3p is derived from the 3' arm, and miR-142-5p is derived from the 5' arm. MicroRNA nomenclature generally follows the above criteria, but exceptions exist.

[0023] In the present invention, the extracellular vesicles may express a clinically significant substance at a higher level than known extracellular vesicles, and the clinically significant substance may be a substance exhibiting an angiogenic effect. For example, the extracellular vesicles of the present invention may express at a higher level one or more angiogenesis-related genes selected from the group consisting of miR-27a, miR-132, miR-146a, miR-146b, miR-184, and miR-210, or at a higher level one or more angiogenesis-related genes selected from the group consisting of vascular endothelial growth factor (VEGF), hypoxia-inducible factor 1-alpha (Hif-1a), and fibroblast growth factor (FGF), compared to extracellular vesicles derived from spheroid-type cell aggregates obtained by three-dimensional dynamic culture of mesenchymal stem cells and extracellular vesicles derived from two-dimensionally cultured mesenchymal stem cells. More preferably, the extracellular vesicles of the present invention may have higher expression of angiogenesis-related miR-27a, miR-132, miR-146a, miR-146b, miR-184, and miR-210, VEGF, Hif-1a, and FGF than extracellular vesicles derived from spheroid-type cell aggregates formed by three-dimensional dynamic culture of mesenchymal stem cells and extracellular vesicles derived from two-dimensionally cultured mesenchymal stem cells.

[0024] Furthermore, the extracellular vesicles may be mixed into cells and internalized when treated in cells, and when mixed into cells and internalized, they may effectively deliver clinically significant substances that are highly expressed in the extracellular vesicles to cells and be highly expressed in the cells.

[0025] In the present invention, extracellular vesicles derived from three-dimensional spheroid cell aggregates that exhibit angiogenesis-promoting effects can be interchangeably referred to as "3D-static-spheroid-EVs." In contrast, extracellular vesicles derived from three-dimensional dynamically cultured spheroid cell aggregates can be interchangeably referred to as "3D-dynamic-PEG-spheroid-EVs."

[0026] In the present invention, "extracellular vesicles derived from spheroid-type cell aggregates obtained by three-dimensional dynamic culture of mesenchymal stem cells" may include, without limitation, extracellular vesicles isolated from spheroid-type cell aggregates obtained by three-dimensional dynamic culture of mesenchymal stem cells, and preferably may be extracellular vesicles disclosed in a registered Korean patent (application number 10-2016-0053026, method for producing extracellular vesicles derived from stem cells).

[0027] In the present invention, "extracellular vesicles derived from two-dimensionally cultured mesenchymal stem cells" can include, without limitation, extracellular vesicles obtained by culturing stem cells according to a conventional two-dimensional culture method and isolating them using a conventional method for isolating extracellular vesicles. In one embodiment of the present invention, the conventional two-dimensional culture method was carried out by culturing stem cells in a cell stack for 3 days, washing them with PBS, exchanging the medium for serum-free medium, and culturing them for an additional 2 days.

[0028] The three-dimensional spheroid cell aggregates of the present invention may have an average diameter of 74.43±7.756 μm, preferably in the size range of 55 to 95.0 μm, and may have an average diameter of 74.43 μm and a coefficient of variation (CV) of 9.59% as a result of measuring the size distribution of 155 of the three-dimensional spheroid cell aggregates. The three-dimensional spheroid cell aggregates of the present invention may have a higher kurtosis of the size distribution than "spheroid cell aggregates obtained by three-dimensional dynamic culture of mesenchymal stem cells." Therefore, the size of the three-dimensional spheroid cell aggregates may be smaller than "spheroid cell aggregates obtained by three-dimensional dynamic culture of mesenchymal stem cells," and may have a relatively uniform size distribution.

[0029] In the present invention, the microwell contains 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(Buthylmethacrylate), PETIA(Pentaerythritol triacrylate), HDDA(Hexan diol diacrylate), EGPEA (Ethylene glycol phenyletheracrylate), BM (Benzylmethacrylate), HPPA (Hydroxyphenoxypropyl The substrate may be coated with any one selected from the group consisting of 2-(4-benzoyl-3-hydroxyphenoxy)ethyl acrylate (BHPEA), 2-(4-benzoyl-3-hydroxyphenoxy)ethyl acrylate (HEMA), 2-(4-hydroxypropyl)methacrylamide (HPMA), and 2-methacryloyloxyethyl phosphorylcholine polymer (MPC), and preferably coated with 2-methacryloyloxyethyl phosphorylcholine polymer (MPC), but is not limited thereto.

[0030] The microwells in the present invention may have a diameter of 200 to 800 μm, preferably 300 to 800 μm, and more preferably 400 to 800 μm.

[0031] Furthermore, the microwells may be microwells with a flat structure with no depth, or in the case of microwells with a depth, they may have a structure with a depth of 100 to 1000 μm, preferably 100 to 900 μm, and more preferably 200 to 900 μm.

[0032] The mesenchymal stem cells cultured using the structure can maintain a high level of viability even after the elapse of the culture period. Preferably, a microarray containing 1,000 to 100,000 of the microwells can be fabricated, thereby increasing the production yield of cell aggregates.

[0033] The production of extracellular vesicles by the "method for producing extracellular vesicles derived from three-dimensional spheroid cell aggregates" of the present invention not only offers the advantages of static culture production, but also enables rapid and efficient mass production of extracellular vesicles with improved clinical applicability, particularly angiogenic activity. In particular, unlike conventional methods for producing extracellular vesicles, such as methods for producing "extracellular vesicles derived from spheroid cell aggregates obtained by three-dimensional dynamic culture of mesenchymal stem cells" or "extracellular vesicles derived from two-dimensionally cultured mesenchymal stem cells," which are not suitable for GMP, the method for producing extracellular vesicles of the present invention is suitable for GMP application and is therefore particularly suitable for the production of pharmaceutical compositions for angiogenesis.

