Nerve regeneration-promoting composition comprising extracellular vesicles derived from three-dimensional spheroid-type cell aggregates

Abstract

To provide a nerve regeneration promoting composition containing extracellular vesicles derived from three-dimensional spheroid cell aggregates produced by a novel production method, and to provide a nerve regeneration promoting method.SOLUTION: Provided is a pharmaceutical composition 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.SELECTED DRAWING: Figure 16

Description

[Technical Field]

[0001] The present invention relates to a nerve regeneration-promoting composition containing extracellular vesicles derived from three-dimensional spheroid cell aggregates produced by a novel production method, and a nerve regeneration-promoting method. [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] Brain neuroplasticity is the phenomenon of structural and functional changes and reorganization of neural pathways. Damage or reduction of neural pathways leads to the development of symptoms in brain injuries such as stroke and head trauma, as well as dementia and many other degenerative neurological diseases. To date, no treatments or methods are available for restoring neural pathways, and they have not been clinically applied to patients with brain diseases such as stroke and dementia. Currently, physical therapy and behavioral therapy are applied to these patients, but their therapeutic effects are limited. Therefore, improving brain neuroplasticity through the promotion of neurogenesis and the restoration of neural circuits is expected to have therapeutic effects on most brain diseases.

[0005] However, mass production and acquisition methods for stem cell-derived extracellular vesicles have not yet been established, and little research has been done on methods to further enhance the efficacy of stem cell-derived extracellular vesicles while maintaining their properties.In particular, there has been little research on extracellular vesicles that can improve brain neuroplasticity.

[0006] 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]

[0007] 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 that can promote neural regeneration and improve neural plasticity are produced, thereby completing the present invention.

[0008] Therefore, an object of the present invention is to produce extracellular vesicles derived from three-dimensional spheroid cell aggregates with improved nerve regeneration promoting ability, and to provide a composition containing the same for preventing, ameliorating, or treating nervous system diseases and injuries. [Means for solving the problem]

[0009] To achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating nervous system diseases and injuries, comprising extracellular vesicles derived from three-dimensional spheroid cell aggregates prepared through the steps of: (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; and (b) isolating extracellular vesicles from the three-dimensional spheroid cell aggregates.

[0010] The present invention also provides a food composition for preventing or ameliorating nervous system diseases and injuries, 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 an in vitro composition for promoting nerve regeneration, 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.

[0012] The present invention also provides a method for producing a composition for promoting nerve regeneration, which contains extracellular vesicles derived from three-dimensional spheroid cell aggregates, 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.

[0013] The present invention also provides a method for preventing or treating nervous system diseases and injuries, 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]

[0014] The extracellular vesicles produced by the novel method of the present invention can induce neurogenesis, neural differentiation, or neural circuit recovery and improve neural plasticity, and can therefore be useful in the treatment of various diseases requiring neural regeneration. [Brief explanation of the drawings]

