A nerve regeneration-promoting composition containing extracellular vesicles derived from three-dimensional spheroid cell aggregates.

Abstract

The present invention relates to a nerve regeneration-promoting composition and a nerve regeneration-promoting method, which contain extracellular vesicles derived from three-dimensional spheroid-type cell aggregates produced by a novel production method. The extracellular vesicles produced by the novel method of the present invention can induce neurogenesis, neurodifferentiation, or neural circuit recovery and improve neural plasticity, and therefore can be usefully used in the treatment of various diseases requiring nerve regeneration.

Description

[Technical Field]

[0001] The present invention relates to a nerve regeneration promoting composition and a nerve regeneration promoting method, which include extracellular vesicles derived from three-dimensional spheroid-type cell aggregates produced by a novel manufacturing method. [Background technology]

[0002] Positive clinical results have been reported for therapies using stem cells, particularly mesenchymal stem cells (MSCs), in a variety of diseases. However, stem cell therapies carry the risk of cell-related side effects such as vascular occlusion, tumor formation, and coagulation disorders, and efficacy verification through clinical trials remains necessary. It is known that the paracrine effect of stem cells induces the regeneration of surrounding skin cells and enhances the ability of blood vessels to regenerate, and extracellular vesicles (EVs) are particularly known as the main effective factor of the paracrine effect.

[0003] Extracellular vesicles are classified into exosomes and microvesicles based on their size. Exosomes have a diameter of 30-150 nm, while microvesicles are 100-1,000 nm in size. Extracellular vesicles are formed when a portion of the cell membrane is 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 the side effects of stem cell use and enhances safety, but also offers advantages in terms of biodistribution and production processes.

[0004] Brain neuroplasticity is the phenomenon in which neural pathways undergo structural and functional changes and reorganization. Damage or reduction of neural pathways causes symptoms not only in brain injuries such as cerebral infarction and head trauma, but also in dementia and many degenerative neurological diseases. To date, there are no effective treatments or methods to restore brain neural pathways, and they have never been clinically applied to patients with brain diseases such as cerebral infarction or dementia. Currently, physical therapy and behavioral therapy are applied to these patients, but their therapeutic effects are limited. Therefore, it is expected that improving brain neuroplasticity through increased neurogenesis and restoration of neural circuits can have a therapeutic effect on most brain diseases.

[0005] However, methods for mass production and acquisition of extracellular vesicles derived from stem cells have not yet been established, and little research has been conducted on methods to further enhance the efficacy of extracellular vesicles while maintaining their characteristics. In particular, extracellular vesicles that can improve brain and neural plasticity have not been widely studied.

[0006] 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]

[0007] 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 by producing three-dimensional spheroid-type cell aggregates (3D-static-spheroids) by static culture using microwells and using these to produce extracellular vesicles that can promote nerve regeneration and improve nerve plasticity.

[0008] Therefore, the object of the present invention is to produce extracellular vesicles derived from three-dimensional spheroid cell aggregates with improved nerve regeneration ability, and to provide compositions containing these vesicles for the prevention, improvement, or treatment of neurological diseases and injuries. [Means for solving the problem]

[0009] To achieve the above objective, the present invention provides a pharmaceutical composition for the prevention or treatment of neurological diseases and injuries 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; and extracellular vesicles derived from the three-dimensional spheroid cell aggregates.

[0010] Furthermore, the present invention provides a food composition for preventing or improving neurological diseases and injuries, 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 including extracellular vesicles derived from the three-dimensional spheroid cell aggregates produced through these steps.

[0011] Furthermore, the present invention provides an in vitro composition for promoting nerve regeneration, 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.

[0012] Furthermore, the present invention provides a method for producing a nerve regeneration promoting composition containing extracellular vesicles derived from three-dimensional spheroid cell aggregates, 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.