[0034] In the present invention, angiogenesis means the formation of new blood vessels (angiogenesis), which refers to the process of inducing or increasing the migration of vascular endothelial cells, promoting tube formation in vascular endothelial cells, and forming new blood vessels from existing blood vessels.

[0035] The extracellular vesicles derived from the three-dimensional spheroid cell aggregates of the present invention highly express various factors that can promote angiogenesis and can induce vascular regeneration, and therefore can be useful in the treatment of various diseases that require vascular regeneration.

[0036] The types of diseases requiring angiogenesis to which extracellular vesicles derived from the three-dimensional spheroid cell aggregates of the present invention can be applied include, but are not limited to, one or more selected from the group consisting of burns, ulcers, ischemia, arteriosclerosis, angina pectoris, myocardial infarction, cardiovascular diseases, cerebrovascular diseases, and alopecia, and particularly cardiovascular diseases or cerebrovascular diseases.

[0037] Furthermore, since the extracellular vesicles derived from the three-dimensional spheroid cell aggregates of the present invention are particularly useful for promoting cerebral angiogenesis, the composition for promoting angiogenesis can be used for promoting cerebral angiogenesis.

[0038] Brain tissue normally receives a large amount of blood flow, but external or internal stimuli can cause cerebral blood vessels to become clogged, reducing the amount of blood flow to the brain and causing the brain to stop functioning normally. If the reduction in cerebral blood flow persists for a certain period of time, the brain tissue will die. When brain tissue becomes necrotic and irreversible, it is called a cerebral infarction, which includes cerebral thrombosis and cerebral embolism. Thrombosis occurs when blood clots locally in a blood vessel, blocking the blood flow. When a clot forms, blood flow to the area of ​​that blood vessel is blocked, and if collateral blood flow from other locations is insufficient, the brain tissue in that area will die.

[0039] Embolism refers to a condition in which a blood clot formed in the heart is carried to the periphery by the blood flow, causing blockage of peripheral blood vessels. When blood vessels are blocked by an embolism, the brain tissue at that site dies. The extracellular vesicles derived from the three-dimensional spheroid cell aggregates of the present invention can promote cerebral angiogenesis and may therefore be useful in restoring reduced cerebral blood flow due to cerebral infarction.

[0040] In addition to the active ingredient, the pharmaceutical composition of the present invention may further contain suitable carriers, excipients and diluents that are commonly used in the manufacture of pharmaceutical compositions. The pharmaceutical composition of the present invention may further contain other pharmaceutically active ingredients or active compounds.

[0041] The pharmaceutical compositions of the present invention can be formulated into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, as well as topical preparations, suppositories, and sterile injectable solutions by conventional methods. Carriers, excipients, and diluents that can be contained in the compositions include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulated, commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants are used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations are prepared by mixing the composition with at least one or more excipients, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

[0042] Oral liquid preparations include suspensions, oral solutions, emulsions, syrups, etc., and may contain various excipients such as wetting agents, sweeteners, flavorings, and preservatives in addition to commonly used simple diluents such as water and liquid paraffin. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Examples of non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Examples of suppository bases include Witepsol, macrogol, Tween 61, cocoa butter, lauric butter, and glycerogelatin.

[0043] The preferred dosage of the pharmaceutical composition of the present invention varies depending on the patient's condition and weight, the severity of the disease, the drug form, the route of administration, and the duration of administration, but can be appropriately selected by those skilled in the art. Administration may be once a day or in several divided doses. The above dosage does not in any way limit the scope of the present invention.

[0044] The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, humans, etc. by various routes, including oral, rectal, or intravenous, intramuscular, subcutaneous, intrauterine, intradural, or intracerebroventricular injection, although any route of administration is contemplated.

[0045] The definitions of the terms excipient, binder, disintegrant, lubricant, flavoring agent, flavoring agent, etc. of the present invention are those described in documents known in the art, and include those having the same or similar functions.

[0046] The present invention also provides a method for promoting angiogenesis, including: (a) three-dimensionally (3D) culturing stem cells in microwells having a diameter of 200 to 800 μm and a depth of 100 to 1000 μm to prepare three-dimensional spheroid cell aggregates; (b) preparing extracellular vesicles derived from the three-dimensional spheroid cell aggregates by isolating extracellular vesicles from the three-dimensional spheroid cell aggregates; and (c) processing the three-dimensional spheroid cell aggregate-derived extracellular vesicles prepared by step (b) into a required object.

[0047] The object is preferably a mammal, including a human, and includes all patients in need of angiogenesis who are undergoing treatment, have undergone treatment, or need to be treated for a disease, and may also include patients who have undergone surgery for angiogenesis.

[0048] Furthermore, the present invention can be used in combination with other drugs or therapeutic methods for angiogenesis. When the present invention is used in combination, it can be used simultaneously or sequentially with other drugs or therapeutic methods for angiogenesis.

[0049] The present invention also provides a food composition for promoting angiogenesis, comprising extracellular vesicles derived from three-dimensional spheroid cell aggregates produced through the steps of: (a) three-dimensionally 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 cell aggregates; and (b) isolating extracellular vesicles from the three-dimensional spheroid cell aggregates.

[0050] The food composition can be similarly referred to in all the descriptions regarding pharmaceutical compositions. The food particularly includes functional health foods. As defined in the present invention, "functional health foods" refer to foods produced and processed using raw materials or ingredients that have functional properties useful to the human body, and "functional" refers to foods that are ingested for the purpose of obtaining beneficial health effects, such as regulating nutrients or physiological effects on the structure and function of the human body. The functional health foods can be in any form, such as tablets, capsules, powders, granules, liquids, or pills.