[0015] [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 miRNA and protein expression related to stroke pathology physiology and EV therapeutic effect, which are highly expressed in 3D-static-spheroid EVs. [Figure 9] Figure 1 shows nerve regeneration-related miRNAs that are highly expressed in 3D-static-spheroid EVs compared to 2D-EVs. [Figure 10a] This figure shows the results of confirming the differences in EV production, EV size, and EV protein content by donor between 2D-EVs and 3D-static-spheroid EVs (WJ-3D EVs). [Figure 10b] Fig. 1 shows the results of confirming donor variation and differences in nerve regeneration-related miRNA expression levels among 2D-cultured WJ-MSCs, 3D-cultured WJ-MSCs, 2D-EVs, and 3D-static-spheroid EVs. [Figure 11]This figure shows the results of examining changes in the expression of VEGF, BDNF, GFG, NGF, HGF, and ANGP1 after treating neural stem cells (pNSCs) with 3D-static-spheroid EVs. [Figure 12] The results confirm that 3D-static-spheroid EVs are internalized into cells (A), and that miR-27a-3p expression was significantly increased in cells treated with 3D-static-spheroid EVs (B). [Figure 13] Figure 13 shows the neuronal differentiation-promoting ability of 3D static spheroid EVs as determined by measuring neurite length. The left side of Figure 13 shows the results of microscopic observation, and the right side of Figure 13 shows the results of neurite length measurement. [Figure 14] This figure shows the results of examining the extent of (a) cerebral infarction lesions and (b) ventricle volume changes 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 immunofluorescence staining to confirm the degree of neurogenesis after treating an animal model with 3D static spheroid EVs. [Figure 16] This figure shows the results of confirming the effect of 3D static spheroid EV injection on motor function recovery in an animal model of cerebral infarction. (a) Internal Capsule, (b) External Capsule. [Figure 17a] Diffusion tensor tractography (DTT) imaging of neural circuit changes was performed 14 days after 3D-static spheroid EV injection. (a) Planar images and color information. Colors indicate the main direction of fiber tracts (red, left-right; green, inferior; blue, anterior-posterior). Representative fiber track graphs from the external capsule (top) and internal capsule (bottom) ROIs on day 14 after 3D-static spheroid EV treatment. (b) PBS alone. (c) White arrows indicate increased neural fibers in 3D-static spheroid EV-treated animals. [Figure 17b]Diffusion tensor tractography (DTT) imaging of neural circuit changes was performed 28 days after 3D-static spheroid EV injection. (a) Planar images and color information. Colors indicate the main direction of fiber tracts (red, left-right; green, inferior; blue, anterior-posterior). Representative fiber track graphs from the external capsule (top) and internal capsule (bottom) ROIs on day 14 after 3D-static spheroid EV treatment. (b) PBS alone. (c) White arrows indicate increased neural fibers in 3D-static spheroid EV-treated animals. [Figure 18] This figure shows the results of resting state functional MRI 28 days after stroke induction (EV group: 3D-static-spheroid EV-treated group). [Figure 19] This figure shows the results of a forelimb asymmetry test (cylinder test) and a limb injury test (grid walking test) conducted to confirm functional recovery due to the neural circuit generation effect of 3D-static-spheroid EVs. [Figure 20] To confirm the relationship between functional recovery and MRI, correlations were confirmed between (a) cylinder test and relative fiber density (rFD) of the internal capsule, (b) cylinder test and interhemispheric resting state functional connectivity (RSFC), (c) grid walk test and rFD of the internal capsule, (d) grid walk test and interhemispheric RSFC of the striatum, and (e) interhemispheric RSFC of the striatum and rFD of the internal capsule. [Figure 21] This figure shows the results of examining the size, runness, and solidity of 3D spheroids produced after varying the diameter and depth of the microwells and the number of cells per well in the production of 3D static spheroid EVs. [Figure 22] This figure shows the results of comparing the expression levels of miRNA-132 in 3D static spheroid EVs and 2D EVs produced under various conditions. BEST MODE FOR CARRYING OUT THE INVENTION

[0016] The present invention provides a pharmaceutical composition for preventing or treating nervous system diseases and injuries, 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.

[0017] The cells of the present invention may be any cells from which extracellular vesicles can be isolated, 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.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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 a nerve regeneration effect. For example, the extracellular vesicles of the present invention may express one or more miR-27a, miR-132, miR-146a, and miR-146b, which are associated with nerve regeneration, 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, or ... or may express one or more miR-146b, which are associated with nerve regeneration, at a higher level than extracellular vesicles derived from spheroid-type cell aggregates formed by three-dimensional dynamic culture of mesenchymal stem cells, or may express one or more miR-146b, which are associated with nerve regeneration, or may express one or more miR-146b, which are associated with nerve regeneration, or may express one or more miR-146b, which are associated with nerve regeneration, or may express one or more miR-146b, which are associated with nerve regeneration, or may express one or more miR-146b, which are associated with nerve regeneration, or may express one or more miR-146b, which are associated with nerve regeneration, or may express one or more More preferably, the extracellular vesicles of the present invention may express miR-27a, miR-132, miR-146a and miR-146b, VEGF, BDNF, FGF, and NGF, which are associated with nerve regeneration, at higher levels 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.