[0013] Furthermore, the present invention provides a method for preventing or treating neurological diseases and injuries, 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 through step (b). [Effects of the Invention]

[0014] The extracellular vesicles produced by the novel method of the present invention can induce neurogenesis, neural differentiation, or the restoration of neural circuits, thereby improving neural plasticity. Therefore, they can be usefully utilized in the treatment of various diseases requiring nerve regeneration. [Brief explanation of the drawing]

[0015] [Figure 1] This diagram illustrates the process of producing 3D spheroid-type cell aggregates and separating extracellular vesicles from them. [Figure 2]Panel A is a diagram showing observation of microwells containing 3D-dynamic-PEG spheroid culture medium. Panel B is a diagram showing observation of microwells containing 3D-static-spheroid culture medium. Panel C is a diagram showing the change in aggregate area depending on the culture period of 3D-static spheroids produced using 3D-dynamic-PEG spheroids and microwells. [Figure 3] This is a diagram comparing the size distribution of 3D-static spheroids and 3D-dynamic-PEG spheroids. [Figure 4] This is an electron micrograph showing the morphology of 3D-static spheroid EVs. [Figure 5] This is a diagram showing the results of nanoparticle tracking analysis (NTA) for confirming the concentration and size distribution of 3D-static spheroid EVs. [Figure 6] This is a diagram showing the results of ELISA and Western blotting for confirming expression markers of 3D-static spheroid EVs. [Figure 7] This is a diagram comparing the production yield of 3D-static spheroid EVs, 3D-dynamic-PEG spheroid EVs, and 2D-EV per origin cell. [Figure 8] This is a diagram showing the expression of miRNAs and proteins associated with the physiology of stroke pathology and the therapeutic effect of EV that are highly expressed in 3D-static spheroid EVs. [Figure 9] This is a diagram showing nerve regeneration-related miRNAs that are highly expressed in 3D-static spheroid EVs compared to 2D-EV. [Figure 10a] This is a diagram showing the results of confirming differences in EV production yield, EV size, and total protein content including EV between 2D-EV and 3D-static spheroid EV (WJ-3D EV) depending on donor. [Figure 10b] This is a diagram showing the results of confirming differences in donor variation and nerve regeneration-related miRNA expression levels between 2D-cultured WJ-MSC, 3D-cultured WJ-MSC, 2D-EV, and 3D-static spheroid EV. [Figure 11]It is a figure showing the results of confirming changes in the expression of VEGF, BDNF, GFG, NGF, HGF, and ANGP1 after treating neural stem cells pNSC with 3D-static-spheroid EVs. [Figure 12] It shows the result (A) confirming that 3D-static-spheroid EVs are internalized into cells, and the result (B) confirming that the expression of miR-27a-3p is significantly increased in cells treated with 3D-static-spheroid EVs. [Figure 13] It is a figure showing the results of confirming the neural differentiation promotion ability of 3D-static-spheroid EVs by measuring neurite length. The left side of Figure 13 is the result of confirming this with a microscope, and the right side of Figure 13 is the figure showing the measurement result of neurite length. [Figure 14] It is a figure showing the results of confirming the degree of volume change of (a) cerebral infarction lesions and (b) ventricles after treating cerebral infarction animal models with PBS as a control group or 3D-static-spheroid EVs as an experimental group. [Figure 15] It is a figure showing the results of confirming the degree of neurogenesis by immunofluorescence staining after treating cerebral infarction animal models with 3D-static-spheroid EVs. [Figure 16] It is a figure showing the results of confirming the recovery effect on motor function loss after injecting 3D-static-spheroid EVs into cerebral infarction animal models. (a): internal capsule, (b): external capsule. [Figure 17a] It is a figure showing the results of investigating neural circuit changes using diffusion tensor tractography (DTT) images on day 14 after 3D-static-spheroid EV injection. (a) Planar image and color information. Colors represent the main direction of fiber tracts (red, left-right; green, anteroposterior; blue, craniocaudal). It is a representative fiber tract graph from the region of interest (ROI) of the external capsule (upper) and internal capsule (lower) on day 14 after treatment with 3D-static-spheroid EVs. (b) PBS alone (c) White arrows indicate increased nerve fibers in animals administered 3D-static-spheroid EVs. [Figure 17b]This figure shows the results of investigating neural circuit changes using diffusion tensor tractography (DTT) images after 3D-static-spheroid EV injection, at 28 days post-injection. (a) Planar image and color information. Colors represent the main direction of the fiber pathway (red, left-right; green, recessive; blue, anterior-posterior). Representative fiber track graphs from the outer capsule (top) and inner capsule (bottom) ROI at 14 days post-treatment with 3D-static-spheroid EV. (b) PBS alone. (c) White arrows indicate increased nerve fibers in animals treated with 3D-static-spheroid EV. [Figure 18] This figure shows the resting state functional MRI results 28 days after inducing a stroke (EV group: 3D-static-spheroid EV treated group). [Figure 19] This figure shows the results of forelimb asymmetry tests (cylinder test) and limb injury tests (grid gait test) conducted to confirm functional recovery through the neural circuit generation effect of 3D-static-spheroid EV. [Figure 20] To confirm the relationship between functional recovery and MRI, we examined the correlations 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 gait test and rFD of the internal capsule, (d) grid gait 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 verifying the size, rundness, and solidity of 3D-static spheroids produced after varying the diameter, depth, and number of cells per well in the manufacturing of 3D-static spheroids EV. [Figure 22] This figure shows the results of comparing the expression levels of miRNA-132 expressed 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 the prevention or treatment of neurological diseases and injuries, 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; and extracellular vesicles derived from the three-dimensional spheroid cell aggregates.