[0051] The food composition of the present invention may also be in the form of a functional ingredient added to various foods, beverages, etc. The food may be in the form of, for example, a beverage, a powdered beverage, a solid, chewing gum, tea, a vitamin complex, or a food additive.

[0052] The present invention also relates to an in vitro composition for promoting angiogenesis, comprising extracellular vesicles derived from three-dimensional spheroid cell aggregates prepared through the steps of: (a) three-dimensionally culturing stem cells in microwells having a diameter of 200 to 800 μm and a depth of 100 to 1000 μm to prepare three-dimensional spheroid cell aggregates; and (b) isolating extracellular vesicles from the three-dimensional spheroid cell aggregates.

[0053] The in vitro composition for promoting angiogenesis of the present invention can be used for experimental purposes and can be a composition intended for treating isolated cells or tissues requiring angiogenesis. The in vitro composition for promoting angiogenesis of the present invention can be a medium composition containing extracellular vesicles derived from three-dimensional spheroid-type cell aggregates. The medium can include, without limitation, media known to those of ordinary skill in the art, such as media containing serum (e.g., fetal bovine serum, horse serum, and human serum). Media that can be used in the present invention include, for example, RPMI series, EMEM, MEM, Iscove's MEM, 199 medium, CMRL 1066, RPMI 1640, F12, F10, DMEM, a mixture of DMEM and F12, Way-mo, McCoy's 5A, or media known in the art suitable for culturing cells requiring angiogenesis.

[0054] The present invention also provides a method for producing an angiogenesis-promoting composition containing extracellular vesicles derived from three-dimensional spheroid cell aggregates, the method comprising: (a) three-dimensionally (3D, 3-dimensionally) 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 cell aggregates; and (b) isolating extracellular vesicles from the three-dimensional spheroid cell aggregates.

[0055] According to the above-described production method, extracellular vesicles derived from three-dimensional spheroid cell aggregates, which have excellent angiogenesis-promoting effects, can be rapidly mass-produced in accordance with GMP standards.

[0056] Duplicate content will be omitted in consideration of the complexity of this specification, and terms not otherwise specified in this specification have the meanings commonly used in the technical field to which this invention belongs.

[0057] The present invention will be described in more detail below 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 to the following examples. [Example]

[0058] 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). The nitrocellulose membrane was then 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 with horseradish peroxidase (HRP)-conjugated secondary antibody (1:1,000, anti-rabbit, Cell Signaling Technology, Beverly, MA, USA) for 2 hours. Proteins were detected using a chemiluminescent substrate from ThermoFisher Scientific, Inc. (Waltham, MA, USA). Labeled proteins were visualized using X-ray film (Agfa, Mortsel, Belgium).

[0059] ELISA ELISA was performed using commercial kits according to the manufacturer's instructions. The following ELISA kits were used: gentamicin (5111GEN, EuroProxima, Arnhem, 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 (ThermoFisher Scientific, Inc.). All kits include standard proteins; therefore, the amount of protein and extracellular vesicles was determined based on the standard curves provided by each kit.

[0060] qPCR Trizol in EV TM RNA was extracted using a nanodrop kit according to the manufacturer's instructions, and RNA was quantified using a nanodrop kit. The RNA was then converted to cDNA via reverse transcription (RT), and real-time PCR was performed using TaqMan probes appropriate for each miRNA and mRNA according to the manufacturer's instructions.

[0061] 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. Excess dye was removed by ultracentrifugation at 100,000 g for 1 hour. Labeled EVs (0.4 μg / ml) were then added to a human NSC cell line (ReNcells®) and cultured for 24 hours. After treatment, cells were washed twice with PBS and stained using standard immunocytochemistry protocols. Cells were then incubated with mouse anti-SMA (1:100, Sigma-Aldrich) antibody overnight at 4°C. Subsequently, cells were washed with PBS and incubated with DyLight-labeled anti-mouse IgG (1:200, 594 nm, Abcam) as a secondary antibody. Vectashield with 1.5 μg / mL 4′-6′diamidino-2-phenylindole (DAPI) (Vector Laboratories) TM After nuclear staining with , cells were imaged using a confocal microscope (LSM 700, Carl Zeiss, Germany).

[0062] Example 1. Isolation of extracellular vesicles through three-dimensional culture of mesenchymal stem cells 1.1 Mesenchymal stem cell preparation Human umbilical cord-derived mesenchymal stem cells (WJ-MSCs) at passage 5 (hereafter referred to as WJ-MSCs, Samsung Medical Center, Seoul, Korea) were obtained and cultured at 37°C in a 5% CO2 incubator. 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-MSCs at passage 6 (passage 6) were used to generate 3D spheroid-type cell aggregates.

[0063] 1.2 Preparation of 3D spheroid-type cell aggregate culture medium WJ-MSCs at passage 6 (as prepared in Example 1.1) were washed with PBS, treated with trypsin (TrypLE™ Express, GIBCO, NY, USA), and incubated in a CO2 incubator for 5 minutes. Fresh serum-free medium was then added to neutralize the trypsin, and the cells were harvested and centrifuged to obtain a cell pellet. Fresh serum-free medium was then added, a cell suspension was prepared, and the cells were counted. After cell counting, 60 ml of cell suspension was uniformly dispensed at a density of 400 cells / well into a microarray containing approximately 69,000 microwells coated with MPC (2-Methacryloyloxyethyl Phosphorylcholine Polymer), each 500 μm in diameter and 200 μm in depth. The cells were then maintained in a static state to induce spontaneous spheroid cell aggregate formation. The cells were then cultured in a CO2 incubator at 37°C for a total of 4 days to produce a 3D spheroid cell aggregate culture medium (hereafter referred to as 3D-static-spheroid culture medium).