[0025] 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.

[0026] In the present invention, extracellular vesicles derived from three-dimensional spheroid cell aggregates that exhibit nerve regeneration-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."

[0027] 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).

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] The "method for producing extracellular vesicles derived from three-dimensional spheroid cell aggregates" of the present invention allows for rapid and efficient mass production of extracellular vesicles that have not only the advantages of static culture but also improved clinical applicability, particularly improved neural regeneration capabilities such as neurogenesis, neural differentiation, and neural circuit restoration. 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 treating or treating nervous system loss and injury.

[0035] In the present invention, neural plasticity refers to the phenomenon in which neural pathways undergo structural and functional changes and reorganization. According to the present invention, treatment with extracellular vesicles derived from three-dimensional spheroid cell aggregates effectively achieves neural regeneration, induction of neurogenesis, and induction of neural differentiation, thereby improving neural plasticity.

[0036] Therefore, the extracellular vesicles derived from the three-dimensional spheroid cell aggregates of the present invention can be applied to various conditions requiring neural system and nerve injury and nerve regeneration. The types of diseases to which the extracellular vesicles of the present invention can be applied include, but are not limited to, one or more diseases or injuries selected from the group consisting of spinal cord injury, Parkinson's disease, stroke, amyotrophic lateral sclerosis, motor nerve injury, peripheral nerve injury due to trauma, nerve injury due to ischemic brain injury, neonatal hypoxic brain injury, cerebral palsy, epilepsy, intractable epilepsy, Alzheimer's disease, congenital metabolic neurological disease, and traumatic brain injury. In particular, the extracellular vesicles derived from the three-dimensional spheroid cell aggregates of the present invention can be used to induce neurogenesis, neurodifferentiation, or neural circuit recovery.

[0037] In the present invention, to confirm whether extracellular vesicles derived from three-dimensional spheroid cell aggregates can induce the recovery of neural circuits, we administered extracellular vesicles derived from three-dimensional spheroid cell aggregates to an animal model and performed MRI DTI (diffusion-tensor imaging) analysis. As a result, we confirmed that neural circuits were regenerated and an increase in nerve fibers was promoted.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] The present invention also provides a method for treating or managing nervous system diseases and injuries, 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.

[0045] The object is preferably a mammal, including a human, and includes patients in need of treatment for a nervous system disease, including patients currently undergoing treatment for a nerve injury or nervous system disease, patients who have been treated for a nerve injury or nervous system disease, and patients who need to be treated for a nerve injury or nervous system disease, and may also include patients who have undergone surgery to treat a nerve injury or nervous system disease.

[0046] Furthermore, the present invention can be used in combination with other existing drugs or therapeutic methods for treating nerve damage or nervous system diseases. When the present invention is used in combination, it can be used simultaneously or sequentially with other drugs or therapeutic methods for treating nerve damage or nervous system diseases.

[0047] The present invention also provides a food composition for preventing or ameliorating nervous system diseases and injuries, 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.

[0048] 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.

[0049] 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.

[0050] The present invention also relates to an in vitro composition for promoting nerve regeneration, 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.

[0051] The in vitro composition for promoting nerve regeneration of the present invention can be used for experimental purposes and can be a composition intended for treating isolated cells or tissues requiring nerve regeneration due to nerve injury. The in vitro composition for promoting nerve regeneration 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 nerve regeneration. The term "neural regeneration" can refer to all of the following: induction of neurogenesis, induction of neural differentiation, and induction of neural circuit recovery.

