Composition for preventing or treating neuronal aging-related diseases or disorders comprising extracellular vesicles

Abstract

The present invention relates to a composition for preventing, alleviating, or treating neuronal aging-related diseases or disorders, the composition comprising extracellular vesicles derived from three-dimensional spheroid-type cell aggregates. The extracellular vesicles according to the present invention can reduce the expression of aging-related proteins and induce reactivation of aged neural stem cells, and thus can be used to treat various neuronal aging-related diseases or disorders by preventing, alleviating, or treating the aging of neurons.

Description

Composition for the prevention or treatment of neuro-aging-related diseases or disorders comprising extracellular vesicles

[0001] The present invention relates to a composition for the prevention, improvement, or treatment of neuro-aging-related diseases or disorders comprising extracellular vesicles derived from three-dimensional spheroid-shaped cell aggregates.

[0002]

[0003] 'Aging' refers to a phenomenon in which cellular functions decline or malfunction as damage to cells or tissues accumulates. In particular, brain tissues, which are composed of post-mitotic cells that no longer undergo mitosis and have completed differentiation, become more sensitive to intracellular damage caused by aging. It is known that genetic pathways and biochemical mechanisms involved in aging are evolutionarily conserved, and various studies are underway to improve age-related diseases by regulating these mechanisms.

[0004] The aging of the nervous system causes the development of various age-related diseases in addition to general aging processes such as muscle loss, reduced sense of balance, and decreased cognitive ability; it can manifest in various forms of geriatric diseases, including cognitive decline resulting from the overall decline in brain function, diseases caused by the selective destruction of nerve cells in specific areas, and diseases caused by cerebrovascular degeneration.

[0005] However, research on treatments that can inhibit the aging of the nervous system, reverse the aging state, or promote the re-division of nerve cells is still lacking.

[0006] Meanwhile, therapies using stem cells, particularly mesenchymal stem cells (MSCs), and positive clinical results have been reported for various diseases. However, stem cell therapies carry a risk of cell-related side effects, such as vascular occlusion, tumor formation, and coagulation disorders, and further verification of efficacy through clinical trials is still required. The paracrine effects of stem cells are known to induce the regeneration of surrounding skin cells and enhance vascular regeneration capabilities, and extracellular vesicles (EVs) are known to be the primary efficacy factors for these paracrine effects.

[0007] Extracellular vesicles are classified into exosomes and microvesicles based on their size; exosomes range in diameter from 30 to 150 nm, while microvesicles range from 100 to 1,000 nm. Extracellular vesicles are formed when a portion of a cell membrane detaches and enters 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 enhances safety by minimizing the side effects associated with stem cell use but is also advantageous in terms of biodistribution and production processes.

[0008] Therefore, there is a need to develop treatments for a wider variety of diseases using extracellular vesicles.

[0009] The inventors confirmed that extracellular vesicles obtained from three-dimensional spheroid-shaped cell aggregates can reverse the aging traits of nerve cells to young cells and achieve an anti-aging effect on nerve cells, and thus completed the present invention.

[0010] Accordingly, the objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of neuro-aging-related diseases or disorders and a health functional food composition for anti-aging, comprising one or more selected from the group consisting of extracellular vesicles obtained from three-dimensional spheroid-shaped cell aggregates or miR-125b and miR-30d identified as active ingredients contained in said extracellular vesicles.

[0011] To achieve the above objective, the present invention provides a pharmaceutical composition for the prevention or treatment of neuro-aging related diseases or disorders, comprising: (a) a step of producing a three-dimensional spheroid-shaped cell aggregate by culturing stem cells in a microwell having a diameter of 200 to 800 μm and a depth of 100 to 1000 μm; and (b) a step of separating extracellular vesicles from the three-dimensional spheroid-shaped cell aggregate.

[0012] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of neuro-aging-related diseases or disorders, comprising one or more selected from the group consisting of miR-125b and miR-30d.

[0013] In addition, the present invention provides a health functional food composition for neuronal anti-aging comprising extracellular vesicles derived from three-dimensional spheroid-shaped cell aggregates, prepared by (a) a step of preparing three-dimensional spheroid-shaped cell aggregates by culturing stem cells in a microwell having a diameter of 200 to 800 μm and a depth of 100 to 1000 μm; and (b) a step of separating extracellular vesicles from the three-dimensional spheroid-shaped cell aggregates.

[0014] In addition, the present invention provides a health functional food composition for anti-aging of nerve cells comprising one or more selected from the group consisting of miR-125b and miR-30d.

[0015] In addition, the present invention provides an in vitro composition for anti-aging of neurons comprising extracellular vesicles derived from three-dimensional spheroid-shaped cell aggregates, prepared by (a) a step of preparing three-dimensional spheroid-shaped cell aggregates by culturing stem cells in a microwell having a diameter of 200 to 800 μm and a depth of 100 to 1000 μm; and (b) a step of separating extracellular vesicles from the three-dimensional spheroid-shaped cell aggregates.

[0016] In addition, the present invention provides a method for improving or treating neuronal cell aging, comprising: (a) a step of producing a three-dimensional spheroid-shaped cell aggregate by culturing stem cells in a microwell having a diameter of 200 to 800 μm and a depth of 100 to 1000 μm; (b) a step of producing an extracellular vesicle derived from the three-dimensional spheroid-shaped cell aggregate by separating extracellular vesicles from the three-dimensional spheroid-shaped cell aggregate; and (c) a step of treating an individual requiring the extracellular vesicle derived from the three-dimensional spheroid-shaped cell aggregate produced through step (b).

[0017] In addition, the present invention provides a method for improving or treating neuronal aging, comprising the step of treating an individual in need with one or more selected from the group consisting of miR-125b and miR-30d.

[0018]

[0019] The extracellular vesicles of the present invention can reduce the expression of aging-related proteins and induce the reactivation of aged neural stem cells, and thus can be utilized in the treatment of various neuronal aging-related diseases or disorders by preventing, improving, or treating neuronal aging.

[0020] Figure 1 shows the results of confirming the change in neurosphere size and cell viability of neural stem cells according to EV treatment ( *p< 0.05, **p< 0.01).