[0064] 1.3 Isolation of extracellular vesicles from 3D spheroid-type cell aggregates The 3D static spheroid culture medium prepared in Example 1.2 was collected, centrifuged at 2,500 g for 10 minutes to remove cellular debris, and filtered through a 0.22 μm syringe filter. The 3D static spheroid culture medium was then passed through a 300 kDa hollow fiber membrane (Pall, NY, USA) using a tangential flow filtration (TFF) system to remove proteins and primarily separate extracellular vesicles. These were then purified again with saline to obtain highly purified extracellular vesicles derived from the 3D static spheroids of the present invention (hereinafter referred to as 3D static spheroid EVs).

[0065] The processes of Examples 1.1 to 1.3 are illustrated in FIG.

[0066] Example 2. Analysis of the properties of 3D spheroid-type cell aggregates The characteristics of the 3D spheroid-type cell aggregates (hereinafter referred to as 3D-static spheroids) present in the 3D-static spheroid culture medium prepared in Example 1.2 were analyzed. The 3D-static spheroid culture medium prepared in Example 1.2 was used as the experimental group. The comparative group used 3D mesenchymal stem cell spheroid-type cell aggregates (hereinafter referred to as 3D-dynamic PEG spheroids) culture medium prepared by culturing mesenchymal stem cells for 5 days according to the dynamic 3D cell culture method disclosed in a Korean patent (Application No. 10-2016-0053026, "Method for Producing Stem Cell-Derived Extracellular Vesicles") . Microwells containing each culture medium were observed under an optical microscope, as shown in Figures 2A and 2B. The change in the area of ​​the spheroid-type cell aggregates after culture compared to the initial culture is shown in Figure 2C.

[0067] As shown in Figure 2, the area of ​​the experimental 3D-static spheroids was compared with that of the comparative 3D-dynamic-PEG spheroids. The results showed that the 3D-static spheroids had a statistically significant decrease in spheroid area at the initial stage of culture compared to the 3D-dynamic-PEG spheroids, due to the densely packed cells and the characteristics of spheroid formation (p=0.0011).

[0068] Example 3. Size analysis of 3D spheroid cell aggregates The size distribution of the 3D-static spheroids prepared in Example 1.2 and the 3D-dynamic PEG spheroids prepared in Example 2 was measured, and the coefficient of variation, skewness, and kurtosis were analyzed based on the measured size distribution data.

[0069] Skewness is a parameter that can tell the direction and degree of inclination of a distribution from the trend of the median; the closer it is to 1, the more the distribution has a long tail to the right, and the closer it is to -1, the more the distribution has a long tail to the left. The skewness of a normal distribution is 0.

[0070] Kurtosis is a parameter that indicates the sharpness of a data distribution. A positive value indicates that a relatively large number of data points are concentrated in the center, and a negative value indicates that a relatively small number of data points are concentrated in the center. For a normal distribution, kurtosis is 0.

[0071] The measured size distribution data is shown in FIG. 3, and the analysis results are shown in Table 1.

[0072] [Table 1]

[0073] As shown in Figure 3 and Table 1, the 3D-static spheroids were found to have an average size of 74.43 μm with a coefficient of variation (CV) of 9.59%. In contrast, the 3D-dynamic PEG spheroids were found to have an average size of 148.66 μm with a coefficient of variation (CV) of 14.1%.

[0074] To compare the measured coefficients of variation, Feltz and Miller's (1996) asymmetric test was performed, resulting in a p-value of 0.01765604, and Krishnamoorthy and Lee's (2014) modified signed-likelihood ratio test was performed, resulting in a p-value of 0.01853969. Therefore, both tests confirmed that the size distributions of 3D-static spheroids and 3D-dynamic PEG spheroids differed to a statistically significant extent.

[0075] Comparing the size distributions of 3D-static spheroids and 3D-dynamic-PEG spheroids, it was confirmed that the kurtosis value of the size distribution of the 3D-static spheroids was relatively large, and that the size distribution of the 3D-static spheroids of the present invention exhibited a tendency to be concentrated around the median. These results confirmed that the production of 3D spheroids by the method of the present invention makes it possible to produce 3D spheroids with a relatively uniform size.

[0076] Example 4. Analysis of extracellular vesicle morphology derived from 3D spheroid-type cell aggregates To observe the morphology of the 3D static spheroid EVs isolated in Example 1.3, transmission electron microscopy (TEM) images were taken. Specifically, 3D static spheroid EVs were fixed for 2 hours with 1% OsO4 dissolved in 0.1 M phosphate buffer (PB). An EM grid was adsorbed, Formvar side down, onto the extracellular vesicle droplet for 1 minute. The grid was then blotted with filter paper and reacted with 2% uranyl acetate for 15 seconds. After removing excess uranyl acetate, the EM grid was observed using a TEM (JEM-1011, JEOL, Japan). The observed images are shown in Figure 4.

[0077] As shown in Figure 4, the 3D static spheroid EVs were confirmed to have a round shape, which is a typical morphology of extracellular vesicles.

[0078] 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 the 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 vesicle-free phosphate buffer solution (PBS). The average size and concentration (particles / mL) were calculated by integrating three recordings, and the results are shown in Figure 5.

[0079] As shown in Figure 5, the mean particle diameter of the 3D static spheroid EVs was confirmed to be 182.5 nm, and the mode diameter was confirmed to be 106.1 nm.