[0052] The present invention also provides a method for producing a composition for promoting nerve regeneration, which contains extracellular vesicles derived from three-dimensional spheroid cell aggregates, 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.

[0053] According to the above-described production method, extracellular vesicles derived from three-dimensional spheroid cell aggregates, which have excellent nerve regeneration-promoting effects, can be rapidly mass-produced in accordance with GMP standards.

[0054] 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.

[0055] 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]

[0056] 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).

[0057] 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.

[0058] 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.

[0059] 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 applied 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).

[0060] 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.

[0061] 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).

[0062] 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).

[0063] The processes of Examples 1.1 to 1.3 are illustrated in FIG.

[0064] 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.

[0065] 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).

[0066] 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.

[0067] 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.

[0068] 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.

[0069] The measured size distribution data is shown in FIG. 3, and the analysis results are shown in Table 1.

[0070] [Table 1]

[0071] 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%.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] [Table 2]

[0082] Example 8. Analysis of 3D-static-spheroid EV-expressed miRNAs and confirmation of neurogenesis effects 8.1 Analysis of miRNA expression within 3D-static-spheroid EVs Sequencing analysis was performed to analyze the miRNA components contained in 3D-static spheroid EVs, which are extracellular vesicles produced by the production method of Example 1. The results of the analysis of the miRNA components contained in 3D-static spheroid EVs are shown in Figure 8.

[0083] As shown in Figure 8, 358 miRNAs were detected in 3D-static spheroid EVs with a normalized RC of 4 or higher, including miRNAs related to stroke pathology and the therapeutic effects of stem cells / EVs. Among these, GO and KEGG analyses were performed on the top 50 expressed miRNAs. GO analysis revealed significant associations between expressed miRNAs and intracellular protein transport and phosphorylation, axon guidance, brain development, glutamatergic synapses and neuronal projections, and DNA binding and transcriptional activation activity. KEGG pathways associated with miRNAs enriched in 3D-static spheroid EVs were identified as pathways related to biological functions, including cancer, axon guidance, signal transduction pathways, and intracellular import. In particular, miRNA-132 was enriched in 3D-static spheroid EVs compared to 2D-static EVs, confirming that 3D-static spheroid EVs can promote new neurogenesis by inhibiting MeCP2 expression in neural stem cells.

[0084] Changes in miRNA expression were profiled using small RNA sequencing. Based on the results of the profiling study, we examined miRNAs that showed significant changes in EVs secreted from stem cells cultured using 3D culture methods compared to 2D-EVs cultured using conventional culture methods. The results are shown in Figure 9.

[0085] As shown in Figure 9, miRNA expression changes were confirmed using TaqMan probes in cell lysates from five donors, and significantly increased expression of miRNAs was identified. Among these, miRNAs with significantly increased expression levels were confirmed to function in vitro in neural stem cells (ReNcells). miR-132 induces neural stem cell proliferation upon transformation into neural stem cells, and miR-132-3p inhibits PSD95 expression and is associated with neural stem cell proliferation. miRNA-27a, miR-146a, and miR-146b are also known to be involved in neurogenesis and angiogenesis. The 3D-static spheroid EVs of the present invention demonstrated significantly elevated expression of miR-132, miR-27a, miR-146a, and miR-146b, which are involved in neural regeneration, confirming their clinical utility as extracellular vesicles in neural regeneration-related fields.

[0086] 8.2. Analysis of miRNA expression 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 10a and 10b.

[0087] As shown in Figure 10a, 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.

[0088] Furthermore, as shown in Figure 10b, 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 miRNAs, particularly miR-27a-3p (1.5-fold), miRNA-146a (2.3-fold), and miRNA-132 (2.6-fold), which are associated with nerve regeneration, than 2D-EVs. Furthermore, miR-181b, another nerve regeneration-related miRNA, was also expressed at a uniform level across donors.

[0089] These results indicate that 3D static spheroid EVs can reduce donor-specific differences in therapeutic active ingredients and demonstrate superior therapeutic efficacy.

