Composition for promoting angiogenesis containing extracellular vesicles derived from three-dimensional spheroid cell aggregates

Abstract

The present invention relates to a composition for promoting angiogenesis, which contains extracellular vesicles derived from three-dimensional spheroid-type cell aggregates produced by a novel production method, and a method for promoting angiogenesis. The extracellular vesicles produced by the novel method of the present invention can highly express various factors capable of promoting angiogenesis and induce vascular regeneration, and therefore can be usefully used in the treatment of various diseases requiring vascular regeneration.

Description

Technical Field

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

[0002] Positive clinical results of therapies using stem cells, particularly mesenchymal stem cells (MSC), for various diseases have been reported. However, stem cell therapeutic agents carry the risk of cell-related side effects such as vascular occlusion, tumor formation, and coagulation disorders as adverse effects, and the fact is that efficacy verification through clinical trials is still required. It is known that the paracrine effect of stem cells induces the regeneration of surrounding skin cells and enhancement of angiogenesis capacity, and in particular, extracellular vesicles (EV) are known to be the main effective factor for the paracrine effect.

[0003] Extracellular vesicles are classified into exosomes and microvesicles according to size, where exosomes have a diameter of 30 to 150 nm, and microvesicles have a size of 100 to 1,000 nm. Extracellular vesicles are formed when a portion of a cell membrane is released into the bloodstream, contain both proteins and nuclear components, and are known to mediate intercellular communication. The use of extracellular vesicles instead of stem cells not only minimizes the side effects caused by the use of stem cells and improves safety, but is also advantageous in terms of biodistribution and production processes.

[0004] However, methods for mass production and obtaining of stem cell-derived extracellular vesicles have not yet been established, and few studies have been conducted on methods that can further enhance the efficacy of extracellular vesicles while maintaining the properties of stem cell-derived extracellular vesicles.

[0005] Therefore, there is a need for extracellular vesicles with further enhanced efficacy and novel therapeutic agents utilizing them. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] Therefore, the inventors of the present invention researched a method for mass-producing three-dimensional spheroid-type cell aggregates or extracellular vesicles derived therefrom that can shorten the culture time. As a result, they completed the present invention by confirming that when three-dimensional spheroid-type cell aggregates (3D-static-spheroids) are produced by static culture using microwells, and extracellular vesicles are produced using these aggregates, extracellular vesicles with improved angiogenesis-promoting ability are produced.

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

[0008] To achieve the above objective, the present invention provides a pharmaceutical composition for promoting angiogenesis, comprising the steps of (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 cell aggregates; and (b) separating extracellular vesicles from the three-dimensional spheroid cell aggregates; and extracellular vesicles derived from the three-dimensional spheroid cell aggregates.

[0009] Furthermore, the present invention provides a food composition for promoting angiogenesis, comprising the steps of (a) culturing stem cells three-dimensionally (3D, 3-dimensional) 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; and including extracellular vesicles derived from the three-dimensional spheroid-type cell aggregates produced through these steps.

[0010] Furthermore, the present invention provides an in vitro composition for promoting angiogenesis, comprising the steps of (a) culturing stem cells three-dimensionally (3D, 3-dimensional) 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) separating extracellular vesicles from the three-dimensional spheroid cell aggregates; and extracellular vesicles derived from the three-dimensional spheroid cell aggregates.

[0011] Furthermore, the present invention provides a method for producing an angiogenesis-promoting composition comprising (a) three-dimensional (3D) culture of 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) separating extracellular vesicles from the three-dimensional spheroid cell aggregates.

[0012] Furthermore, the present invention provides an angiogenesis promotion method comprising: (a) three-dimensional (3D) culture of 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; (b) producing extracellular vesicles derived from the three-dimensional spheroid cell aggregates by separating extracellular vesicles from the three-dimensional spheroid cell aggregates; and (c) processing an object requiring the extracellular vesicles derived from the three-dimensional spheroid cell aggregates produced through step (b). [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, thus making them useful in the treatment of various diseases that require vascular regeneration. [Brief explanation of the drawing]