[0021] Figure 2a shows morphological changes and SA-β-gal staining results after treatment with DPBS (control) or extracellular vesicles (EV) in Passage 2 (P2) and Passage 8 (P8) (Top: Morphology of suspension-cultured neurospheres (bright-field), Middle: SA-β-gal staining of 3D neurospheres, Bottom: SA-β-gal staining of 2D monolayer-cultured NSCs).

[0022] Figure 2b shows the results of quantifying the percentage of positive cells (%) after SA-β-gal staining in 2D monolayer cultured NSCs and 3D neuroglobules (**p< 0.01, ***p< 0.001, ****p< 0.0001).

[0023] Figure 3 shows the results of confirming changes in the expression of aging-related markers through immunoblotting and quantification (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; #p<0.05, ##p<0.01, ###<0.001 (P8-PBS vs P8-EV comparison); ns, non-representative).

[0024] Figure 4 shows the results confirming the cell cycle arrest alleviation and recovery effects following EV treatment.

[0025] Figure 5 shows the results of confirming the effect of EV administration on alleviating aging indicators in the aged hippocampus in the young, old, and EV administration groups (*p<0.05, **p<0.01, ***p<0.001).

[0026] Figure 6 shows the SA-β-gal staining results of the elderly hippocampus following EV administration in the young, old, and EV administration groups (Old + EV).

[0027] Figure 7 shows the results of confirming changes in p53 and p21 expression following miR-125b overexpression (mimic) and inhibitor treatment.

[0028] Figure 8 shows the results of confirming changes in p53 and p21 expression following miR-30d overexpression (mimic) and inhibitor treatment.

[0029]

[0030] The present invention provides a pharmaceutical composition for the prevention or treatment of neuro-aging-related diseases or disorders, comprising: (a) a step of producing a three-dimensional spheroid-shaped cell aggregate by culturing stem cells in a microwell having a diameter of 200 to 800 μm and a depth of 100 to 1000 μm; and (b) a step of separating extracellular vesicles from the three-dimensional spheroid-shaped cell aggregate.

[0031] In the present invention, the cell may be of any type of animal or plant origin, including human and non-human mammals, and may be various types of immune cells, tumor cells, or stem cells; preferably, the stem cell may be a mesenchymal stem cell, a pluripotent stem cell, an induced pluripotent stem cell, or an embryonic stem cell.

[0032] In the present invention, the three-dimensional culture refers to culturing in a state in which a three-dimensional arrangement is formed within a test tube. Unlike two-dimensional culture, cell growth in three-dimensional culture allows the cells to grow in all directions in vitro, and this may be more similar to the cellular environment in vivo.

[0033] In the present invention, the three-dimensional culture of step (a) can be performed by any three-dimensional cell culture technique known in the art to which the present invention belongs, for example, a cell culture using a microwell array culture, porous microsphere 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 a static culture. In the present invention, when the three-dimensional culture of step (a) is performed as a static culture, the devices required for shaking culture are not required, making the culture easier, and there is an advantage of enabling mass culture in a GMP (Good Manufacturing Practices) facility.

[0034] In the present invention, the three-dimensional culture of step (a) may be cultured for 1 to 10 days, preferably for 2 to 4 days. In the present invention, when the culture of step (a) is cultured for 2 to 4 days, the viability of cells present in the three-dimensional spheroid-shaped cell aggregate is maintained at a high level, and the culture time is relatively shorter compared to the conventional process for manufacturing three-dimensional spheroid-shaped cell aggregates, so three-dimensional spheroid-shaped cell aggregates and extracellular vesicles derived therefrom can be rapidly manufactured.

[0035] In the present invention, the three-dimensional culture of step (a) may be cultured by dispensing mesenchymal stem cells into microwells at a density of 100 to 1000 cells / well, preferably at a density of 100 to 600 cells / well, and more preferably at a density of 100 to 500 cells / well, 100, 200, 300, 400, and 500 cells / well.

[0036] In the present invention, the step of separating extracellular vesicles in step (b) may be prepared 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 that applies external force to the cells, and preferably, the separation may be performed by a tangential flow filtration (TFF) method, but is not limited thereto.

[0037] The extracellular vesicles of the present invention may have a high expression of a clinically significant substance compared to known extracellular vesicles, and said clinically significant substance may be a substance that prevents, improves, or treats the aging of nerve cells. Preferably, the extracellular vesicles of the present invention may include one or more selected from the group consisting of miR-125b and miR-30d, and when the extracellular vesicles of the present invention are administered, miR-125b or miR-30d contained in the extracellular vesicles is delivered to nerve cells to prevent, improve, or treat the aging of nerve cells. Accordingly, the extracellular vesicles of the present invention may have a high expression of miR-125b and miR-30d compared to extracellular vesicles derived from spheroid-type cell aggregates cultured in three-dimensional dynamic culture and extracellular vesicles derived from mesenchymal stem cells cultured in two dimensions.

[0038] In addition, for example, the extracellular vesicles of the present invention may be highly expressing one or more selected from the group consisting of miR-27a, miR-132, miR-146a, and miR-146b compared to extracellular vesicles derived from spheroid-type cell aggregates cultured in three-dimensional dynamic culture of mesenchymal stem cells and extracellular vesicles derived from mesenchymal stem cells cultured in two dimensions, and may be highly expressing 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).

[0039] In addition, the above extracellular vesicles may be incorporated into the cell and internalized upon treatment with the cell, and in the case of internalization, clinically significant substances highly expressed in the extracellular vesicles may be effectively delivered to the cell, resulting in high expression in the cell.

[0040] In the present invention, the extracellular vesicle derived from a three-dimensional spheroid-shaped cell aggregate can be used interchangeably with "3D-static-spheroid-EV" or "EV".

[0041] In the present invention, "extracellular vesicles derived from spheroid-shaped cell aggregates obtained by three-dimensional dynamic culture of mesenchymal stem cells" may include, without limitation, extracellular vesicles isolated from spheroid-shaped cell aggregates obtained by three-dimensional dynamic culture of mesenchymal stem cells, and preferably, may be extracellular vesicles disclosed in a registered patent (Application No. 10-2016-0053026, Method for producing extracellular vesicles derived from stem cells).