[0090] Example 8.3 Comparison of changes in nerve regeneration-related protein expression after treatment of neural stem cells To further confirm the usefulness of 3D static spheroid EVs in promoting nerve regeneration, we treated target cells, neural stem cells (pNSCs), with EVs for 6 hours, and then confirmed the expression levels of mRNA related to nerve regeneration in the neural stem cells by qPCR.

[0091] As shown in Figure 11, it was confirmed that the gene expression of VEGF, BDNF, FGF, and NGF was increased in neural stem cells treated with 3D-static-spheroid EVs compared to neural stem cells treated with 2D-EVs, confirming that 3D-static-spheroid EVs contained more VEGF, BDNF, FGF, and NGF mRNA than 2D-EVs.

[0092] Taking all of the above results into consideration, it was confirmed that the 3D-static-spheroid EVs of the present invention have significantly increased levels of miR-27a, miR-132, miR-146a, and miR-146b, which are miRNAs related to nerve regeneration, compared to WJ-2D-EVs and 3D-dynamic-spheroid EVs, and contain higher amounts of VEGF, BDNF, FGF, and NGF mRNA. Therefore, it can be expected that the 3D-static-spheroid EVs of the present invention will have a superior effect on nerve regeneration compared to previously reported EVs.

[0093] Example 9. Evaluation of cellular uptake capacity of 3D-static-spheroid EVs To evaluate the cellular uptake of 3D-static spheroid EVs, CFSE-labeled 3D-static spheroid EVs were administered to human NSC cell lines (ReNcells). After 24 hours of culture, the cells were examined using a confocal microscope (LSM 700, Carl Zeiss, Germany). At 12 hours, miR-27a-3p expression in ReNcells was also confirmed by qPCR. The results are shown in Figure 12.

[0094] As shown in Figure 12A, when cells were treated with 3D-static spheroid EVs, they were taken up and internalized by the cells. Furthermore, as shown in Figure 12B, the expression of miR-27a-3p was significantly increased in cells treated with 3D-static spheroid EVs compared to the control group.

[0095] This confirmed that when 3D-static spheroid EVs were administered to target neurons, neurogenesis-promoting substances, such as miR-27a-3p, which are highly expressed in 3D-static spheroid EVs, were effectively delivered to the cells, indicating that 3D-static spheroid EVs can be used as a therapeutic agent for neural regeneration.

[0096] Example 10. Confirmation of the ability of 3D-static-spheroid EVs to promote neural differentiation To investigate the neural differentiation potential of extracellular vesicles (2D-EVs) and 3D-static spheroid EVs derived from two-dimensionally cultured stem cells, 5 × 10 2D-EVs and 3D-static spheroid EVs were added to primary cultured neural stem cells (NSCs) from the cerebral cortex isolated from day 14.5 SD rat (rat) embryos. 8 After treatment with 2D-EVs, 3D-static spheroid EVs, NGF, and basal medium, the neural differentiation potential was compared with that of a control group treated with basal medium alone or a nerve growth factor (NGF)-treated group. The neural differentiation potential of 2D-EVs, 3D-static spheroid EVs, NGF, and basal medium was confirmed by measuring the neurite length under a microscope on day 4 of treatment culture, and the results are shown in Figure 13.

[0097] As shown in Figure 13, the length of neurites increased in the NGF, 2D-EV, and 3D-static-spheroid EV treated groups compared to the control group, confirming that neural differentiation had occurred. In particular, it was confirmed that the 3D-static-spheroid EV treated group was able to induce neural differentiation at a significantly higher level than the NGF or 2D-EV treated groups.

[0098] Example 11. Effect of 3D-static-spheroid EVs on stroke animal models 11.1 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 Inc., 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.