[0017] The cells in this invention can be used without limitation as long as they are cells 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.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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 nerve regeneration effects. For example, preferably, the extracellular vesicles of the present invention may express one or more selected from the group consisting of miR-27a, miR-132, miR-146a, and miR-146b, which are associated with nerve regeneration, 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-dimensionally cultured mesenchymal stem cells, or they may express 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). 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, in a higher degree 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.

[0025] 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.

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

[0027] 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).

[0028] 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.

[0029] 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."

[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 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.

[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 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.

[0033] 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.

[0034] By producing extracellular vesicles using the "method for producing extracellular vesicles derived from three-dimensional spheroid cell aggregates" according to 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 nerve regeneration capabilities such as neurogenesis, neural differentiation, and neural circuit restoration. 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 according to the present invention is suitable for GMP application, and is therefore particularly suitable for the production of pharmaceutical compositions for the treatment of nervous system loss and injury.

[0035] In this invention, neural plasticity refers to the phenomenon in which neural pathways undergo structural and functional changes and reorganization. According to this invention, nerve regeneration, induction of neurogenesis, and induction of neural differentiation can be effectively achieved by processing extracellular vesicles derived from three-dimensional spheroid cell aggregates, thereby improving neural plasticity.

[0036] Therefore, the extracellular vesicles derived from the three-dimensional spheroid cell aggregates of the present invention are applicable to a variety of nervous systems and nerve injuries, and conditions requiring nerve regeneration. The types of diseases to which the extracellular vesicles of the present invention can be applied are not limited to these, but may include 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 diseases, and traumatic brain injury. In particular, the extracellular vesicles derived from the three-dimensional spheroid cell aggregates of the present invention can be used for inducing neurogenesis, inducing neural differentiation, or inducing the restoration of neural circuits.

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

[0038] 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.

[0039] 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.

[0040] 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.

[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. 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.

[0042] 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.

[0043] The definitions of the terms excipients, binders, disintegrants, lubricants, flavoring agents, and other similar terms in this invention include those described in literature known to the industry and having the same or similar functions.

[0044] Furthermore, the present invention provides a method for treating or managing neurological diseases and injuries, 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).

[0045] The object is preferably a mammal including humans, and includes all patients who require treatment for a neurological disorder, including patients currently undergoing treatment for nerve injury or a neurological disorder, patients who have previously received treatment for nerve injury or a neurological disorder, and patients who require treatment for nerve injury or a neurological disorder, and may also include patients who have undergone surgery for the treatment of nerve injury or a neurological disorder.

[0046] Furthermore, the present invention can be used in combination with other existing drugs or treatment methods for the treatment of nerve injury or neurological disorders. When the present invention is used in combination, it can be used simultaneously with or sequentially with drugs or treatment methods for the treatment of nerve injury or neurological disorders.

[0047] Furthermore, the present invention provides a food composition for preventing or improving neurological diseases and injuries, 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.

[0048] 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.

[0049] 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.

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

[0051] The nerve regeneration-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 that require nerve regeneration due to nerve damage. The nerve regeneration-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 technicians of the 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 nerve regeneration. The term "nerve regeneration" can be used in the sense of encompassing all of the following: induction of neurogenesis, induction of neural differentiation, or induction of neural circuit restoration.