[0080] Example 6. Analysis of markers expressed in extracellular vesicles derived from 3D spheroid-type cell aggregates Experiments were conducted to confirm the expression markers of EVs in the 3D-static spheroids isolated in Example 1.3. Cell lysate and secretome were used as controls. Cell lysate was prepared by washing 3D-static spheroids with PBS, treating them with trypsin, and then harvesting the cells. The harvested cells were then centrifuged to obtain a cell pellet. Secretome was prepared by obtaining the cell pellet, isolating EVs from the supernatant culture medium using the procedure in Example 1.3, and obtaining the remaining culture secretome. Marker analysis was performed using ELISA to quantify the extracellular vesicle-specific positive markers CD9, CD63, CD81, and HSP70, as well as specific contaminating protein markers calreticulin, histone H2A.Z, cytochrome C, albumin, and antibiotics. In addition, we used Western blot to quantify specific contaminating protein markers of extracellular vesicles, such as histone H2A.Z, histone H3, lamin A / C, and calreticulin, and we also quantified flotillin-1, a positive marker for extracellular vesicles. The results of the ELISA analysis and Western blot are shown in Figure 6.

[0081] As shown in Figure 6, 3D-static spheroid EVs expressed all of the extracellular vesicle-specific positive markers CD9, CD63, CD81, and HSP70, while the contaminating protein markers highly expressed in cell lysates and secretomes, calreticulin, histone H2A.Z, histone H3, cytochrome C, albumin (BSA, bovine serum albumin), lamin A / C, and antibiotics, were almost completely absent. In particular, the extracellular vesicle-positive marker flotilin-1, which is expressed very low in cell lysates, was found to be expressed relatively highly in 3D-static spheroid EVs.

[0082] Example 7. Analysis of extracellular vesicle production from 3D spheroid-type cell aggregates An experiment was conducted to compare the production yields of 3D-static spheroid EVs (extracellular vesicles) prepared by the manufacturing method in Example 1, extracellular vesicles isolated from 3D-dynamic PEG spheroids (3D-dynamic PEG spheroid EVs) prepared in Example 2, and extracellular vesicles isolated from stem cells cultured using a conventional 2D culture method (2D-EVs). 2D-EVs were prepared as follows: Stem cells cultured in a cell stack for 3 days were washed with PBS, replaced with serum-free medium, and cultured for an additional 2 days. The culture medium was collected, and cellular debris was removed sequentially using centrifugation and a 0.2 μm filter. The culture medium was then passed through a hollow fiber membrane using a TFF system to remove proteins, and EVs were first separated and purified again with 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 the results are shown in Table 2 and Figure 7.

[0083] [Table 2]

[0084] Example 8. Analysis of expressed microRNAs (miRNAs) in extracellular vesicles derived from 3D spheroid cell aggregates To confirm differences in the characteristics of 3D-static spheroid EVs (extracellular vesicles produced by the production method of Example 1), 3D-dynamic-PEG spheroid EVs (extracellular vesicles isolated from 3D-dynamic-PEG spheroids in Example 2), and 2D-EVs produced in Example 7, miRNA and protein expression were measured by qPCR. The miRNAs and proteins that were more highly expressed in 3D-static spheroid EVs compared to 2D-EVs are shown in Figure 8, and the angiogenesis-related miRNAs and proteins that were more highly expressed in 3D-static spheroid EVs compared to 3D-dynamic-PEG spheroid EVs are shown in Figure 9.

[0085] As shown in Figure 8, compared to 2D-EVs, 3D-static-spheroid EVs were confirmed to express higher levels of miR-146a, miR-27a, and miR-146b, which are miRNAs that are effective in angiogenesis / neurogenesis and immune regulation, as well as integrin 1 / 2 and VEGF / R2 (vascular endothelial growth factor / R2), which are proteins that are effective in angiogenesis / neurogenesis.

[0086] As shown in Figure 9, compared to 3D-dynamic-PEG spheroid EVs, 3D-static-spheroid EVs were confirmed to have higher expression of miR-146a, miR-27a, miR-132, miR-184, and miR-210, which are miRNAs that are effective in angiogenesis.

[0087] These results confirm that the 3D-static spheroid EVs, which are extracellular vesicles produced by the production method of Example 1, are novel extracellular vesicles that differ in miRNA and protein expression compared to 2D-EVs or 3D-dynamic-PEG spheroid EVs. In particular, they highly express miRNAs related to angiogenesis / neurogenesis, immunomodulation, rejuvenation, or anti-tumor, making them clinically useful extracellular vesicles.

[0088] Example 9. Confirmation of the angiogenic effect of extracellular vesicles derived from 3D spheroid-type cell aggregates 9.1. Comparison of angiogenesis-related miRNA and Ephrin A3 expression In Example 8, it was confirmed that 3D-static-spheroid EVs, which are extracellular vesicles prepared by the manufacturing method in Example 1, are novel extracellular vesicles with miRNA and protein expression patterns that differ from existing 2D-EVs or 3D-dynamic-PEG spheroid EVs. To confirm the usefulness of 3D-static-spheroid EVs in promoting angiogenesis, they were treated with target HUVEC cells, and the expression changes of miRNAs and Ephrin A3 (eph-related receptor tyrosine kinase ligand 3), which are associated with angiogenic effects, were confirmed by qPCR and Western blot. The results are shown in Figure 10.

[0089] As shown in Figure 10, qPCR results confirmed that miR-210 expression was significantly increased in the 3D-static spheroid EVs of the present invention compared to conventional 2D-EVs. Furthermore, it was confirmed that Ephrin A3 protein expression was significantly inhibited after treatment of HUVECs with 3D-static spheroid EVs compared to the 2D-EV treatment group. miR-210 targets Ephrin A3, which is known to promote endothelial cell migration and form capillary-like structures. These results further demonstrate that 3D-static spheroid EVs can effectively induce angiogenesis.