[0042] In the present invention, the microwell is made of TMSPMA (3-(Trimetoxysily) propylmethacrylate), HEA (Hydroxyethyl acrylate), GMA (Glycidyl methacrylate), EGDMA (diethyleneglycol dimethacrylate), THFA (Tetrahydrofurfuryl acrylate), HMAA (Hydroxymethul acrylamide), and PEA (Phenyl acrylate). epoxyacrylate), HOFHA (6-Hydroxy-2, 2,3,3,4,4,5,5-octafluoro), EOPT (Polyethoxylated(4)pentaerythritoltetraacrylate), HPA (Hydroxypropyl acrylate), BMA (Buthylmethacrlate), PETIA (Pentaerythritol triacrylate), HDDA (Hexan) diol diacrylate), EGPEA (Ethyleneglycol phenyletheracrylate), It may be coated with any one selected from the group consisting of BM (Benzylmethacrylate), HPPA (Hydroxyphenoxypropyl acrylate), BHPEA (2-(4-Benzoyl-3-hydroxyphenoxy)ethylacrylate), HEMA (Hydroxyethyl methacrylate), HPMA (N-(2-Hydroxypropyl) methacrylamide) and MPC (2-Methacryloyloxyethyl Phosphorylcholine Polymer), preferably coated with MPC (2-Methacryloyloxyethyl Phosphorylcholine Polymer), but is not limited thereto.

[0043] The microwell of the present invention may have a diameter of 200 to 800 μm, preferably 300 to 800 μm, more preferably 400 to 800 μm, for example, a diameter of 400, 500, 600, 700, or 800 μm.

[0044] In addition, the microwell may be a flat microwell with no depth, or, in the case of a microwell that forms depth, may have a structure of 100 to 1000 μm, preferably 100 to 900 μm, and more preferably 200 to 900 μm.

[0045] Mesenchymal stem cells cultured by the above structure may maintain a high viability even after a period of culture. Preferably, a microarray containing 1,000 to 100,000 of the above microwells can be fabricated to increase the production yield of cell aggregates.

[0046] In the present invention, neuro-aging-related diseases or disorders may include, without limitation, various diseases or disorders that may occur due to the aging of nerve cells, and preferably, may be one or more selected from the group consisting of senile degeneration of brain, senile degeneration of the brain, dementia, delirium, dizziness, and geriatric headache.

[0047] In the present invention, it was confirmed that extracellular vesicles derived from three-dimensional spheroid-shaped cell aggregates induce one or more activities selected from the group consisting of neuronal proliferation, inhibition of aging marker protein expression, reduction of β-galactosidase activity, and restoration of cell cycle arrest in aged neural stem cells, and through this, it was confirmed that the characteristics of aging neuronal cells can be induced into the characteristics of young cells.

[0048] In addition, excellent anti-aging substances contained in extracellular vesicles derived from three-dimensional spheroid-shaped cell aggregates were isolated and identified. It was confirmed that miR-125b or miR-30d contained in said extracellular vesicles can directly regulate the p53 signaling axis and regulate the expression of its downstream factor, p21, thereby inducing the alleviation of aging and the reactivation of the cell cycle in neural stem cells. Therefore, the extracellular vesicles of the present invention may contain one or more selected from the group consisting of miR-125b and miR-30d, or may be extracellular vesicles that express these substances at a higher level compared to naturally derived extracellular vesicles, extracellular vesicles obtained through two-dimensional culture or three-dimensional dynamic culture. In addition, since the present invention has confirmed the p53 / p21 pathway modulating effect of miR-125b or miR-30d analogs (mimics), the present invention provides a pharmaceutical composition for the prevention or treatment of neuro-aging-related diseases or disorders comprising one or more selected from the group consisting of miR-125b and miR-30d. One or more selected from the group consisting of miR-125b and miR-30d may be administered or delivered in a state in which they are included or loaded in an EV.

[0049] The pharmaceutical composition of the present invention may further comprise, in addition to the active ingredient, a suitable carrier, excipient, and diluent commonly used in the preparation of pharmaceutical compositions. The pharmaceutical composition of the present invention may further comprise other pharmaceutical active ingredients or active mixtures.

[0050] The pharmaceutical composition of the present invention may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, as well as external preparations, suppositories, and sterile injectable solutions, according to conventional methods. Carriers, excipients, and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulating, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid dosage forms for oral administration include tablets, pills, powders, granules, and capsules, and these solid dosage forms are prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the above composition. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

[0051] Liquid formulations for oral administration include suspensions, oral liquids, emulsions, and syrups; in addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients, such as humectants, sweeteners, flavorings, and preservatives. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspensions. Witepsol, Macrogol, Tween 61, cocoa paste, laurin paste, and glycerogelatin may be used as bases for suppositories.

[0052] 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 form of the drug, the route of administration, and the duration, but can be appropriately selected by those skilled in the art. Administration may be performed once a day or divided into several doses. The above dosage does not limit the scope of the present invention in any way.

[0053] The pharmaceutical composition of the present invention may be administered to mammals, such as rats, mice, livestock, and humans, by various routes. All modes of administration are expected, for example, by transdermal, oral, rectal or intravenous, intramuscular, subcutaneous, intradural, or intracerebroventricular injection.

[0054] The definitions of terms for the excipients, binders, disintegrants, lubricants, synergists, flavorings, etc. of the present invention include those described in literature known in the art that have the same or similar functions.

[0055] In addition, the present invention provides, in another aspect, a health functional food composition for anti-aging of nerve cells comprising: (a) a step of producing a three-dimensional spheroid-shaped cell aggregate by culturing stem cells in a microwell having a diameter of 200 to 800 μm and a depth of 100 to 1000 μm; and (b) a step of separating extracellular vesicles from the three-dimensional spheroid-shaped cell aggregate; or one or more selected from the group consisting of miR-125b and miR-30d.

[0056] One or more selected from the group consisting of the above miR-125b and miR-30d may be administered or delivered in a state in which they are included or loaded in an EV.