[0014] [Figure 1] This diagram illustrates the process of producing 3D spheroid-type cell aggregates and separating extracellular vesicles from them. [Figure 2] Figure A shows an observation of a microwell containing 3D-dynamic-PEG spheroid culture medium. Figure B shows an observation of a microwell containing 3D-static-spheroid culture medium. Figure C shows the change in aggregate area of ​​3D-dynamic-PEG spheroids and 3D-static-spheroids produced using microwells over time. [Figure 3] This figure compares the size distribution of 3D static spheroids and 3D dynamic PEG spheroids. [Figure 4] This is a diagram showing the morphology of a 3D static spheroid EV observed with an electron microscope. [Figure 5] This figure shows the results of nanoparticle tracking analysis (NTA) to confirm the concentration and size distribution of 3D static spheroid EVs. [Figure 6] The figure shows the results of ELISA and Western blot to confirm the expression markers of 3D-static spheroid EVs. [Figure 7] This figure compares the production of 3D-static-spheroid EV, 3D-dynamic-PEG-spheroid EV, and 2D-EV per derived cell. [Figure 8] This figure shows miRNAs and proteins that are highly expressed in 3D-static-spheroid EV compared to 2D-EV. [Figure 9] This figure shows angiogenesis-related miRNAs that are highly expressed in 3D-static spheroid EV compared to 3D-dynamic PEG spheroid EV. [Figure 10]This figure shows the results of comparing the expression of miR-210, an angiogenesis-related miRNA, in 2D-EV and 3D-static spheroid EV; the results of confirming GAPDH in HUVEC cells treated with 2D-EV and 3D-static spheroid EV; and the results of confirming the effect of the EVs on Ephrin A3 expression inhibition. [Figure 11a] This figure shows the results of confirming differences in EV production yield, EV size, and the amount of protein contained in EV among different donors for 2D-EV and 3D-static spheroid EV (WJ-3D EV). [Figure 11b] This figure shows the results of confirming donor variation and differences in the expression level of angiogenesis-related miRNA in 2D-cultured WJ-MSC, 3D-cultured WJ-MSC, 2D-EV, and 3D-static spheroid EV. [Figure 12] This figure shows the results of confirming changes in the expression of angiogenesis-related factors VEGF, Hif-1a, and FGF after treating vascular endothelial cells (HUVECs) with 3D-static spheroid EV. [Figure 13] This figure shows the results of comparing the tube formation effect after treating vascular endothelial cells (HUVECs) with PBS (control), VEGF, 2D-EV, and 3D-static spheroid EV. [Figure 14] This figure shows the results of confirming the degree of volume changes in cerebral infarct lesions and ventricles after treating cerebral infarction animal models with PBS as a control group or 3D-static spheroid EV as an experimental group. [Figure 15] This figure shows the results of confirming the effect of neurovascular generation after treating cerebral infarction animal models with PBS as a control group or 3D-static spheroid EV as an experimental group. [Figure 16] This figure shows the results of confirming the recovery effect on motor function loss after injecting 3D-static spheroid EV into cerebral infarction animal models. [Figure 17]This figure shows the results of verifying the size, roundness, and solidity of 3D spheroids produced in 3D static spheroid EV manufacturing, after varying the diameter, depth, and number of cells per well of the microwells. [Figure 18] This figure shows the results of comparing the expression levels of miRNA-132 and miRNA-210 expressed in 3D-static-spheroid EVs and 2D-EVs produced under various conditions. [Figure 19] This figure shows the results of tube formation experiments to confirm the angiogenic capacity of 3D-static-spheroid EVs and 2D-EVs manufactured under various conditions. [Best Mode for Carrying Out the Invention]

[0015] The present invention provides a pharmaceutical composition for promoting angiogenesis, comprising the steps of (a) culturing stem cells in three dimensions (3D) 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) separating extracellular vesicles from the three-dimensional spheroid cell aggregates; and extracellular vesicles derived from the three-dimensional spheroid cell aggregates.

[0016] The cells in this invention can be used without limitation as long as they are capable of separating extracellular vesicles, and may be cells isolated from objects of organisms in nature. Furthermore, the cells may be derived from any type of animal, including humans and non-human mammals, or from 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] In this invention, the three-dimensional culture means that cells are cultured in a three-dimensional arrangement within a test tube. Unlike two-dimensional culture, in three-dimensional culture, cell growth allows cells to grow in any direction outside the body (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 carried out by any three-dimensional cell culture technique known in the art to which the present invention belongs, for example, cell culture using microwell array culture, porous microparticle culture, hanging drop culture, low attachment plate culture, membrane-based cell-detachment culture, thermal lifting culture, centrifugation culture, semisolid medium culture, etc. Preferably, the three-dimensional culture may be dynamic culture or static culture, and more preferably static culture. In the present invention, when the three-dimensional culture in step (a) is performed as static culture, the equipment necessary for shaking culture is not required, which makes the culture even easier and has the advantage of enabling large-scale culture in GMP (Good Manufacturing Practices, standards for manufacturing management and quality control of pharmaceuticals and quasi-drugs) manufacturing plants.

[0019] In the present invention, the three-dimensional culture in step (a) may be cultured for 1 to 10 days, and preferably for 2 to 4 days. In the present invention, when the culture in step (a) is cultured for 2 to 4 days, the viability of cells present in the three-dimensional spheroid cell aggregate is maintained at a high level, and the culture time is relatively shorter compared to existing three-dimensional spheroid cell aggregate manufacturing processes, allowing for the rapid production of three-dimensional spheroid cell aggregates and extracellular vesicles derived therefrom.

[0020] In the present invention, the three-dimensional culture in step (a) above may involve dispensing mesenchymal stem cells into microwells at a density of 100 to 1000 cells / well and culturing them, preferably dispensing at a density of 100 to 600 cells / well and culturing them, and more preferably dispensing at a density of 100 to 500 cells / well and culturing them.

[0021] In the present invention, the step of separating the extracellular vesicles in step (b) above can be carried out using a method selected from the group consisting of extrusion, sonication, cell lysis, homogenization, freeze-thaw, electroporation, chemical treatment, mechanical decomposition, and treatment with physical stimulation by applying external force to the cells, preferably by tangential flow filtration (TFF) method, but not limited thereto.

[0022] In this invention, microRNAs are named by prefixing them with "mir" followed by a hyphen and a number. The number often indicates the order in which they were named; for example, mir-123 was named before mir-156 and is therefore presumed to have been discovered earlier. "mir-" indicates pre-microRNA, while "miR" (with a capital letter) signifies mature microRNA. MicroRNAs with nearly identical sequences, except for one or two, are named by adding a lowercase letter. For example, miR-121a and miR-121b are produced by their respective precursors, mir-121a and mir-121b, and their sequences are very similar. Pre-microRNAs, which are the same mature microRNA but located in different parts of the genome, are further named by adding a hyphen 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 in different parts of the genome. MicroRNA nomenclature is indicated by the species. For example, hsa-miR-123 is human (Homo sapiens) microRNA, and oar-miR-123 is sheep (Ovis aries) microRNA. "v" means viral (miRNA encoded by a viral genome), and "d" means Drosophila microRNA. If two mature microRNAs originate from different arms (3' arm or 5' arm) of the same pre-microRNA, "-3p" or "-5p" is added to the end of the name. miR-142-3p originates from the 3' arm, and miR-142-5p originates from the 5' arm. MicroRNA nomenclature is generally based on the above criteria, but there are exceptions.