[0052] Furthermore, the present invention provides a method for producing a nerve regeneration-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.

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

[0054] 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.

[0055] 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]

[0056] 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).

[0057] 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.

[0058] 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.

[0059] 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 used to treat human NSC cell lines (ReNcells) 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).

[0060] 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.

[0061] 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).

[0062] 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).

[0063] The processes of the above-mentioned Examples 1.1 to 1.3 are illustrated in Figure 1.

[0064] 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 3D mesenchymal stem cell spheroid-type cell aggregate (hereinafter referred to as 3D-dynamic PEG spheroid) culture medium 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 ​​the spheroid-type cell aggregates after culturing compared to the initial stage of culture.

[0065] 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 3D-static spheroids compared to the 3D-dynamic PEG spheroids showed a statistically significant reduction in spheroid-to-area ratio during the initial stages of culture (p=0.0011).

[0066] 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.

[0067] 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.

[0068] 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.

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

[0070] [Table 1]

[0071] 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%.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] As shown in Figure 4, the 3D-static spheroid EV was confirmed to have a round shape, which is typical of extracellular vesicles.

[0076] 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.

[0077] 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.

[0078] 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.

[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 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.

[0080] 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.

[0081] [Table 2]

[0082] Example 8.3 D-static spheroid EV expression miRNA analysis and confirmation of neurogenesis effect 8.1 Analysis of miRNA expression in 3D-static spheroid EVs Sequencing analysis was performed to analyze the miRNA components contained in the 3D-static-spheroid EV, which is an extracellular vesicle produced by the manufacturing method of Example 1. The results of the analysis of the miRNA components contained in the 3D-static-spheroid EV are shown in Figure 8.

[0083] As shown in Figure 8, 358 miRNAs, including those related to the physiology of stroke pathology and the therapeutic effects of stem cells / EVs, were detected in 3D-static spheroid EVs with normalized RC values ​​of 4 or higher. Of these, the top 50 expressed miRNAs were subjected to GO and KEGG analyses. GO analysis showed a significant correlation between expressed miRNAs and intracellular protein transport and phosphorylation, axon guidance, brain development, glutamate synapses and neuronal protrusion, and DNA-binding transcriptional activation activity. KEGG pathways associated with the abundant miRNAs in 3D-static spheroid EVs included pathways related to biological functions, such as cancer, axon guidance, signaling pathways, and intracellular translocation. In particular, 3D-static spheroid EVs contained more miRNA-132 than 2D-EVs, confirming that 3D-static spheroid EVs can promote neoneurogenesis by inhibiting MeCP2 expression in neural stem cells.

[0084] Changes in miRNA expression were profiled using small RNA sequencing. Based on the profiling results, miRNAs showing significant changes in EVs secreted from stem cells cultured using the 3D culture method were examined compared to 2D EVs cultured using existing culture methods, and the results are shown in Figure 9.

[0085] As shown in Figure 9, changes in miRNA expression were examined using a TaqMan probe in cell lysates from five donors, and significantly increased expression of miRNAs was identified. Among these, the in vitro function of miRNAs with high levels of expression change was confirmed in neural stem cells (ReNcell). miR-132 was found to induce proliferation of neural stem cells upon transformation, and miR-132-3p was found to inhibit PSD95 expression and be involved in the proliferation of neural stem cells. miRNA-27a, miR-146a, and miR-146b are also known to be involved in neurogenesis and angiogenesis. The 3D-static spheroid EV of the present invention showed remarkably high expression of miR-132, miR-27a, miR-146a, and miR-146b, which are involved in nerve regeneration, thus confirming that it is an extracellular vesicle that can be clinically useful in nerve regeneration-related fields.

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

[0087] As shown in Figure 10a, 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.

[0088] Furthermore, as shown in Figure 10b, 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 uniform amounts of miRNAs that can demonstrate nerve regeneration effects, such as miR-27a-3p (1.5 times), miRNA-146a (2.3 times), and miRNA-132 (2.6 times), compared to 2D-EVs. In addition, miR-181b, another nerve regeneration-related miRNA, was also confirmed to show uniform levels of expression among the donors.