[0090] 9.2. Analysis of miRNA expression differences by donor Stem cell therapeutics are known to have the problem of donor variation, whereby components vary depending on the donor. An experiment was conducted to determine 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 provided by Samsung Medical Center (Seoul, Korea). The number of EVs, EV protein levels, and miRNA profiles produced by each donor were compared for 2D-cultured WJ-MSCs and 3D-cultured WJ-MSCs (Example 1.1) and 3D-static spheroid EVs (extracellular vesicles) produced by the manufacturing method of Example 1. MiRNA profiling was performed using small RNA sequencing, and the results are shown in Figures 11a and 11b.

[0091] As shown in Figure 11a, 2D-EVs derived from 2D-cultured WJ-MSCs showed variability in the quantity, size, and amount of EV-produced proteins produced by each donor, whereas 3D-static-spheroid EVs showed consistent results without significant variability between donors.

[0092] Furthermore, as shown in Figure 11b, 2D-cultured WJ-MSCs and 2D-EVs showed significant donor-specific variation in miRNA composition, whereas 3D-cultured WJ-MSCs and 3D-static spheroid EVs showed reduced donor-specific variation. In particular, 3D-static spheroid EVs exhibited a consistent miRNA profile without donor-specific variation, and contained significantly more angiogenic miRNAs (miR-27a-3p (1.5-fold), miR-146a-5p (2.3-fold), miR-210 (2.6-fold), and miR-132 (2.6-fold)) than 2D-EVs. Furthermore, other angiogenesis-related miRNAs (miR-199a, miR-125b, miR-26a, let-7, miR-125a, miR-181b, and miR-92a) were also uniformly expressed across donors.

[0093] These results indicate that 3D static spheroid EVs can reduce donor-specific differences in therapeutic active ingredients and demonstrate superior therapeutic efficacy.

[0094] Taking all of the above results into consideration, the 3D-static-spheroid EVs of the present invention showed significantly increased levels of miR-27a, miR-132, miR-146a, miR-146b, miR-184, and miR-210, which are miRNAs related to angiogenesis, compared to WJ-2D-EVs and 3D-dynamic-spheroid EVs. Furthermore, they inhibited Ephrin A3 protein expression, suggesting that they may be expected to exhibit superior angiogenesis effects compared to previously reported EVs, reduce donor-related differences, and potentially become an excellent therapeutic agent.

[0095] Example 10. Confirmation of changes in angiogenesis-related factor expression by treatment with extracellular vesicles derived from 3D spheroid-type cell aggregates 3D-static-spheroid EVs, which are extracellular vesicles produced by the manufacturing method of Example 1, and 2D-EVs, which are extracellular vesicles isolated from stem cells cultured using a conventional 2D culture method, were treated with vascular endothelial cells (HUVECs), and changes in the expression 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 Figure 12.

[0096] As shown in Figure 12, it was confirmed that the expression of VEGF (Vascular endothelial growth factor), Hif-1a (Hypoxia-inducible factor 1-alpha), and FGF (Fibroblast growth factor) was increased in vascular endothelial cells (HUVECs) treated with 3D-static spheroid EVs compared to cells treated with 2D-EVs.

[0097] VEGF and Hif-1a are angiogenesis-related proteins, and FGF is a protein that regulates biological functions related to cell proliferation, survival, and differentiation, indicating that 3D-static-spheroid EVs are excellent extracellular vesicles for clinical application in angiogenesis.

[0098] Example 11. Confirmation of neurovascularization effects by EV treatment of 3D static spheroids To demonstrate the angiogenic growth effect of extracellular vesicles obtained by 3D stem cell culture, HUVECs attached to Matrigel were treated with WJ-2D-EV and the 3D-static spheroid EV of the present invention, and the tube formation effect was confirmed. Specifically, HUVECs were cultured in M199 medium (Gibco) supplemented with 20% FBS, 5 U / mL heparin, and 3 ng / mL bFGF. 1.7 × 10 cells were cultured. 4Cells were seeded onto growth factor-reduced Matrigel Matrix (BD Bioscience, MA, USA) in μ-Slides Angiogenesis (ibidi, Graefelfing, Germany) at a density of 1000 μg / ml and allowed to form tubes for 4 hours in a humidified chamber at 37°C and 5% CO2. Images were taken with a phase-contrast microscope (Olympus), and the number of tube-like structures was quantified per microscopic field (4x magnification) using ImageJ software. The results are shown in Figure 13.

[0099] As can be seen from Figure 13, the experimental group treated with the 3D-static-spheroid EV of the present invention showed significantly increased tube formation compared to the group treated with VEGF, an angiogenesis-related protein, and also showed superior tube formation effects compared to the WJ-2D-EV treatment group.

[0100] Example 12. Effect of 3D-static-spheroid EVs on stroke animal models 12.1 Creation of a stroke model To create an animal stroke model, a photothrombotic (PT) cerebral infarction model was established. PT stroke was induced in the right sensorimotor cortex of adult male C57BL / 6J mice (Orient Bio Inc., Seongnam, South Korea) weighing 20–25 g (8–12 weeks old). Briefly, mice were anesthetized with a mixture of ketamine (100 mg / kg, Yumiko Yoko, Seoul, South Korea) and xylazine (10 mg / kg, Rompon® inj., Bayer, Berlin, Germany) administered intraperitoneally and placed in a stereotaxic apparatus (KOPF Instruments, Tujunga, CA, USA). A midline incision was made along the scalp from the eye to the neck using a scalpel, and the periosteum was removed to expose the skull. Rose Bengal solution (30 mg / kg, 10 mg / mL saline, Sigma-Aldrich, St. Louis, MO, USA) was administered intravenously through the jugular vein. Five minutes later, a 100 mW, 532 nm diode-pumped solid-state green laser (Dongwoo Optron Co., Ltd., Gwangju) was positioned 2.5 mm lateral to the bregma, providing a 3 mm diameter illumination. The laser was activated in the region of interest (ROI) for 15 minutes, and the incision was sutured using 6-0 monofilament sutures. The rat's body temperature was maintained at 37.0–37.5°C throughout the surgery.