[0023] In the present invention, the extracellular vesicles may express clinically significant substances at a higher level compared to known extracellular vesicles, and the clinically significant substances may be substances that exhibit angiogenic effects. For example, preferably, the extracellular vesicles of the present invention may express one or more substances selected from the group consisting of miR-27a, miR-132, miR-146a, miR-146b, miR-184, and miR-210, which are associated with angiogenesis, at a higher level compared to extracellular vesicles derived from spheroid cell aggregates obtained by three-dimensional dynamic culture of mesenchymal stem cells and extracellular vesicles derived from two-dimensional cultured mesenchymal stem cells, or they may express one or more substances selected from the group consisting of VEGF (Vascular endothelial growth factor), Hif-1a (Hypoxia-inducible factor 1-alpha), and FGF (Fibroblast growth factor). More preferably, the extracellular vesicles of the present invention may express miR-27a, miR-132, miR-146a, miR-146b, miR-184, and miR-210, VEGF, Hif-1a, and FGF, which are associated with angiogenesis, more highly than extracellular vesicles derived from spheroid cell aggregates obtained 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 can be internalized upon application to cells, and when internalized, they can effectively deliver clinically significant substances highly expressed in the extracellular vesicles to the cells, leading to their high expression within the cells.

[0025] In this invention, extracellular vesicles derived from three-dimensional spheroid cell aggregates exhibiting angiogenesis-promoting effects can be used interchangeably with "3D-static-spheroid-EV". In contrast, extracellular vesicles derived from three-dimensionally dynamically cultured spheroid cell aggregates can be used interchangeably with "3D-dynamic-PEG-spheroid-EV".

[0026] In the present invention, "extracellular vesicles derived from spheroid cell aggregates obtained by three-dimensional dynamic culture of mesenchymal stem cells" may include, without limitation, any extracellular vesicles isolated from spheroid cell aggregates obtained by three-dimensional dynamic culture of mesenchymal stem cells, and preferably, the extracellular vesicles disclosed in the Korean Registered Patent (Application No. 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, any extracellular vesicles that are cultured according to a conventional two-dimensional culture method and isolated by a conventional method for separating extracellular vesicles. In one embodiment of the present invention, the conventional two-dimensional culture method was carried out by washing stem cells cultured in a cell stack for 3 days with PBS, replacing the medium with serum-free medium, and culturing for a further 2 days.

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

[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 material may be coated with any of the following selected materials: acrylate, BHPEA (2-(4-Benzoyl-3-hydroxyphenoxy)ethyl acrylate), 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 it is not limited to these.

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

[0032] The aforementioned structure allows mesenchymal stem cells cultured with this structure to maintain a high level of viability even after a period of culture time. Preferably, a microarray containing 1,000 to 100,000 microwells can be prepared to increase the production yield of cell aggregates.

[0033] By producing extracellular vesicles using the "method for producing extracellular vesicles derived from three-dimensional spheroid cell aggregates" in the present invention, in addition to the advantages of production by static culture, it is possible to rapidly and efficiently mass-produce extracellular vesicles with improved clinical applicability, particularly angiogenic ability. In particular, unlike conventional methods for producing extracellular vesicles, such as the method for producing "extracellular vesicles derived from spheroid cell aggregates obtained by three-dimensional dynamic culture of mesenchymal stem cells" or the method for producing "extracellular vesicles derived from two-dimensionally cultured mesenchymal stem cells," the method for producing extracellular vesicles in the present invention is suitable for GMP application, and is therefore particularly suitable for the production of pharmaceutical compositions for angiogenesis.

[0034] In this invention, angiogenesis refers to the formation of new blood vessels, which involves inducing or increasing the migration of vascular endothelial cells, promoting tube formation in vascular endothelial cells, and resulting in the formation of 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 a variety of factors that can promote angiogenesis and induce vascular regeneration, and therefore can be usefully used in the treatment of various diseases requiring vascular regeneration.

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

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

[0038] Brain tissue normally receives a large supply of blood, but external or internal stimuli can cause blockages in the cerebral blood vessels. When the amount of blood supplied to the brain decreases, the brain tissue ceases to function normally. If the decrease in cerebral blood flow persists for a certain period of time, the brain tissue will die. When the brain tissue dies to an irreversible state, it is called a cerebral infarction, which includes cerebral thrombosis and cerebral embolism. Thrombosis is when blood clots form locally in a blood vessel, blocking it. When a blood clot forms, blood flow is impaired in that area, and if collateral pathways from other parts of the body are insufficient to compensate, 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 bloodstream, blocking peripheral blood vessels. When a blood vessel is blocked by an embolism, the brain tissue in that area dies. The extracellular vesicles derived from the three-dimensional spheroid-type cell aggregates of the present invention can promote the formation of new cerebral angiogenesis and may therefore be useful in restoring reduced cerebral blood flow caused by cerebral infarction.

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

[0041] The pharmaceutical compositions of the present invention can be prepared by conventional methods into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, as well as topical preparations, suppositories, and sterile injection solutions. Examples of carriers, excipients, and diluents that may be included 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, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulation, the compositions are prepared using commonly used fillers, bulking agents, binders, wetting agents, disintegrants, surfactants, and other diluents or excipients. Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc. Such solid dosage forms are prepared by mixing the composition with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

[0042] Liquid formulations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients may be included, such as humectants, sweeteners, fragrances, and preservatives. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injection esters such as ethyl oleate. Suppository bases may include witepsol, macrogol, Tween 61, cocoa butter, lauric acid butter, and glycerol gelatin.

[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. The administration may be once a day or divided into several doses. The aforementioned dosage does not limit the scope of the present invention in any way.