[0057] The above anti-aging may include, without limitation, preventing young cells from progressing into senescent cells, preventing already aged cells from aging further, causing aged cells to have a younger phenotype, or producing more young cells through cell division.

[0058] The above description of the health functional food composition may be cited in the same way as the description of the pharmaceutical composition. "Health functional food" as defined in the present invention refers to a food manufactured and processed using raw materials or ingredients having functional properties useful to the human body, and "functionality" means consuming for the purpose of obtaining effects useful for health purposes, such as regulating nutrients or physiological actions regarding the structure and function of the human body. The above health functional food may have any one of the forms of tablets, capsules, powders, granules, liquids, or pills.

[0059] In addition, the food composition of the present invention may be a food composition in which a functional ingredient is added to various foods or beverages, etc. The food may have any one of the following forms, for example: beverages, powdered beverages, solids, chewing gum, tea, vitamin complexes, and food additives.

[0060] In addition, the present invention relates to an in vitro composition for anti-aging of neurons comprising: (a) a step of producing a three-dimensional spheroid-shaped cell aggregate by culturing stem cells in a microwell having a diameter of 200 to 800 μm and a depth of 100 to 1000 μm; and (b) a step of separating extracellular vesicles from the three-dimensional spheroid-shaped cell aggregate; or one or more selected from the group consisting of miR-125b and miR-30d.

[0061] One or more selected from the group consisting of the above miR-125b and miR-30d may be administered or delivered in a state in which they are included or loaded in an EV.

[0062] The in vitro composition for anti-aging of neurons according to the present invention may be used for experimental purposes and may be capable of reversing the phenotype of aged neurons to that of young cells, suppressing the expression of aging-related proteins or genes, or activating a suspended cell cycle. The in vitro composition may be a culture medium composition, and the culture medium may include, without limitation, any culture medium known to a person skilled in the art, such as a culture medium containing serum (e.g., fetal bovine serum, horse serum, and human serum). The culture media that may be used in the present invention may include, for example, the 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 any culture medium known in the art suitable for culturing neurons or neural stem cells.

[0063] In addition, the present invention relates to a method for improving or treating neuronal cell aging, comprising: (a) a step of producing a three-dimensional spheroid-shaped cell aggregate by culturing stem cells in a microwell having a diameter of 200 to 800 μm and a depth of 100 to 1000 μm; (b) a step of producing an extracellular vesicle derived from the three-dimensional spheroid-shaped cell aggregate by separating extracellular vesicles from the three-dimensional spheroid-shaped cell aggregate; and (c) a step of treating an individual requiring the same with one or more selected from the group consisting of the extracellular vesicle derived from the three-dimensional spheroid-shaped cell aggregate produced through step (b); or miR-125b and miR-30d.

[0064] One or more selected from the group consisting of the above miR-125b and miR-30d may be administered or delivered in a state in which they are included or loaded in an EV.

[0065] The individual requiring the above may be an individual that needs to prevent young cells from progressing into senescent cells, prevent already aged cells from aging further, cause aged cells to have a younger phenotype, or produce more young cells through cell division, and may be an individual having one or more diseases or conditions selected from the group consisting of, for example, senile degeneration of brain, senile degeneration of the brain, dementia, delirium, dizziness, and senile headache.

[0066] Redundant content is omitted out of consideration for the complexity of this specification, and terms not otherwise defined in this specification have the meanings commonly used in the technical field to which this invention belongs.

[0067] The present invention will be explained in detail below by way of examples. However, the following examples are merely illustrative of the present invention, and the scope of the present invention is not limited by the following examples.

[0068]

[0069] Example 1. Isolation of extracellular vesicles through three-dimensional culture of mesenchymal stem cells

[0070] 1.1 Preparation of Mesenchymal Stem Cells

[0071] Human umbilical cord-derived mesenchymal stem cells (hereinafter WJ-MSC, Samsung Medical Center, Seoul, Korea) at the passage 5 stage 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 at the passage 6 stage were used to construct 3D spheroid-shaped cell aggregates.

[0072]

[0073] 1.2 Preparation of 3D Spheroidal Cell Aggregate Culture Medium

[0074] The WJ-MSCs from passage 6 prepared in Example 1.1 above were washed with PBS, treated with trypsin (TrypLE™ Express, GIBCO, NY, USA), and reacted in a CO2 incubator for 5 minutes. Afterward, fresh serum-free medium was added to neutralize the trypsin and recover the cells, 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 counting the cells, 60 ml of cell suspension was uniformly dispensed into a microarray containing approximately 69,000 microwells coated with MPC (2-Methacryloyloxyethyl Phosphorylcholine Polymer) with diameters and depths of 500 μm × 200 μm, at a density of 400 cells / well, and spontaneous spheroid-shaped cell aggregate formation was induced while maintaining a static state and cultured in a CO2 incubator at 37°C for a total of 4 days to prepare a 3D spheroid-shaped cell aggregate culture medium (hereinafter referred to as 3D-static-spheroid culture medium).

[0075]

[0076] 1.3 Isolation of extracellular vesicles derived from 3D spheroidal cell aggregates

[0077] The 3D-static-spheroid culture medium prepared in Example 1.2 was recovered, centrifuged at 2,500g for 10 minutes to remove cellular foreign matter, and filtered through a 0.22 μm syringe filter. Subsequently, the 3D-static-spheroid culture medium was passed through a 300 kDa hollow fiber membrane (Pall, NY, USA) using a Tangential Flow Filtration (TFF) system to remove proteins and isolate extracellular vesicles. The culture medium was then purified once more with physiological saline to obtain high-purity extracellular vesicles derived from the 3D-static-spheroid of the present invention (hereinafter referred to as EV).