[0101] 12.2 Confirmation of the effect of improving lesions in a stroke model The stroke animal model prepared in Example 12.1 was treated with PBS as the control group or 3D-static-spheroid EV as the experimental group at 6x10 8 After intravascular injection of EV / mouse, MRI T2-weighted images (T2WI) were taken 3 days (PBS group, n = 20; EV group, n = 23), 14 days (PBS group, n = 8; EV group, n = 10), and 28 days (PBS group, n = 8; EV group, n = 6) to compare the extent of cerebral infarction and ventricular volume changes between the experimental and control groups. The results are shown in Table 3 and Figure 14.

[0102] [Table 3]

[0103] As can be seen from Table 3 and Figure 14, the cerebral infarction lesions and ventricular volume were significantly reduced in the experimental group injected with 3D-static-spheroid EVs compared to the PBS-treated control group.

[0104] 12.3 Confirmation of angiogenic effects through immunohistochemical analysis Immunohistochemical analysis was performed to confirm whether injection of the 3D-static spheroid EVs of the present invention into an animal model of stroke could effectively generate neurovascularization. The animal model of stroke prepared in Example 12.1 was injected with 0.3 x 10 PBS as a control group or 0.3 x 10 3D-static spheroid EVs as an experimental group. 8 EV / mouse~30×10 8 EV / mouse was intravenously injected, and 4 weeks after the stroke, immunofluorescence analysis was performed on the brain tissue using collagen IV, a blood vessel marker, to confirm the number of blood vessels. The results are shown in Figure 15.

[0105] As shown in Figure 15, angiogenesis was observed in the stroke animal model injected with the 3D static spheroid EVs of the present invention compared to the PBS-treated control group, and this was confirmed to be dependent on the number of EVs injected.

[0106] 12.4 Confirmation of angiogenic effects through behavioral analysis It was confirmed that injection of the 3D-static spheroid EVs of the present invention into an animal model of cerebral infarction effectively induced angiogenesis. A forelimb asymmetry test (cylinder test) and a limb loss test (grid walking test) were performed to confirm whether such angiogenesis could restore motor function loss induced in the animal model of cerebral infarction. More specifically, the 3D-static spheroid EVs (6 x 10 8EV / mouse) was injected into the blood vessels of rats, and analysis was performed on days 14 (PBS group, n = 8; 3D-static-spheroid-EV-treated group, n = 10) and 28 (PBS group, n = 8; 3D-static-spheroid-EV-treated group, n = 6). The results are shown in Figure 16.

[0107] As shown in Figure 16, injection of the 3D static spheroid EVs of the present invention improved both the cylinder test and grid walking test indices. These results demonstrate that treatment with the 3D static spheroid EVs of the present invention can induce angiogenesis in an animal model of stroke, thereby improving impaired motor function.

[0108] Example 13: Verification of the effect of 3D static spheroid EVs on manufacturing conditions 13.1 Experimental methods and conditions The above examples confirmed the excellent effects of the 3D static spheroid EVs of the present invention. To confirm whether similar effects could be achieved in EVs produced using the same manufacturing method as in Example 1 but with different microwell specifications and cell numbers, the cell count per microwell was changed from 400 cells / well to 200 cells / well, and the microwell specifications were changed from 500 μm x 200 μm in diameter and depth to 500 μm x 600 μm, or to flat, 800 μm diameter wells.

[0109] The experimental conditions changed according to the manufacturing method of Example 1 are shown in Table 4 below.

[0110] [Table 4]

[0111] WJ-MSCs at passage 6 (as prepared in Example 1.1) were washed with PBS, treated with trypsin (TrypLE™ Express, GIBCO, NY, USA), and incubated in a CO2 incubator for 5 minutes. Fresh serum-free medium was then added, the trypsin was neutralized, and the cells were recovered. A cell pellet was obtained using a centrifuge. Fresh serum-free medium was then added, a cell suspension was prepared, and the cells were counted. After cell counting, the cells were evenly dispensed into microwells under the same conditions as in Table 4, maintained in a static state, and spontaneous spheroid cell aggregate formation was induced. The cells were then cultured in a CO2 incubator at 37°C for a total of 4 days to prepare 3D spheroid cell aggregate culture medium.

[0112] 13.2 Confirmation of 3D spheroid-type cell aggregate morphology It was confirmed that uniform spheroids were formed under the conditions of Experimental Examples 1 to 3, as in Example 1. The culture medium of the 3D spheroid-type cell aggregates 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 roundness and solidity of the spheroids were also confirmed. The results are shown in Figure 17.

[0113] 17, it was confirmed that the sizes of all 3D spheroid cell aggregates produced in Experimental Examples 1 to 3 were formed within the range of 55 to 131 μm, which is the size range of the cell aggregates produced in Example 1, and that the average value of the size distribution was 70.34 to 99.51 μm. Furthermore, when the roundness and solidity were confirmed, it was confirmed that the spheroid cell aggregates in Experimental Examples 1 to 3 also showed average roundness values ​​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 cell aggregates in Example 1, and similar to the average solidity value of 0.9488 (CV 2.64%) of the 3D spheroid cell aggregates in Example 1, similar to the average solidity value of 0.9488 (CV 2.64%) of the 3D spheroid cell aggregates in Experimental Examples 1 to 3 also showed average values ​​of 0.9744, 0.1, and 0.9752, respectively.

[0114] These results indicate that 3D spheroid cell aggregates with 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.