[0044] The pharmaceutical compositions of the present invention can be administered to mammals such as rats, mice, livestock, and humans via a variety of routes. All methods of administration are conceivable, but for example, they can be administered orally, rectally or intravenously, intramuscularly, subcutaneously, intrauterine dura mater, or intraventricularly.

[0045] The definitions of the terms excipients, binders, disintegrants, lubricants, flavoring agents, etc., in this invention include those described in literature known to the industry and whose functions are the same or similar.

[0046] Furthermore, the present invention provides an angiogenesis promotion method comprising: (a) three-dimensional (3D) culture of 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; (b) a step of producing extracellular vesicles derived from the three-dimensional spheroid cell aggregates by separating extracellular vesicles from the three-dimensional spheroid cell aggregates; and (c) a step of processing an object requiring the extracellular vesicles derived from the three-dimensional spheroid cell aggregates produced in step (b).

[0047] The aforementioned object is preferably a mammal, including humans, and includes all patients who require angiogenesis and are currently being treated, have been treated in the past, or need treatment 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 drugs or treatments for angiogenesis. When the present invention is used in combination, it can be used simultaneously with or sequentially with other drugs or treatments for angiogenesis.

[0049] Furthermore, the present invention provides a food composition for promoting angiogenesis, comprising the steps of (a) culturing stem cells in three dimensions (3D) 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) separating extracellular vesicles from the three-dimensional spheroid cell aggregates; and extracellular vesicles derived from the three-dimensional spheroid cell aggregates.

[0050] The food composition can be described in the same manner as the description relating to the pharmaceutical composition. The food includes, in particular, functional foods for health. As defined in this invention, "functional foods for health" means foods manufactured and processed using raw materials or ingredients that have functional properties useful to the human body, and "functional" means being taken for the purpose of obtaining effects useful for health purposes, such as regulating nutrients or physiological effects on the structure and function of the human body. The functional foods for health may be in the form of tablets, capsules, powders, granules, liquids, or pills.

[0051] Furthermore, the food composition of the present invention may be a food composition in which functional ingredients are added to various foods or beverages. The food can take any of the following forms: a beverage, a powdered beverage, a solid, chewing gum, tea, a vitamin complex, or a food additive.

[0052] Furthermore, the present invention relates to an in vitro composition for promoting angiogenesis, comprising the steps of (a) culturing stem cells in three dimensions (3D) 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) separating extracellular vesicles from the three-dimensional spheroid cell aggregates; and including extracellular vesicles derived from the three-dimensional spheroid cell aggregates.

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

[0054] Furthermore, the present invention provides a method for producing an angiogenesis-promoting composition containing extracellular vesicles derived from three-dimensional spheroid cell aggregates, comprising the steps of (a) culturing stem cells in three dimensions (3D, 3-dimensional) 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) separating extracellular vesicles from the three-dimensional spheroid cell aggregates.

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

[0056] Repetitive information has been omitted in consideration of the complexity of this specification, and terms used herein, unless otherwise specified, have the meanings commonly used in the art to which the present invention pertains.

[0057] The present invention will be described in 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. [Examples]

[0058] Western blot Cells and extracellular vesicles were dissolved in a 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 a protease inhibitor). 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 cultured overnight at 4°C with primary antibodies against histone H2A / Z, histone H3, lamin A / C, flutirin-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, nitrocellulose membranes were cultured for 2 hours with 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). Labeled proteins were visualized using X-ray film (Agfa, Mortsel, Belgium).

[0059] ELISA ELISA was performed using commercial kits according to the individual manufacturers' manuals. 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 included standard proteins; therefore, the amounts of protein and extracellular vesicles were determined based on the standard curve for each kit.

[0060] qPCR Trizol in EV TM RNA was extracted using the manufacturer's guidelines, and the RNA was quantified using nanodrop. The RNA was then synthesized into 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 manual.

[0061] EV labeling and cellular absorption Purified extracellular viable cells (EVs) were stained with CFSE (5-(and-6)-Carboxyfluorescein Diacetate, Succinimidyl Ester) labeling dye (c-1157, Invitrogen) according to the manufacturer's guidelines. Excess dye was removed by ultracentrifugation at 100,000 g per hour. Labeled EVs (0.4 μg / ml) were treated with human NSC cell lines (ReNcells (trademark)) and cultured for 24 hours. After treatment, the cells were washed twice with PBS and stained using a standard immunocytochemistry protocol. The cells were cultured overnight at 4°C with mouse anti-SMA (1:100, Sigma Aldrich) antibody. Subsequently, the cells were washed with PBS and cultured with a secondary antibody, DyLight-labeled anti-mouse IgG (1:200, 594 nm, Abcam) antibody. 1.5 μg / mL 4'-6'-2-phenylindole (DAPI) (Vector Laboratories) along with Vectashield TM After nuclear staining using [a specific method], the 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 Preparation of Mesenchymal Stem Cells Human umbilical cord-derived mesenchymal stem cells (WJ-MSCs, Samsung Medical Center, Seoul, South Korea) in passage 5 were obtained and cultured in a 37°C, 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 in passage 6 were used to create 3D spheroid cell aggregates.

[0063] 1.2 Preparation of 3D spheroid cell aggregate culture medium WJ-MSCs at the 6th passage stage, 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. 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 cell suspension was uniformly dispensed into a microarray containing approximately 69,000 microwells, each 500 μm in diameter and 200 μm in depth, coated with MPC (2-Methacryloyloxyethyl Phosphorylcholine Polymer), to a density of 400 cells / well. A static state was maintained to induce the formation of spontaneous spheroid cell aggregates, and the cells were cultured in a CO2 incubator at 37°C for a total of 4 days to produce a 3D spheroid cell aggregate culture medium (hereinafter referred to as 3D-static-spheroid culture medium).