[0078]

[0079] Example 2. Confirmation of anti-aging effect due to EV treatment

[0080] 2.1 Preparation of Aged Mouse Embryonic Neural Stem Cells and Detection of Aging Markers

[0081] 2.1.1 Preparation of Mouse Embryo-Derived Neural Stem Cells

[0082] An experiment was conducted to determine whether the EV prepared in Example 1 induces cell cycle recovery and neurogenesis in aged neural stem cells (NSCs). To construct aged mouse embryonic neural stem cells for the experiment, neural stem cells were isolated from mouse embryos, and a 3D neurosphere suspension culture system was established. Neural stem cells cultured using the 3D neurosphere suspension method can stably maintain their self-renewal ability and stem cell characteristics for a long period, making them suitable for precisely comparing the progression of cellular aging and anti-aging effects.

[0083] For neural stem cell culture, Neurobasal medium was prepared containing Glutamax 100x, Non-Essential amino acid 100x, N2 supplement 100x, B-27 w / o Vita A 50x, EGF 25ng / ml, and FGF 25ng / ml. On E12.5 days of C57BL / 6 mouse embryos, the fetus and placenta were separated, and the fetal brain was obtained through dissection. Only the subventricular zone was isolated and stored in a medium containing growth factors (4℃). In a clean bench, the tissue was physically divided into small pieces using a Dorco sieve, and the cells were chemically isolated using TrypLE at 37℃ for 15 minutes. After filtration in a 40µm strainer and centrifugation at 110g for 5 minutes, the supernatant was removed and the cells were washed with growth factor medium. After repeating the centrifugation twice, cell counting was performed using Trypone Blue.

[0084] Primary neural stem cells isolated from mice formed neurospheres under suspension culture conditions. During the passage culture process from passage 2 (P2) to passage 10 (P10), neurospheres with high sphericity and a neat and compact appearance were observed in the early passages, but as the passages progressed, the diameter of the spheres gradually decreased and the morphological heterogeneity increased.

[0085] To verify whether the cultured cells exhibited the characteristics of neural stem cells, immunofluorescence staining was performed using Sox2 and Nestin, representative markers of neural stem cells. Neural stem cell markers were identified by staining the obtained neurospheres whole mounts or by performing 2D monolayer culture in which the neurospheres were attached to and stained on culture dishes coated with PLO / LMN (Poly-L-ornithine (PLO) 10 µg / ml and Laminin 10 µg / mL). Since SOX2 was detected in the nuclei and Nestin in the cytoplasm of almost all cells, it was verified that the cells constituting the 3D and 2D neurospheres were neural stem cells.

[0086] 2D or 3D culture of mouse embryonic neural stem cells was performed in the following manner.

[0087] - PLO&LMN coating for 2D culture

[0088] Coating was performed with Poly-L-ornithine (PLO: 10 µg / ml) at 37°C for 1 hour, and the Poly-L-ornithine (PLO) was washed with PBS. Subsequently, open-door coating was performed using Laminin 10 µg / mL at 4°C. After washing the Laminin with PBS, the plates were used immediately or stored at 4°C. Using the above method, 1 x 10⁶ plates were coated with PLO and Laminin. 5 Mouse embryonic neural stem cells were cultured at / mL, and when culturing neural bulbs, they were cultured on standard plates. For the group treated with EV after 2D culture, 6x10 starting from passage 2 8 / mL of EV was treated, and half of the culture was replaced daily.

[0089]

[0090] - 3D neural bulb passage culture

[0091] Subculture of the 3D neural bulb was performed in the following manner.

[0092] Subculture was performed when the average diameter of the neuron reached 150–200 µm, and the neurons were collected in conical tubes. After centrifuging at 110 g for 5 minutes, the supernatant was removed, washed with DBPS, and centrifuged once. After centrifugation, the cells were isolated as single cells in TripLE at 37°C and 5% for 10 minutes. After centrifuging at 110 g for 5 minutes, the supernatant was removed, and cell counting was performed using trypan blue. Subsequently, the medium was replaced with growth factor medium, seeded, and used for the experiment.

[0093]

[0094] - 2D neural stem cell passages

[0095] Subculture was performed when the confluency of 2D neural stem cells reached 80–90%. First, the culture medium was removed and washed with DPBS. After inoculating with TripLE, the cells were detached in a 37°C, 5% CO2 incubator for 10 minutes. Five times the amount of culture medium as TripLE was added to stop enzymatic activity, and the cells were scraped with a pipette and collected in a conical tube. After centrifuging at 110g for 5 minutes, the supernatant was removed, and cells were counted using trypan blue and used for subsequent experiments.

[0096]

[0097] 2.1.2. Detection of aging markers in mouse embryonic-derived neural stem cells

[0098] To evaluate the aging process of mouse neural stem cells during passage and the anti-aging effects of EV treatment in Example 1, p53 and p21, representative markers of aging, were identified through protein electrophoresis. All samples were collected on the day of passage and stored frozen at -80°C; the experiment was performed after finally obtaining the 10th passage sample. Proteins were extracted using RIPA buffer, and after quantifying the total protein concentration using a BCA protein assay, 10 µg of protein was separated by SDS-PAGE. Once protein merging was complete, the proteins were transferred to a nitrocellulose membrane via Turbo Transfer. Blocking was performed for one day at 4°C using 5% skim milk in TBS-T. On the following day, the primary antibodies [p53 (1:2500), p21 (1:2000), p16 (1:1000), gamma-H2AX (1:1000), β-actin (1:5000)] were diluted in blocking buffer and locked at room temperature for 2 hours. The samples were washed three times with 1X TBS-T for 10 minutes each, and then locked with HRP-Conjugated secondary antibodies (2x dilution of the primary antibodies) at room temperature for 1 hour. Three washes were performed with 1X TBS-T for 10 minutes each. Signal detection was performed using the ECL™ Prime Western Blotting System, and quantitative analysis of band signal intensity was conducted using ImageJ.

[0099]

[0100] 2.2 Evaluation of Anti-aging Activity According to EV Treatment - Evaluation of Neuroglobule Formation Efficiency

[0101] Mouse embryonic neural stem cells prepared in Example 2.1 undergo aging as passages progress, resulting in decreased neuroglobule formation efficiency and reduced cell viability. During the passage process, the EVs from Example 1 were treated, and their effects on neuroglobule formation efficiency and cell viability were evaluated. The EVs were applied starting from passage 2 at 6x10 8 It was treated at / mL, and the control group was treated with PBS.