[0089] These results indicate that 3D-static spheroid EV can reduce differences in therapeutic active ingredients among donors 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 3D-static-spheroid EVs for 6 hours, and then confirmed the mRNA expression levels related to nerve regeneration in the neural stem cells by qPCR.

[0091] As shown in Figure 11, increased gene expression of VEGF, BDNF, FGF, and NGF was observed in neural stem cells treated with 3D-static-spheroid EV compared to neural stem cells treated with 2D-EV, confirming that 3D-static-spheroid-EV contains higher levels of VEGF, BDNF, FGF, and NGF mRNA compared to 2D-EV.

[0092] Based on a comprehensive analysis of all the above results, the 3D-static spheroid EVs of the present invention show significantly increased levels of miRNAs related to nerve regeneration, such as miR-27a, miR-132, miR-146a, and miR-146b, compared to WJ-2D-EVs and 3D-dynamic spheroid EVs. Furthermore, they contain higher levels of VEGF, BDNF, FGF, and NGF mRNA. Therefore, it is expected that they will exhibit superior nerve regeneration effects compared to previously reported EVs.

[0093] Example 9.3 Evaluation of the cellular uptake ability of D-static spheroid EVs To evaluate the cellular uptake ability of 3D-static spheroid EVs, CFSE-labeled 3D-static spheroid EVs were used to treat human NSC cell lines (ReNcells). After 24 hours of culture, the cells were examined using a confocal microscope (LSM 700, Carl Zeiss, Germany). Simultaneously, the expression of miR-27a-3p within ReNcells was confirmed by qPCR at 12 hours, and the results are shown in Figure 12.

[0094] As shown in Figure 12A, when 3D-static-spheroid EV was treated with cells, it was confirmed that it was taken up by the cells and internalized. Furthermore, as shown in Figure 12B, a significant increase in miR-27a-3p expression was confirmed in cells treated with 3D-static-spheroid EV compared to the control group.

[0095] This confirmed that when 3D-static-spheroid EV is used to treat target neurons, neurogenesis-promoting substances highly expressed in 3D-static-spheroid EV, such as miR-27a-3p, are effectively delivered to the cells. This result suggests that 3D-static-spheroid EV can be used as a therapeutic agent for nerve regeneration.

[0096] Example 10.3 Confirmation of the neuronal differentiation-promoting ability of D-static spheroid EVs To investigate the neural differentiation potential of extracellular vesicles (2D-EVs) and 3D-static-spheroid EVs derived from two-dimensionally cultured stem cells, 2D-EVs and 3D-static-spheroid EVs were introduced into 5 × 10⁻¹⁶ neural stem cells (NSCs) primarily cultured from the cerebral cortex isolated from 14.5-day-old SD rat embryos. 8 After treating each sample at a rate of / ml, the neural differentiation potential was compared with a control group (treated with base medium only) or a nerve growth factor (NGF) treated group. The neural differentiation potential of 2D-EV, 3D-static-spheroid EV, NGF, and base medium treatments was confirmed by measuring the length of neurites under a microscope on day 4 of culture, and the results are shown in Figure 13.

[0097] As shown in Figure 13, compared to the control group, the NGF, 2D-EV, and 3D-static-spheroid EV treatment groups showed increased neurite length and confirmed neural differentiation. In particular, the 3D-static-spheroid EV treatment group was found to induce neural differentiation at a significantly higher level than the groups treated with NGF or 2D-EV.

[0098] Example 11. Effects of 3D-static-spheroid EV on a stroke animal model. 11.1 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 Yanghag, Seoul, South Korea) and xylazine (10 mg / kg, Rompon 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.