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

[0065] The processes of the above-mentioned Examples 1.1 to 1.3 are illustrated in Figure 1.

[0066] Example 2. Analysis of the properties of 3D spheroid cell aggregates The properties of 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 experimental group used the 3D-static spheroid culture medium prepared in Example 1.2, while the comparison group used a culture medium of 3D mesenchymal stem cell spheroid-type cell aggregates (hereinafter referred to as 3D-dynamic PEG spheroids) prepared by culturing mesenchymal stem cells for 5 days according to the dynamic 3D cell culture method disclosed in a Korean registered patent (application number 10-2016-0053026, method for producing extracellular vesicles derived from stem cells). Microwells containing each culture medium were observed using an optical microscope and are shown in Figures 2A and 2B. Figure 2C shows the change in the area of ​​spheroid-type cell aggregates after culturing compared to the initial stage of culture.

[0067] As shown in Figure 2, a comparison of the area of ​​the experimental group (3D-static spheroids) and the comparison group (3D-dynamic PEG spheroids) revealed that, due to the characteristics of cell condensation and spheroid formation, the spheroid-to-spheroid area in the 3D-static spheroid group was statistically significantly reduced compared to the 3D-dynamic PEG spheroid group in the initial stages of culture (p=0.0011).

[0068] Example 3. Analysis of the size of 3D spheroid cell aggregates The size distribution of the 3D-static spheroids manufactured in Example 1.2 and the 3D-dynamic PEG spheroids manufactured 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 allows us to understand the direction and degree of the slope of a distribution from the trend of the median. A value closer to 1 indicates a distribution with a longer tail to the right, while a value closer to -1 indicates a distribution with a longer tail to the left. The skewness of a normal distribution is 0.

[0070] Kurtosis is a parameter that indicates the degree of peaking in a data distribution. A positive value means that a relatively large number of data points are concentrated in the central region, while a negative value means that a relatively small number of data points are concentrated in the central region. For a normal distribution, the kurtosis is 0.

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

[0072] [Table 1]

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

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

[0075] A comparison of the size distributions of 3D-static spheroids and 3D-dynamic PEG spheroids revealed that the kurtosis value of the 3D-static spheroid size distribution was relatively large, and the size distribution of the 3D-static spheroids of the present invention showed a concentration of values ​​around the median. These results confirm that the manufacturing method of 3D-static spheroids of the present invention makes it possible to produce 3D spheroids with relatively consistent size.

[0076] Example 4. Analysis of extracellular vesicle morphology derived from 3D spheroid cell aggregates To observe the morphology of the 3D-static spheroid EVs separated in Example 1.3, images were taken using a transmission electron microscope (TEM). Specifically, the 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 onto a droplet of extracellular vesicles with the Formvar side facing downwards for 1 minute. Then, it was blotted with filter paper and reacted with 2% uranyl acetate for 15 seconds. After removing the excess uranyl acetate, the EM grid was observed using TEM (JEM-1011, JEOL, Japan), and the observed image is shown in Figure 4.

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

[0078] Example 5.3 Analysis of the concentration and size of extracellular vesicles derived from D-spheroid cell aggregates. To confirm the concentration and size distribution of 3D-static spheroid EVs separated in Example 1.3, nanoparticle tracking analysis (NTA) was performed using NanoSight NS300 (Malvern, Worcestershire, UK). For optimal analysis, the 3D-static spheroid EVs were pre-diluted in vesicle-free phosphate buffer (PBS). The average size and concentration (particles / mL) were calculated by integrating three records, and the results are shown in Figure 5.

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

[0080] Example 6. Analysis of extracellular vesicle expression markers derived from 3D spheroid cell aggregates Experiments were conducted to confirm the expression markers of 3D-static spheroid EVs isolated in Example 1.3. Cell lysate and secretome were used as control groups. Cell lysate was prepared by washing 3D-static spheroids with PBS, treating them with trypsin, collecting the cells, and obtaining a cell pellet using a centrifuge. Secretome was prepared by obtaining the cell pellet, separating EVs in the culture medium (the supernatant) through the process of Example 1.3, and obtaining the remaining cultured secretion. Marker analysis was performed by quantifying extracellular vesicle-specific positive markers CD9, CD63, CD81, and HSP70 using ELISA, and quantifying specific contaminating protein markers such as calreticulin, histone H2A, Z, cytochrome C, albumin, and antibiotics. Furthermore, using Western blotting, histone H2A, Z, histone H3, lamin A / C, and calreticulin, which are specific contaminating protein markers of extracellular vesicles, were quantified, and flotilin-1, an extracellular vesicle-positive marker, was quantified. The results of the ELISA analysis and Western blotting 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 contaminating protein markers highly expressed in cell lysates and secretomes, such as 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 at very low levels in cell lysates, was found to be relatively highly expressed in 3D-static spheroid EVs.

[0082] Example 7.3 Analysis of extracellular vesicle production from D-spheroid cell aggregates Experiments were conducted to compare the production yields of extracellular vesicles (EVs) produced by the manufacturing method of Example 1 (3D-static-spheroid EVs), extracellular vesicles isolated from 3D-dynamic-PEG spheroids of Example 2 (3D-dynamic-PEG spheroid EVs), and extracellular vesicles isolated from stem cells cultured using a standard 2D culture method (2D-EVs). 2D-EVs were prepared using the following procedure: Stem cells cultured in a cell stack for 3 days were washed with PBS, replaced with serum-free medium, and cultured for another 2 days. The culture medium was collected, and cell debris was continuously removed using centrifugation and a 0.2 μm filter. Subsequently, the culture medium was passed through a hollow fiber membrane using a TFF system to remove proteins, and the EVs were primarily separated. These were then purified again with physiological saline to obtain high-purity 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.