[0102] Neuroglobule formation efficiency was confirmed by measuring the diameter of 5 neuroglobules per image after capturing images in 3 different wells on day 6 after passage culture, and cell viability was evaluated using the CCK-8 assay (OD 450 nm) in the same passage. The results of neuroglobule formation efficiency and cell viability are shown in Figure 1. Data were expressed as the mean ± SEM of at least 3 independent experiments, and statistical analysis was performed using two-way ANOVA followed by post-hoc testing.

[0103] In neural stem cells from Passages 2-6, a stable spherical structure was maintained regardless of EV treatment; however, from Passage 7 onwards, the size and density of neural spheres decreased rapidly in the PBS-treated groups, and in Passages 9-10, the structure collapsed and numerous cell fragments were observed. In contrast, the EV-treated groups maintained a spherical structure even under the same passaging conditions, and exhibited a relatively healthy form as intercellular connections did not loosen.

[0104] As shown in Figure 1, quantitative analysis results showed that the average diameter of the nerve bulb decreased to approximately 40 to 60 μm in the PBS group during passages 8 to 10, whereas the EV-treated group maintained a significantly larger diameter of 70 to 90 μm (p < 0.01). In other words, compared to the PBS-treated group, the EV-treated group mitigated the decrease in nerve bulb size as passages progressed, and showed a significant size preservation effect, particularly in the P7 to P10 range.

[0105] Cell viability also remained statistically significantly higher in the EV-treated group, and after 7 passages, the proportion of surviving cells increased to approximately 1.3-1.5 times compared to the PBS group (p < 0.01).

[0106] The above results suggest that EVs partially restore the reduced proliferative capacity, decreased metabolic activity, and reduced viability caused by aging of mouse stem cells during long-term culture. In other words, it is highly likely that various growth factors, microRNAs, and protein regulators present in EVs contributed to maintaining the neurosphere reformation and viability of aging neural stem cells by activating intracellular metabolic pathways and signaling (e.g., Akt, CREB, BDNF pathways).

[0107]

[0108] 2.3. Evaluation of Anti-aging Activity According to EV Treatment - Lysosome Activity

[0109] SA-β-gal is an enzyme used as a representative indicator of senescence, referring to β-galactosidase that is specifically activated under pH 6.0 conditions. In aged cells, β-gal activity within lysosomes increases and is detected as a cyan staining reaction, which allows for the visual distinction of the irreversible cessation of cell proliferation (senescence). Since SA-β-gal staining is utilized as the most common and reliable biological indicator for quantitatively and qualitatively evaluating the degree of senescence, the anti-aging activity of neural stem cells following EV treatment in Example 1 was confirmed through SA-β-gal staining. Experiments were performed on both PLO / LMN-coated culture plates and 3D neural sphere models; for the 2D model, the number of stained cells was counted in a total of three independent experiments, while for the 3D neural sphere, the OD value was measured using Image J after staining. Figure 2a shows the results of confirming the morphology of neuroglobules and SA-β-gal staining in EV-treated or untreated cells at passages 2 and 8, and Figure 2b shows the results of quantifying positive cells by calculating the relative light density (ROD) from the SA-β-gal staining images. Data were expressed as the mean ± SEM of at least three independent experiments, and statistical analysis was performed using one-way ANOVA (Tucky post-hoc test).

[0110] As shown in Figure 2a, almost no β-galactosidase staining signal was observed in young cells from passage 2, whereas strong blue staining and high SA-β-gal activity were observed in senescent cells from passage 8 (P8-DPBS). This indicates that lysosomal enzyme activity and intracellular senescence markers (SA-β-gal) increased with aging. On the other hand, in EV-treated passage 8 cells (Passage 8(P8)-EV), staining intensity was significantly reduced, and the cell morphology maintained a denser and more spherical structure compared to the DPBS group. This indicates that EV treatment brought the metabolic activity of the cells closer to a steady state and suppressed excessive lysosomal activity (i.e., cellular senescence).

[0111] As shown in Figure 2b, quantitative analysis results showed that the proportion of 2D β-gal positive cells decreased significantly from approximately 45-50% in the P8-DPBS group to approximately 20% or less in the P8-EV group (p < 0.01), and SA-β-gal activity (ROD value) in 3D spheroids also decreased by approximately 40% compared to P8-PBS (p < 0.001).

[0112] Through the above results, it was confirmed that EV induces the recovery of regenerative capacity by inhibiting the lysosome pathway overactivated due to aging, thereby alleviating both intracellular aging signals (p53-p21-p16 axis) and β-gal activity.

[0113]

[0114] 2.4. Evaluation of Anti-aging Activity Following EV Treatment - Confirmation of Changes in Aging Markers

[0115] As the aging phenotype of 2D and 3D neural stem cells was significantly confirmed at passage 8, the expression of aging-related proteins and genes was examined in the samples from passage 8. Protein expression of p53, p21, and p16, which are aging markers and cell cycle arrest markers, and Sirt1, an upstream gene of p53, was confirmed via electrophoresis. In addition, changes in the expression of the gene BAX, which is associated with apoptosis that increases during aging, were evaluated. The results of measuring changes in each aging-related marker are shown in Figure 3.

[0116] As shown in Figure 3, compared to the PBS group (P8-PBS), the EV-treated group (P8-EV) showed increased Sirt1 expression and decreased p53 and p21 expression, while there was no significant change in p16. β-Actin was used as a loading control. Additionally, RT-qPCR analysis results showed that the EV-treated group had significantly reduced p53, p21, p16, and BAX expression compared to P8-PBS, suggesting that EV alleviates aging-related signals. Specifically, in Western blot and quantitative graphs, the aging-induced group (P8-PBS) showed approximately 3-5 times higher expression of p53, p21, and p16, respectively, compared to the P2-control. In contrast, the expression of these genes was significantly reduced in the P8-EV treated group; p53 and p21 decreased by more than 50% compared to P8-PBS (p < 0.01 or 0.001), and p16 also decreased (p < 0.01), confirming that EV treatment alleviates cellular senescence signals by suppressing the overexpression of senescence markers. As a result of changes in gene expression related to senescence and cell death (quantification via qPCR), apoptotic / senescence-related genes such as p53, p21, p16, Bax, and Caspase3 were significantly elevated in the P8-PBS group. In the P8-EV group, the expression of all these genes decreased, and a distinct decrease was observed in p21 and Bax, which are downstream genes of the p53 pathway (p < 0.01 to 0.001). Conversely, the expression of the cell proliferation marker PCNA relatively recovered in the EV group, confirming that EV treatment has the effect of returning the expression of genes related to cellular senescence and cell death to normal levels. Data were expressed as mean ± SEM (n ≥ 3 independent experiments), and statistical analysis was performed using one-way ANOVA (Tucky's post-hoc test).