[0099] 11.2 Morphological analysis using animal MRI studies In Example 11.1, 3D-static spheroid EV was administered to a photothrombotic cerebral infarction model rat. To investigate morphological changes by MRI, the infarct lesion volume and ventricular volume were measured using T2-weighted images (T2WI). Specifically, 3D-static spheroid EV 6x10 was administered to the photothrombotic cerebral infarction model rat. 8EV / mouse was injected into the blood vessels of rats, and T2-weighted MRI (T2WI) images were taken at 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 degree of cerebral infarction lesions and ventricular volume changes was 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 above, there was a statistically significant difference in ischemic lesion volume between the control group and the 3D-static-spheroid EV treatment group on day 3 post-stroke (p=0.030). At day 14 post-stroke, the lesion volume in the 3D-static-spheroid EV treatment group was significantly reduced compared to the control group (p=0.034). Ventricular volume was measured in the lateral and dorsal third ventricle. At days 14 and 28 post-stroke, ventricular volume was significantly lower in the 3D-static-spheroid EV 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 a lumen occlusion method. Eight-week-old male Sprague-Dawley rats (270-300g) were anesthetized with 1.5% isoprolan during surgery, and their body temperature was maintained at 37-37.5°C using a heating pad during the surgery and occlusion period. A round-ended 4-0 surgical monofilament nylon suture was moved from the left common carotid artery to the lumen of the internal carotid artery until the origin of the middle cerebral artery was occluded. Reperfusion was performed 90 minutes after tMCAo.

[0103] 11.4 Confirmation of neurogenesis-promoting effect through immunofluorescence staining To confirm the neurogenesis effect, ischemic stroke animals prepared in Example 11.3 are intravenously injected with 3D static spheroid EVs at three doses (0.3×10 10 / rat, 1.5×10 10 / rat, 3.0×10 10 / rat). Two weeks after stroke onset, immunofluorescence staining was performed on brain tissue using DCX, a positive neuronal marker, and Ki67, a cell proliferation marker, as observation indicators for neurogenesis. The results of immunofluorescence staining and the quantified Ki67 / DCX results for each dose experimental group are shown in Figure 15.

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

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

[0106] [Table 4]

[0107] As shown in Table 4 and Figure 16, at 14 days post-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 post-stroke, the rFA values ​​in the 3D-static-spheroid EV group were significantly higher than those in the control group in both IC and EC ROI (p=0.027 and p<0.001, respectively), confirming a greater degree of neural circuit recovery.

[0108] 3D-Static-Spheroid EV(6×10 8Changes in neural circuits after injecting EV / mouse into the blood vessels of rats were investigated using diffusion tensor tractography (DTT) imaging. Diffusion tensor tractography (DTT) images were acquired by MRI 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 neural circuit change in the treatment group and the control group was compared. The results are shown in Figure 17.

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

[0110] Example 13: Confirmation of the neural circuit generation effect of 3D-static spheroid EVs: 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 difference analysis of resting-state functional connectivity between the control group and the 3D-static-spheroid EV group was performed for 16 regions of interest. Six cortical regions and two cortical areas were selected for analysis from both ipsilesional and contralesional perspectives. The results of the analysis 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 administration group compared to the control group (p=0.008). This result indicates that 3D-static-spheroid EV induced enhancement of neural circuits in a stroke model.

[0112] 14.3 Confirmation of functional recovery through neural circuit generation in D-static spheroid EVs In Example 11.1, it was confirmed in the previous example that injecting the 3D-static-spheroid EV of the present invention into the cerebral infarction animal model effectively induced neural circuit formation. To confirm whether such neurogenesis could restore motor function loss induced in the cerebral infarction animal model, forelimb asymmetry tests (cylinder test) and limb loss tests (grid gait test) were performed. More specifically, the 3D-static-spheroid EV (6x10) was injected into the fabricated photothrombotic cerebral infarction model. 8 EV / mouse) was injected into the blood vessels of rats, and difference analysis was performed at 14 days (PBS group, n=8; 3D-static-spheroid EV treatment group, n=10) and 28 days (PBS group, n=8; 3D-static-spheroid EV treatment group, n=6). The results are shown in Figure 19.

[0113] As shown in Figure 19, it was confirmed that both the cylinder test index and the grid gait test index improved when the 3D-static-spheroid EV of the present invention was injected. These results indicate that the 3D-static-spheroid EV treatment of the present invention induces neural circuit regeneration in an animal model of cerebral infarction, and as a result, damaged motor function can be improved.