[0099] 11.2 Morphological analysis using animal MRI studies After administering 3D-static spheroid EVs to the rat photothrombotic cerebral infarction model prepared in Example 11.1, the infarct volume and ventricle volume were measured using T2-weighted images (T2WI) to examine morphological changes by MRI. Specifically, 6x10 3D-static spheroid EVs were administered to the photothrombotic cerebral infarction model. 8EV / mouse was injected into the blood vessels of rats, and T2-weighted MRI 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). The extent of cerebral infarction and ventricular volume changes were compared between the experimental and control groups. The results are shown in Table 3 and Figure 14.

[0100] [Table 3]

[0101] As can be seen from Table 3 and Figure 14, there was a statistically significant difference in ischemic lesion volume between the control group and the 3D-static spheroid EV-treated group on day 3 after cerebral infarction (p=0.030). At day 14 after stroke, the lesion volume in the 3D-static spheroid EV-treated group was significantly reduced compared to the control group (p=0.034). Ventricle volume was measured in the lateral and dorsal third ventricles. At days 14 and 28 after stroke, ventricular volume was significantly lower in the 3D-static spheroid EV-treated group compared to the control group.

[0102] 11.3 Creation of an animal model of ischemic stroke Transient middle cerebral artery occlusion (tMCAo) was induced using the luminal occlusion method. Eight-week-old male Sprague-Dawley rats (270–300 g) were anesthetized with 1.5% isoprolan during surgery, and their body temperature was maintained at 37–37.5°C using a heating pad throughout the surgery and occlusion period. A blunt-ended 4-0 surgical monofilament nylon suture was passed from the left common carotid artery to the lumen of the internal carotid artery until it blocked the origin of the middle cerebral artery. tMCAo was performed 90 min after reperfusion.

[0103] 11.4 Confirmation of neurogenesis-promoting effects through immunofluorescence staining To confirm the effect of neurogenesis, three doses (0.3 × 10 10 / rat, 1.5×10 10 / rat, 3.0×10 10 After intravenous injection of 3D static spheroid EVs (1 / rat), two weeks after stroke, immunofluorescence staining was performed using DCX, a neuron-positive marker, and Ki67, a cell proliferation marker, as indicators of neurogenesis in the brain tissue. The results of immunofluorescence staining and quantified Ki67 / DCX ratios for each dose experimental group are shown in Figure 15.

[0104] As shown in Figure 15, it was confirmed that neurogenesis was significantly increased in all experimental groups administered with 3D-static-spheroid EVs compared to the control group administered with PBS.

[0105] Example 12. Confirmation of neural circuit generation effect of 3D static spheroid EVs The photothrombotic cerebral infarction model prepared in Example 11.1 was injected with 3D-static spheroid EVs (6 × 10 8 EVs (6x10) were injected into the blood vessels of rats, and changes in brain microstructure and connectivity were examined using diffusion-tensor imaging (DTI). In a photothrombotic cerebral infarction model, 3D-static spheroid EVs (6x10) were injected into the blood vessels of rats. 8EV / mouse) was injected into the blood vessels of rats, and MRI diffusion tensor imaging (DTI) and fiber tractography images 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). The therapeutic effect was demonstrated 14 and 28 days after stroke using relative changes in fractional anisotropy (rFA) and fiber density (rFD) values ​​from the external capsule (EC) and internal capsule (IC) regions of interest (ROIs). The results of DTI and fiber tractography are shown in Figure 16, and group-averaged rFA and rFD values ​​are shown in Table 4.

[0106] [Table 4]

[0107] As shown in Table 4 and Figure 16, at 14 days after stroke, the 3D-static-spheroid-EV group showed a statistically significant increase in rFA values ​​in both IC and EC compared to the control group (p=0.003 and p=0.045, respectively). At 28 days, the 3D-static-spheroid-EV group showed significantly higher rFA values ​​in both IC and EC ROIs compared to the control group (p=0.027 and p<0.001, respectively), confirming a higher degree of neural circuit recovery.