[0117]

[0118] 2.5. Evaluation of Anti-aging Activity Following EV Treatment - Cell Cycle Changes

[0119] Through Example 2.4 above, it was confirmed that EV significantly inhibits the expression of aging marker proteins (p53, p21, p16), suggesting that EV contributes to alleviating cell cycle arrest and aging signals. Therefore, to confirm the effect of EV treatment on unlocking cell cycle arrest, changes in the cell cycle following EV treatment were measured using flow cytometry. The results of the cell cycle analysis are shown in Figure 4.

[0120] As shown in Figure 4, the P8-PBS group showed an increase in the G0 / G1 phase and a decrease in the S and G2 / M phases, confirming cell cycle arrest. On the other hand, the P8-EV group showed a decrease in the G0 / G1 ratio and a significant increase in the S phase ratio, and the G2 / M phase was also partially recovered (p < 0.05 to 0.01). This result demonstrates that EV treatment alleviates cell cycle arrest caused by aging and restores entry into the DNA synthesis phase (S phase). This result is consistent with the results of Example 2.4, which showed a decrease in the expression of cell cycle arrest and aging-related proteins such as p53, p21, p27, and p16, indicating that EV is effective in alleviating cellular aging.

[0121]

[0122] Example 3. Confirmation of anti-aging effects in aged mice according to EV treatment

[0123] The anti-aging effect of EV prepared by the method of Example 1 was confirmed in an aged mouse model. Aged mice (18 to 22 months of age) were transdermally injected with EV at a dose of 6 x 10^8 into the skin around the neck once; the mice were sacrificed 7 days later, and the hippocampus was extracted to confirm the expression of aging genes. Specifically, the subventricular zone (SVZ) of the brain was isolated, and hippocampal proteins were extracted from the young group (Young - 8 weeks), the aged group (Old), and the EV-administered group (Old + EV). The effects on the p53-p21 axis and the p16-gamma H2AX axis were confirmed through Sirt1, p53, p21, p16, and γH2AX immunoblotting and quantification. β-actin was used as a loading control.

[0124] In addition, to examine changes in lysosomal activation that occur during aging, β-galactosidase staining, an aging-related marker, was performed on hippocampal sections of the young, old, and EV-treated groups. Data were expressed as mean ± SEM (n = 5-6), and statistical analysis was performed using one-way ANOVA (Tucky post-hoc test).

[0125] The results of measuring changes in aging gene expression in aging mice are shown in Fig. 5, and the results of β-galactosidase staining are shown in Fig. 6.

[0126] As shown in Figure 5, p53, p21, p16, and γH2AX increased and Sirt1 decreased in the aging group; it was confirmed that Sirt1 was restored and the expression of aging-related proteins decreased upon EV administration. Compared to the aging group (Old), p53 expression decreased by more than 70% in the EV-treated group (Old+EV) (p < 0.01), and p21 and p16 proteins also decreased by more than 50%, respectively (p < 0.01–0.001). Furthermore, γH2AX, a DNA damage indicator, was also significantly attenuated (p < 0.05–0.01). This clearly demonstrates that EV inhibits the intracellular DNA damage response and the p53 signaling axis (p53-p21-p16), thereby alleviating cell cycle arrest and aging signals.

[0127] In addition, as shown in Figure 6, SA-β-gal staining results of the hippocampus revealed that the staining in the hippocampus of aged mice was darker than in young mice, while the staining tended to be lighter in the EV-treated group. This indicates that while strong blue staining signals were observed in aged mice (Old), confirming increased lysosomal activity and the accumulation of senescent cells, the staining intensity was significantly lower in the EV-administered group (Old+EV), suggesting that a reduction in senescent cells and the normalization of metabolic activity within the hippocampal tissue were induced. These changes are consistent with the anti-aging phenotype observed in in vitro cell experiments, suggesting that EV induces aging mitigation and functional recovery not only at the cellular level but also at the tissue and organ levels.

[0128]

[0129] Example 4. Analysis of EV anti-aging active ingredients

[0130] Through Examples 2 and 3, it was confirmed that EV exhibits anti-aging activity in vitro and in vivo. Furthermore, experiments were conducted to determine which factors influence the alleviation of aging and cell cycle reactivation of neural stem cells through the analysis of active components contained in EV. As a result of predicting the complementary binding sequence between miR-125b and TP53 3' UTR, conserved seed regions were identified in humans (hTp53), mice (mTp53), and zebrafish (zTp53). Hypothesizing that miR-125b-5p and miR-30d-5p contained in EV regulate p53 expression and thereby achieve anti-aging effects, experiments were conducted to confirm the anti-aging effects of miR-125b-5p and miR-30d-5p. Mimics and inhibitors of miR-125b-5p and miR-30d-5p were purchased from Bioneer (miR-125b-5p: MI0000470, miR-30d-5p: MI0000255) and used.

[0131] The sequence information used for each experiment is as follows:

[0132] miR-125b-5p mimics : 5′-UCCCUGAGACCCUAACUUGUCA-3′ (Sequence No. 1),

[0133] miR-125b-5p inhibitor : 5′-UCACAAGUUAGGGUCUCAGGGA-3′ (SEQ No. 2)

[0134] miR-30d-5p mimics : 5′-UGUAAACAUCCCCGACUGGAAG-3′ (sequence number 3),

[0135] miR-30d-5p inhibitor : 5′-CUUCCAGUCGGGGAUGUUUACA-3′ (SEQ No. 4).