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

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

[0116] Example 15. Verification of 3D-static-spheroid EV effect under manufacturing conditions. 15.1 Experimental methods and conditions The above examples confirmed the excellent effects of the 3D-static spheroid EV of the present invention. To confirm whether similar effects could be observed in EVs produced using the same manufacturing method as in Example 1 but with different microwell specifications and cell counts, the cell count per microwell was changed from 400 cells / well to 200 cells / well, and the microwell specifications were changed from 500 μm × 200 μm in diameter and depth to 500 μm × 600 μm or to flat, shallow wells with a diameter of 800 μm, and cells were cultured.

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

[0118] [Table 5]

[0119] 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 5, kept static to induce spontaneous spheroid cell aggregate formation, and cultured for a total of 4 days in a CO2 incubator at 37°C to produce a 3D spheroid cell aggregate culture medium.

[0120] 15.2 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 3D spheroid-type cell aggregate culture medium 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 21.

[0121] As shown in Figure 21, in all of Experimental Examples 1 to 3, it was confirmed that the size of the manufactured 3D spheroid cell aggregates was within the size range of 55 to 131 μm, which is the same as the size range of the cell aggregates manufactured 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.

[0122] 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 using the method of Example 1.

[0123] 15.3 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 comparison of miRNA expression patterns was confirmed using extracellular vesicles derived from spheroid-type cell aggregates produced using the methods of Experimental Examples 1 and 2, which used different microwell conditions. The expressed miRNA analysis was confirmed 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 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.

[0125] In summary, the 3D-static-spheroid EV of the present invention exhibits high expression of miRNAs and neurogenesis-related factors, and has been confirmed to have excellent neurogenesis, neural circuit formation, and associated stroke improvement effects both in vitro and in vivo. Therefore, it is expected to show excellent effects in the prevention or treatment of various diseases requiring nerve damage and nerve regeneration.

[0126] 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 the prevention or treatment of neurological diseases and injuries, comprising extracellular vesicles derived from three-dimensional spheroid cell aggregates produced through [method / 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, and miR-146b in a higher expression than the extracellular vesicles derived from two-dimensionally cultured mesenchymal stem cells. A pharmaceutical composition for the prevention or treatment of neurological diseases and injuries, wherein the manufacturing process does not involve a shaking culture step.

2. The pharmaceutical composition for the prevention or treatment of neurological diseases and injuries according to claim 1, wherein when the extracellular vesicles separated in step (c) are treated with neural stem cells, the extracellular vesicles separated in step (c) cause the neural stem cells to express one or more selected from the group consisting of VEGF, BDNF, FGF, and NGF at a higher level than extracellular vesicles derived from mesenchymal stem cells cultured in two dimensions.

3. The pharmaceutical composition for the prevention or treatment of neurological diseases and injuries according to claim 1, wherein the neurological 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 disease, and traumatic brain injury.

4. The pharmaceutical composition for the prevention or treatment of neurological diseases and injuries according to claim 1, wherein the composition is for inducing neurogenesis, inducing neural differentiation, or inducing the restoration of neural circuits.

5. The induction of the aforementioned neural circuit recovery is confirmed by MRI DTI (diffusion-tensor image), a pharmaceutical composition for the prevention or treatment of neurological diseases and injuries according to claim 4.

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; A food composition for the prevention or improvement of neurological diseases and injuries, comprising extracellular vesicles derived from three-dimensional spheroid cell aggregates produced through [method / process], The extracellular vesicles isolated in step (c) above express one or more selected from the group consisting of miR-27a, miR-132, miR-146a, and miR-146b in a higher expression than the extracellular vesicles derived from two-dimensionally cultured mesenchymal stem cells. A food composition in which 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; An in vitro composition for promoting nerve regeneration, comprising extracellular vesicles derived from three-dimensional spheroid-type cell aggregates manufactured through [method / process], The extracellular vesicles isolated in step (c) above express one or more selected from the group consisting of miR-27a, miR-132, miR-146a, and miR-146b in a higher expression than the extracellular vesicles derived from two-dimensionally cultured mesenchymal stem cells. An in vitro composition for promoting nerve regeneration, wherein the manufacturing process does not include a shaking culture step.

8. (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 a nerve regeneration 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, and miR-146b in a higher expression than the extracellular vesicles derived from two-dimensionally cultured mesenchymal stem cells. A manufacturing method wherein the method does not include a shaking culture step.

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