[0108] 3D-static-spheroid EVs (6 × 10 8After intravascular injection of EV (EV / mouse) into rats, changes in neural circuits were examined using diffusion tensor tractography (DTT) images. MRI diffusion tensor tractography (DTT) images were taken 14 days (PBS group, n = 8; EV group, n = 10) and 28 days (PBS group, n = 8; EV group, n = 6). Figure 17 shows a comparison of the extent of changes in neural circuits between the treatment and control groups.

[0109] As shown in Figure 17a and Figure 17b, 3D-static-spheroid EV administration confirmed increased nerve fibers, indicated by white arrows, at both 14 and 28 days, respectively.

[0110] Example 13: Confirmation of neural circuit generation effect of 3D static spheroid EV: Functional MRI To confirm the enhancement of neural circuits by extracellular vesicles, resting-state functional MRI was performed 28 days after stroke induction in the cerebral infarction animal model prepared in Example 11.1. A differential analysis of resting-state functional connectivity between the control group and the 3D-static-spheroid EV group was performed on 16 regions of interest. Six cortical and two cortical regions from the ipsilesional and contralesional aspects were selected for analysis. The results are shown in Figure 18.

[0111] As shown in Figure 18, inter-hemispheric striatal functional connectivity was significantly increased in the 3D-static spheroid EV-treated group compared to the control group (p=0.008), indicating that 3D-static spheroid EVs induced an enhancement of neural circuits in the cerebral infarction model.

[0112] 14. Confirmation of functional recovery by neural circuit generation in 3D static spheroid EVs The previous example confirmed that the injection of the 3D-static spheroid EVs of the present invention into the cerebral infarction animal model prepared in Example 11.1 effectively induced neural circuit formation. To confirm whether such neurogenesis could restore motor function loss induced in the cerebral infarction animal model, a forelimb asymmetry test (cylinder test) and a limb loss test (grid walking test) were performed. More specifically, 3D-static spheroid EVs (6 x 10 8 EV / mouse) was injected into the blood vessels of rats, and differential analysis was performed at 14 days (PBS group, n = 8; 3D-static spheroid EV-treated group, n = 10) and 28 days (PBS group, n = 8; 3D-static spheroid EV-treated group, n = 6). The results are shown in Figure 19.

[0113] As shown in Figure 19, injection of the 3D static spheroid EVs of the present invention improved both the cylinder test index and the grid walking test index. These results indicate that treatment with the 3D static spheroid EVs of the present invention induces regeneration of neural circuits in an animal model of cerebral infarction, thereby improving impaired motor function.

[0114] To confirm the relationship between functional recovery and MRI, we performed a correlation analysis between two behavioral tests (cylinder test and grid walking test) and the rs-fMRI results, and the results are shown in Figure 20.

[0115] As shown in Figure 20, 14 days after stroke induction, there was no statistically significant correlation between behavioral tests and MRI parameters (p > 0.05). 28 days after stroke induction, there was a significant correlation between the results of the cylinder test and the rFD of the IC ROI (r = -0.553, p = 0.040). A trend was also found between the results of the cylinder test and the interhemispheric striatum (r = -0.460, p = 0.098). Statistically significant correlations were observed between the limb injury score of the grid walking test and the rFD of the IC ROI (r = -0.684, p = 0.007) and the interhemispheric RSFC of the striatum (r = -0.691, p = 0.006). The interhemispheric RSFC of the striatum further showed a significant correlation with the rFD of the internal capsule (r = 0.776, p = 0.001).

[0116] Example 15. Verification of the effect of 3D static spheroid EVs on manufacturing conditions 15.1 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 with 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, depthless wells with a diameter of 800 μm.

[0117] The experimental conditions changed in the manufacturing method of Example 1 are shown in Table 5 below.

[0118] [Table 5]

[0119] 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 5, 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.

[0120] 15.2 Confirmation of 3D spheroid-type cell aggregate morphology It was confirmed that spheroids were uniformly formed under the conditions of Experimental Examples 1 to 3, as in Example 1. The 3D spheroid-type cell aggregate culture medium 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 21.