[0083] [Table 2]

[0084] Example 8.3 Analysis of microRNAs (miRNAs) expressed in extracellular vesicles derived from D-spheroid cell aggregates. To confirm the differences in characteristics between 3D-static spheroid EVs, which are extracellular vesicles produced by the manufacturing method of Example 1, 3D-dynamic PEG spheroid EVs, which are extracellular vesicles isolated from 3D-dynamic PEG spheroids of Example 2, and 2D-EVs produced in Example 7, miRNA expression and protein expression were measured by qPCR. Figure 8 shows miRNAs and proteins that are highly expressed in 3D-static spheroid EVs compared to 2D-EVs, and Figure 9 shows angiogenesis-related miRNAs and proteins that are highly expressed in 3D-static spheroid EVs compared to 3D-dynamic PEG spheroid EVs.

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

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

[0087] These results confirm that 3D-static-spheroid EVs, which are extracellular vesicles produced by the manufacturing 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 associated with angiogenesis / neurogenesis, immunomodulation, rejuvenation, or antitumor, and are confirmed to be extracellular vesicles that can be used clinically.

[0088] Example 9.3 Confirmation of the angiogenesis-promoting effect of extracellular vesicles derived from D-spheroid cell aggregates 9.1. Comparison of angiogenesis-related miRNA and Ephrin A3 expression Through Example 8, it was confirmed that 3D-static-spheroid EVs, which are extracellular vesicles produced by the manufacturing method of Example 1, are novel extracellular vesicles with miRNA and protein expression patterns distinct from existing 2D-EVs or 3D-dynamic-PEG spheroid EVs. To confirm the usefulness of 3D-static-spheroid EVs in promoting angiogenesis, HUVEC cells, which are target cells, were treated with 3D-static-spheroid EVs. Changes in the expression of miRNAs and Ephrin A3 (eph-related receptor tyrosine kinase ligand 3) associated with the angiogenic effect were then confirmed by qPCR and Western blotting, and the results are shown in Figure 10.

[0089] As shown in Figure 10, qPCR results confirmed a significant increase in miR-210 expression in the 3D-static-spheroid EV of the present invention compared to existing 2D-EVs. Furthermore, after treatment of 3D-static-spheroid EVs with HUVEC, Ephrin A3 protein expression was significantly inhibited compared to the 2D-EV treated group. miR-210 is known to promote the migration of vascular endothelial cells and form capillary-like structures, and targets Ephrin A3. In other words, these results once again demonstrate that 3D-static-spheroid EVs can effectively induce angiogenesis.

[0090] 9.2. Analysis of differences in miRNA expression among donors Stem cell therapies are known to have the problem of donor variation, where the components differ depending on the donor. Experiments were 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 from Samsung Medical Center (Seoul, South Korea). The number of EVs produced, the amount of EV protein, and the miRNA profile were compared for each donor of 2D-cultured WJ-MSCs and 3D-cultured WJ-MSCs from Example 1.1, and 3D-static spheroid EVs, which are extracellular vesicles produced by the manufacturing method of Example 1. The miRNAs were profiled using the Small RNA sequencing method, 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 amount, size, and amount of EV-producing protein produced among different donors, while 3D-static-spheroid EVs showed consistent results with little variation among donors.

[0092] Furthermore, as shown in Figure 11b, while 2D cultured WJ-MSCs and 2D-EVs showed significant differences in miRNA composition depending on the donor, 3D cultured WJ-MSCs and 3D-static-spheroid EVs showed reduced differences depending on the donor. In particular, 3D-static-spheroid EVs showed no differences depending on the donor, exhibiting a consistent miRNA profile, and were found to contain more uniformly expressed miRNAs that can demonstrate angiogenesis compared to 2D-EVs, such as miR-27a-3p (1.5 times), miR-146a-5p (2.3 times), miR-210 (2.6 times), and miR-132 (2.6 times). In addition, other angiogenesis-related miRNAs such as miR-199a, miR-125b, miR-26a, let-7, miR-125a, miR-181b, and miR-92a were also confirmed to show uniform levels of expression among the donors.

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

[0094] Based on a comprehensive analysis of all the above results, the 3D-static spheroid EVs of the present invention, compared to WJ-2D-EVs and 3D-dynamic spheroid EVs, show a significant increase in miRNAs related to angiogenesis, namely miR-27a, miR-132, miR-146a, miR-146b, miR-184, and miR-210. Furthermore, they inhibit Ephrin A3 protein expression, suggesting they may exhibit superior angiogenesis compared to previously reported EVs, reducing donor-dependent differences and potentially becoming a superior therapeutic agent.

[0095] Example 10. Confirmation of changes in angiogenesis-related factor expression by treatment of extracellular vesicles derived from 3D spheroid cell aggregates. After treating vascular endothelial cells (HUVECs) with 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 standard 2D culture method, we confirmed the 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), and the results are shown in Figure 12.

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

[0097] VEGF and Hif-1a are angiogenesis-related proteins, and FGF is a factor that regulates biological functions related to cell proliferation, survival, and differentiation. This demonstrates that 3D-static spheroid EVs are extracellular vesicles that are clinically superior in angiogenesis.

[0098] Example 11.3 Confirmation of neurovascularization effect by D-static spheroid EV treatment To demonstrate the neovascularization 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. Cell size was 1.7 × 10⁶. 4Cell density was measured using μ-Slides Angiogenesis (ibidi, Graefelfing, Germany) to reduce growth factors, and the cells were inoculated into Matrigel Matrix (BD Bioscience, MA, USA). The cells were then inoculated into a humidified chamber at 37°C with 5% CO2 for 4 hours to form tubes. Images were taken using a phase-contrast microscope (Olympus), and the number of tubular structures was quantified using ImageJ software at the microscope field (4x magnification). The results are shown in Figure 13.