[0115] 13.3 Comparison of miRNA expression patterns of EVs derived from 3D spheroid types Since it was confirmed that spheroid cell aggregates with morphology similar to that of Example 1 could be produced under the conditions of Experimental Examples 1 to 3, extracellular vesicles were isolated from these aggregates using the method of Example 1.3 to confirm whether the isolated EVs also exhibited similar miRNA expression patterns. Comparison of miRNA expression patterns was performed using extracellular vesicles derived from spheroid cell aggregates produced by the methods of Experimental Examples 1 and 2, which differed in microwell conditions. Expression miRNA analysis was performed using the same qPCR method as in Example 8. The results are shown in Figure 18. The miRNA expression patterns were compared with those of 2D-EVs produced in Example 7.

[0116] As shown in Figure 18, even when the microwell diameter was changed to 500 or 800 μm, the expression of miR-132 and miR-210, miRNAs that are effective in angiogenesis / neurogenesis and immunomodulation, was significantly increased compared to existing 2D-EVs, as was the case with the EVs produced in Example 1. These results demonstrate that EVs derived from three-dimensional spheroid cell aggregates obtained by the method of the present invention using microwells with diameters of 200 to 800 μm exhibit common miRNA marker expression characteristics. Therefore, these EVs are sometimes referred to as 3D-static-spheroid EVs.

[0117] 13.4 Confirmation of the angiogenic effect of EVs derived from 3D spheroids An experiment was conducted using microwells with diameters of 200 to 800 μm to confirm the angiogenic effect of 3D static spheroid EVs obtained by the method of the present invention. The experimental groups were 3D static spheroid EVs produced under the conditions of Experimental Examples 1 and 2 and Example 1, and 2D EVs produced in Example 7. The tube-forming effect was confirmed using the same method as in Example 11. The results are shown in Figure 19.

[0118] As shown in Figure 19, all 3D-static-spheroid EVs prepared under the conditions of Experimental Examples 1 and 2 and Example 1 showed significantly superior tube formation effects compared to the control group and 2D-EVs.

[0119] In summary, the 3D-static-spheroid EVs of the present invention highly express miRNAs and angiogenic factors related to angiogenesis, and have been confirmed to have excellent angiogenic ability and associated stroke-improving effects in vitro and in vivo. Therefore, they are expected to be highly effective in preventing or treating various diseases that require vascular damage and angiogenesis.

[0120] Although the present invention has been described in detail above, it will be obvious to those skilled in the art that the specific description is merely a preferred embodiment and does not limit the scope of the present invention. Therefore, the true scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. (a) culturing stem cells three-dimensionally (3D) 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) isolating extracellular vesicles from the three-dimensional spheroid cell aggregates; A pharmaceutical composition for promoting angiogenesis, comprising extracellular vesicles derived from three-dimensional spheroid cell aggregates produced through the method described above.

2. The pharmaceutical composition for promoting angiogenesis according to claim 1, wherein 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 promoting angiogenesis according to claim 1, wherein the three-dimensional culture in step (a) is a static culture.

4. 2. The pharmaceutical composition for promoting angiogenesis according to claim 1, wherein the three-dimensional culture in step (a) comprises dispensing mesenchymal stem cells into microwells at a density of 200 to 600 cells / well and culturing the cells.

5. The pharmaceutical composition for promoting angiogenesis according to claim 1, wherein the extracellular vesicles express one or more types selected from the group consisting of miR-27a, miR-132, miR-146a, miR-146b, miR-184, and miR-210 at a higher level than extracellular vesicles derived from spheroid-type cell aggregates formed by three-dimensional dynamic culture of mesenchymal stem cells and extracellular vesicles derived from two-dimensionally cultured mesenchymal stem cells.

6. The pharmaceutical composition for promoting angiogenesis according to claim 1, wherein the extracellular vesicles express at least one selected from the group consisting of VEGF (Vascular endothelial growth factor), Hif-1a (Hypoxia-inducible factor 1-alpha), and FGF (Fibroblast growth factor) at a higher level than extracellular vesicles derived from spheroid-type cell aggregates obtained by three-dimensional dynamic culture of mesenchymal stem cells and extracellular vesicles derived from two-dimensionally cultured mesenchymal stem cells.

7. The pharmaceutical composition for promoting angiogenesis according to claim 1, wherein the composition for promoting angiogenesis is for the prevention or treatment of one or more conditions selected from the group consisting of burns, ulcers, ischemia, arteriosclerosis, angina pectoris, myocardial infarction, cardiovascular disease, cerebrovascular disease, and alopecia.

8. The pharmaceutical composition according to claim 1, wherein the composition for promoting angiogenesis is for promoting cerebral angiogenesis.

9. (a) culturing stem cells three-dimensionally (3D) 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) isolating extracellular vesicles from the three-dimensional spheroid cell aggregates; A food composition for promoting angiogenesis, comprising extracellular vesicles derived from three-dimensional spheroid cell aggregates produced through the method.

10. (a) culturing stem cells three-dimensionally (3D) 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) isolating extracellular vesicles from the three-dimensional spheroid cell aggregates; An in vitro composition for promoting angiogenesis, comprising extracellular vesicles derived from three-dimensional spheroid cell aggregates produced through the method.

11. (a) culturing stem cells three-dimensionally (3D) 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) isolating extracellular vesicles from the three-dimensional spheroid cell aggregates; A method for producing a composition for promoting angiogenesis, comprising extracellular vesicles derived from three-dimensional spheroid cell aggregates, comprising:

12. (a) three-dimensionally (3D) culturing stem cells in microwells having a diameter of 200 to 800 μm and a depth of 100 to 1000 μm to prepare three-dimensional spheroid-type cell aggregates; (b) preparing extracellular vesicles derived from the three-dimensional spheroid-type cell aggregates by separating the extracellular vesicles from the three-dimensional spheroid-type cell aggregates; and (c) processing the extracellular vesicles derived from the three-dimensional spheroid-type cell aggregates prepared through step (b) into a desired object; A method for promoting angiogenesis, comprising:

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