[0121] 21 , it was confirmed that the size of the 3D spheroid cell aggregates produced in all of Experimental Examples 1 to 3 was formed within the range of 55 to 131 μm, which is the size range of the cell aggregates produced in Example 1, and the average value of the size distribution was confirmed to be 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.

[0122] 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.

[0123] 15.3 Comparison of miRNA expression patterns of EVs derived from 3D spheroid types Since it was confirmed that spheroid cell aggregates with a 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 used different microwell conditions. Expression miRNA analysis was performed using the same qPCR method as in Example 8. The results are shown in Figure 22. The miRNA expression patterns were compared with those of 2D-EVs produced in Example 7.

[0124] As shown in Figure 22, 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 can exhibit common miRNA marker expression characteristics. Therefore, these EVs may be referred to as 3D-static-spheroid EVs.

[0125] Taking the above into consideration, the 3D static spheroid EVs of the present invention have been shown to highly express miRNAs and neurogenesis factors related to neurogenesis, and have been shown to have excellent neurogenesis capabilities, neural circuit formation, and the associated stroke improvement effects in vitro and in vivo. These findings suggest that the EVs are likely to be effective in preventing or treating various diseases requiring nerve damage and nerve regeneration.

[0126] While certain aspects of the present invention have been described in detail above, it will be obvious to those skilled in the art that such specific descriptions are merely preferred embodiments and are not intended to 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 preventing or treating nervous system diseases and injuries, comprising extracellular vesicles derived from three-dimensional spheroid cell aggregates produced through the method of claim 1.

2. 2. The pharmaceutical composition for preventing or treating nervous system diseases and injuries 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 preventing or treating nervous system diseases and injuries according to claim 1, wherein the three-dimensional culture in step (a) is a static culture.

4. 2. The pharmaceutical composition for preventing or treating nervous system diseases and injuries 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 them.

5. The pharmaceutical composition for preventing or treating nervous system diseases and injuries 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, and miR-146b 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. 2. The pharmaceutical composition for preventing or treating nervous system diseases and injuries according to claim 1, wherein the extracellular vesicles express at a higher level one or more selected from the group consisting of VEGF (vascular endothelial growth factor), BDNF (brain-derived neurotrophic factor), FGF (brain-derived neurotrophic factor), and NGF (brain-derived neurotrophic factor) 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. 2. The pharmaceutical composition for preventing or treating nervous system diseases and injuries according to claim 1, wherein the nervous system diseases and injuries are one or more diseases and injuries selected from the group consisting of spinal cord injury, Parkinson's disease, stroke, amyotrophic lateral sclerosis, motor nerve injury, peripheral nerve injury due to trauma, nerve injury due to ischemic brain injury, neonatal hypoxic brain injury, cerebral palsy, epilepsy, intractable epilepsy, Alzheimer's disease, congenital metabolic neurological diseases, and traumatic brain injury.

8. The pharmaceutical composition for preventing or treating nervous system diseases and injuries according to claim 1, wherein the composition is for inducing neurogenesis, for inducing neurodifferentiation, or for inducing neural circuit recovery.

9. The pharmaceutical composition for preventing or treating nervous system diseases and injuries according to claim 8, wherein the induction of neural circuit recovery is confirmed by MRI DTI (diffusion-tensor image).

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; A food composition for preventing or ameliorating nervous system diseases and injuries, 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; An in vitro composition for promoting nerve regeneration, comprising extracellular vesicles derived from three-dimensional spheroid cell aggregates produced through the method.

12. (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 nerve regeneration, comprising extracellular vesicles derived from three-dimensional spheroid cell aggregates, comprising:

13. (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 cell aggregates by separating cells and extracellular vesicles from the three-dimensional spheroid 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; 2. A method for preventing or treating nervous system diseases and injuries, comprising:

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