[0099] As can be seen in Figure 13, the experimental group treated with the 3D-static-spheroid EV of the present invention showed a significant increase in tube formation compared to the VEGF-treated group, which is an angiogenesis-related protein, and demonstrated a superior tube formation effect compared to the WJ-2D-EV-treated group.

[0100] Example 12. Effects of 3D-static-spheroid EV on a stroke animal model. 12.1 Creation of a stroke model To create an animal model of stroke, a photothrombotic (PT) stroke model was developed. Using 20-25g (8-12 week old) adult male C57BL / 6J mice (Orient Bio Inc., Seongnam, South Korea), PT stroke was induced in the sensorimotor cortex of the right hand of the mouse. In short, a mixture of ketamine (100 mg / kg, Yuen Trading Co., Seoul, South Korea) and xylazine (10 mg / kg, Rompon (trademark registered) inj., Berlin Bayer, Germany) was administered intraperitoneally to anesthetize the mice, and they were placed in a stereotactic device (KOPF Instruments, Tujunga, CA, USA). A midline incision was made along the scalp from the eye to the neck using a scalpel, the periosteum was removed, and the skull was exposed. Rose bengal solution (30 mg / kg, 10 mg / mL physiological saline, Sigma-Aldrich, St. Louis, MO, USA) was administered intravenously via the jugular vein. Five minutes later, a 100 mW, 532 nm diode-pumped solid-state green laser (Dongwoo Optron, Gwangju) providing a 3 mm diameter illumination was positioned 2.5 mm laterally to the bregma. The laser was activated in the region of interest (ROI) for 15 minutes, and the incision site was sutured with 6-0 monofilament sutures. The rats were kept at a body temperature of 37.0–37.5°C during the surgery.

[0101] 12.2 Confirmation of the effect of improving lesions in stroke models The stroke animal model prepared in Example 12.1 was used as the control group for PBS, or as the experimental group for 3D-static-spheroid EV, in a 6x10x2 8 After intravascular injection using EV / mouse, MRI T2-weighted images (T2WI) were acquired 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), and the degree of cerebral infarction lesions and ventricular volume changes were compared between the experimental group and the control group. The results are shown in Table 3 and Figure 14.

[0102] [Table 3]

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

[0104] 12.3 Confirmation of angiogenic effects through immunohistochemical analysis Immunohistochemical analysis was performed to confirm whether neurovascular structures could be effectively generated when 3D-static-spheroid EV of the present invention was injected into stroke animal models. Stroke animal models prepared in Example 12.1 were injected with PBS as a control group or 3D-static-spheroid EV as an experimental group (0.3 × 10⁻⁶). 8 EV / mouse~30×10 8 The drug was injected intravenously using EV / mouse, and four weeks after the stroke, immunofluorescence analysis was performed on brain tissue using collagen IV, a vascular 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 it was confirmed that this was dependent on the number of EVs injected.

[0106] 12.4 Confirmation of angiogenic effects through behavioral analysis It was confirmed that injecting the 3D-static-spheroid EV of the present invention into an animal model of cerebral infarction effectively induces angiogenesis. To confirm whether such angiogenesis restores motor function loss induced in the animal model of cerebral infarction, forelimb asymmetry tests (cylinder test) and limb loss tests (grid gait test) were performed. More specifically, 3D-static-spheroid EV (6x10) was injected into a fabricated photothrombotic cerebral infarction model. 8EV / mouse) was injected into the blood vessels of rats, and analysis was performed on day 14 (PBS group, n=8; 3D-static-spheroid EV treatment group, n=10) and day 28 (PBS group, n=8; 3D-static-spheroid EV treatment group, n=6). The results are shown in Figure 16.

[0107] As shown in Figure 16, it was confirmed that injecting the 3D-static-spheroid EV of the present invention improved all cylinder test indices and grid gait test indices. These results indicate that the 3D-static-spheroid EV treatment of the present invention induces angiogenesis in stroke animal models, and as a result can improve damaged motor function impairment.

[0108] Example 13 Verification of 3D-static-spheroid EV effect under manufacturing conditions 13.1 Experimental methods and conditions The above examples confirmed the excellent effects of the 3D-static spheroid EV of the present invention. In order to confirm whether similar effects could be observed in EVs produced using the same method as in Example 1, but with different microwell specifications and cell count conditions, the cell count per microwell was changed from 400 cells / well to 200 cells / well, and the microwell specifications were changed to 500 μm × 600 μm, or to flat wells with no depth and a diameter of 800 μm, respectively, and cells were cultured.

[0109] The experimental conditions modified by the manufacturing method in Example 1 are shown in Table 4 below.

[0110] [Table 4]

[0111] WJ-MSCs at the 6th passage stage, 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 collected. 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, the cells were uniformly dispensed into microwells under the same conditions as in Table 4, kept static to induce spontaneous spheroid cell aggregate formation, and cultured in a CO2 incubator at 37°C for a total of 4 days to produce a 3D spheroid cell aggregate culture medium.

[0112] 13.2 Confirmation of 3D spheroid cell aggregate morphology Under the conditions of Experimental Examples 1-3, it was confirmed that spheroids were uniformly formed in the same manner 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 using 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 further confirmed. The results are shown in Figure 17.

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

[0114] These results demonstrate that even when the number of cells per microwell changes to 200 or 400, and the diameter of the microwell changes to 400-800, 3D spheroid-type cell aggregates with similar morphology can be effectively formed by the method of Example 1.