[0136]

[0137] miR-125b directly conserved with P53 was transfected into neural stem cells, and p53 and p21 expression were analyzed by immunoblot and RT-PCR after 48 hours. Samples were taken 24 hours after treatment with 50 nM of MicroRNA-125b-5p mimic and 100 nM of its inhibitor, and p53 protein and gene expression were confirmed. β-Actin was used as a loading control, and quantitative data were expressed as relative changes compared to the NC mimic (mean ± SEM, n ≥ 3). Statistical analysis was performed using one-way ANOVA (Tucky post-hoc test). The regulatory effect of microRNA 125b-5p on p53 is shown in Figure 7.

[0138] Changes in protein expression 48 hours after transduction of negative control (NC) mimic, miR-30d-5p mimic 50 nM, NC inhibitor, or miR-30d-5p inhibitor 100 nM were confirmed by immunoblot and RT-PCR. β-Actin was used as a loading control. Data were presented as mean ± SEM (n ≥ 3 independent experiments), and statistical analysis was performed using one-way ANOVA (Tucky post-hoc test). The p53 regulatory effect following the introduction of microRNA 30d is shown in Figure 8.

[0139] As shown in Figure 7, miR-125b overexpression (mimic) reduced p53 and p21 expression, and miR-125b inhibitor treatment increased their expression.

[0140] As shown in Figure 8, miR-30d-5p overexpression (mimic) also reduced p53 and p21 protein expression, while miR-30d-5p inhibition (inhibitor) increased p53 and p21 expression. In quantitative analysis, the p53 / β-Actin ratio was significantly decreased in the miR-30d-5p overexpression group, whereas it increased in the inhibitor-treated group. RT-qPCR results indicate that miR-30d-5p expression was successfully regulated after mimic / inhibitor treatment.

[0141] Through the above results, it was confirmed that miR-125b and miR-30d act directly on p53, and that p53 expression is regulated when corresponding miR-125b and miR-30d analogs and inhibitors are transfected into neural stem cells. This result demonstrates that miR-125b and miR-30d directly regulate the p53 signaling axis and supports the fact that microRNAs such as miR-125b and miR-30d, delivered via EVs, can induce senescence mitigation and cell cycle restart in neural stem cells mediated by the p53 / p21 pathway.

[0142]

[0143] Foregoing, specific parts of the present invention have been described in detail. It will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

1. (a) a step of producing a three-dimensional spheroid-shaped cell aggregate by culturing stem cells in a microwell having a diameter of 200 to 800 μm and a depth of 100 to 1000 μm; and (b) a step of separating extracellular vesicles from the three-dimensional spheroid-shaped cell aggregates; a pharmaceutical composition for the prevention or treatment of neuro-aging-related diseases or disorders, comprising extracellular vesicles derived from three-dimensional spheroid-shaped cell aggregates.

2. A pharmaceutical composition for the prevention or treatment of neuro-aging-related diseases or disorders, wherein the stem cells are one or more selected from the group consisting of mesenchymal stem cells, pluripotent stem cells, induced pluripotent stem cells, and embryonic stem cells.

3. A pharmaceutical composition for the prevention or treatment of neuro-aging-related diseases or disorders, wherein the three-dimensional culture of step (a) is static culture in accordance with claim 1.

4. A pharmaceutical composition for the prevention or treatment of a neuro-aging-related disease or disorder according to claim 1, wherein the neuro-aging-related disease or disorder is one or more selected from the group consisting of senile degeneration of brain, senile degeneration of the brain, dementia, delirium, dizziness, and senile headache.

5. A pharmaceutical composition for the prevention or treatment of neuroaging-related diseases or disorders, wherein, in claim 1, the extracellular vesicles induce the phenotype of neuroaging cells to the phenotype of young cells through one or more activities selected from the group consisting of neuronal cell proliferation, inhibition of aging marker protein expression, reduction of β-galactosidase activity, and restoration of cell cycle arrest.

6. A pharmaceutical composition for the prevention or treatment of neuro-aging-related diseases or disorders, wherein the extracellular vesicles of claim 1 comprise one or more selected from the group consisting of miR-125b and miR-30d.

7. A pharmaceutical composition for the prevention or treatment of neuro-aging-related diseases or disorders, comprising one or more selected from the group consisting of miR-125b and miR-30d.

8. (a) a step of producing a three-dimensional spheroid-shaped cell aggregate by culturing stem cells in a microwell having a diameter of 200 to 800 μm and a depth of 100 to 1000 μm; and (b) a step of separating extracellular vesicles from the above three-dimensional spheroid-shaped cell aggregates; a health functional food composition for neuronal anti-aging comprising extracellular vesicles derived from three-dimensional spheroid-shaped cell aggregates.

9. A health functional food composition for neuronal anti-aging comprising one or more selected from the group consisting of miR-125b and miR-30d.

10. (a) a step of producing a three-dimensional spheroid-shaped cell aggregate by culturing stem cells in a microwell having a diameter of 200 to 800 μm and a depth of 100 to 1000 μm; and (b) a step of separating extracellular vesicles from the three-dimensional spheroid-shaped cell aggregates; an in vitro composition for neuronal anti-aging comprising extracellular vesicles derived from three-dimensional spheroid-shaped cell aggregates prepared through the above step. 11.(a) A step of producing a three-dimensional spheroid-shaped cell aggregate by culturing stem cells in a microwell having a diameter of 200 to 800 μm and a depth of 100 to 1000 μm; (b) a step of preparing extracellular vesicles derived from three-dimensional spheroid-shaped cell aggregates through a step of separating extracellular vesicles from the three-dimensional spheroid-shaped cell aggregates; and (c) a step of treating an individual in need with an extracellular vesicle derived from a three-dimensional spheroid-shaped cell aggregate prepared through step (b) above; a method for improving or treating neuronal cell aging comprising:

12. A method for improving or treating neuronal aging comprising the step of treating an individual in need with one or more selected from the group consisting of miR-125b and miR-30d.

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