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

[0116] As shown in Figure 18, even when the microwell conditions were changed to diameters of 500 and 800 μm, it was confirmed that miR-132 and miR-210, miRNAs that exhibit efficacy in angiogenesis / neurogenesis and immunomodulation, similar to those produced in Example 1, showed a significant increase in expression compared to existing 2D-EVs. These results indicate 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 can commonly exhibit miRNA marker expression characteristics. Therefore, these can all be referred to as 3D-static-spheroid EVs.

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

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

[0119] In summary, the 3D-static-spheroid EV of the present invention exhibits high expression of angiogenesis-related miRNAs and angiogenic factors, and demonstrates excellent angiogenic ability and associated stroke-improving effects both in vitro and in vivo. Therefore, it is expected to show excellent effects in preventing or treating various diseases that require vascular damage and angiogenesis.

[0120] Having described in detail certain aspects of the present invention, it will be obvious to those with ordinary skill in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Therefore, the substantial scope of the invention is defined by the appended claims and their equivalents.

Claims

1. (a) A step of producing three-dimensional spheroid cell aggregates by three-dimensional (3D) culture of mesenchymal stem cells seeded in microwells having a diameter of 200–800 μm and a depth of 100–1000 μm, which is carried out by dispensing mesenchymal stem cells into microwells at a density of 200–600 cells / well and culturing them; (b) The step of 3D static culture of the three-dimensional spheroid cell aggregates prepared in the microwells; and (c) The step of separating extracellular vesicles from the three-dimensional spheroid cell aggregate; A pharmaceutical composition for promoting angiogenesis, comprising extracellular vesicles derived from three-dimensional spheroid-type cell aggregates manufactured through [method], The extracellular vesicles isolated in step (c) above express one or more selected from the group consisting of miR-27a, miR-132, miR-146a, miR-146b, miR-184, and miR-210 in a higher expression than extracellular vesicles derived from spheroid cell aggregates obtained by three-dimensional dynamic culture of mesenchymal stem cells and extracellular vesicles derived from two-dimensional culture of mesenchymal stem cells. A pharmaceutical composition for promoting angiogenesis, wherein the manufacturing process does not include a shaking culture step.

2. The extracellular vesicles separated in step (c) express one or more selected from the group consisting of Vascular endothermic growth factor (VEGF) / R2 and integrin 1 / 2 in a higher degree than extracellular vesicles derived from mesenchymal stem cells cultured in two dimensions, according to claim 1, for promoting angiogenesis.

3. The angiogenesis-promoting pharmaceutical composition according to claim 1, wherein the angiogenesis-promoting composition 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 diseases, cerebrovascular diseases, and alopecia.

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

5. (a) A step of producing three-dimensional spheroid cell aggregates by three-dimensional (3D) culture of mesenchymal stem cells seeded in microwells having a diameter of 200–800 μm and a depth of 100–1000 μm, which is carried out by dispensing mesenchymal stem cells into microwells at a density of 200–600 cells / well and culturing them; (b) The step of 3D static culture of the three-dimensional spheroid cell aggregates prepared in the microwells; and (c) The step of separating extracellular vesicles from the three-dimensional spheroid cell aggregate; A food composition for promoting angiogenesis, comprising extracellular vesicles derived from three-dimensional spheroid-type cell aggregates manufactured through, The extracellular vesicles isolated in step (c) above express one or more selected from the group consisting of miR-27a, miR-132, miR-146a, miR-146b, miR-184, and miR-210 in a higher expression than extracellular vesicles derived from spheroid cell aggregates obtained by three-dimensional dynamic culture of mesenchymal stem cells and extracellular vesicles derived from two-dimensional culture of mesenchymal stem cells. A food composition for promoting angiogenesis, wherein the manufacturing process does not include a shaking culture step.

6. (a) A step of producing three-dimensional spheroid cell aggregates by three-dimensional (3D) culture of mesenchymal stem cells seeded in microwells having a diameter of 200–800 μm and a depth of 100–1000 μm, which is carried out by dispensing mesenchymal stem cells into microwells at a density of 200–600 cells / well and culturing them; (b) The step of 3D static culture of the three-dimensional spheroid cell aggregates prepared in the microwells; and (c) The step of separating extracellular vesicles from the three-dimensional spheroid cell aggregate; blood vessels containing extracellular vesicles derived from three-dimensional spheroid cell aggregates produced through An in vitro composition for promoting neogenesis, The extracellular vesicles isolated in step (c) above express one or more selected from the group consisting of miR-27a, miR-132, miR-146a, miR-146b, miR-184, and miR-210 in a higher expression than extracellular vesicles derived from spheroid cell aggregates obtained by three-dimensional dynamic culture of mesenchymal stem cells and extracellular vesicles derived from two-dimensional culture of mesenchymal stem cells. An in vitro composition for promoting angiogenesis, wherein the manufacturing process does not include a shaking culture step.

7. (a) A step of producing three-dimensional spheroid cell aggregates by three-dimensional (3D) culture of mesenchymal stem cells seeded in microwells having a diameter of 200–800 μm and a depth of 100–1000 μm, which is carried out by dispensing mesenchymal stem cells into microwells at a density of 200–600 cells / well and culturing them; (b) The step of 3D static culture of the three-dimensional spheroid cell aggregates prepared in the microwells; and (c) The step of separating extracellular vesicles from the three-dimensional spheroid cell aggregate; A method for producing an angiogenesis-promoting composition containing extracellular vesicles derived from three-dimensional spheroid cell aggregates, The extracellular vesicles isolated in step (c) above express one or more selected from the group consisting of miR-27a, miR-132, miR-146a, miR-146b, miR-184, and miR-210 in a higher expression than extracellular vesicles derived from spheroid cell aggregates obtained by three-dimensional dynamic culture of mesenchymal stem cells and extracellular vesicles derived from two-dimensional culture of mesenchymal stem cells. A manufacturing method wherein the method does not include a shaking culture step.

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