Composition for treatment of lipid metabolism disorder diseases, comprising stem cell-derived extracellular vesicles having enhanced efficacy

Abstract

The present invention relates to a use of extracellular vesicles derived from a three-dimensional spheroid-type cell aggregate prepared by a method of the present invention, for the prevention, alleviation, or treatment of lipid metabolism disorder diseases. It has been confirmed that extracellular vesicles derived from a three-dimensional spheroid-type cell aggregate, according to the present invention, in a disease model, increase cholesterol efflux-related protein expression, and improve lipid metabolism. This means that the extracellular vesicles derived from a three-dimensional spheroid-type cell aggregate may mitigate the progression of atherosclerosis by inhibiting lipid accumulation in macrophages and promoting cholesterol removal, and thus the extracellular vesicles derived from a three-dimensional spheroid-type cell aggregate of the present invention may be utilized in various ways in the field of prevention or treatment of lipid metabolism disorder diseases including arteriosclerosis.

Description

Composition for treating lipid metabolism disorders comprising stem cell-derived extracellular vesicles with enhanced efficacy

[0001] The present invention relates to the use of extracellular vesicles derived from three-dimensional spheroid-shaped cell aggregates produced by the method of the present invention for the prevention, improvement, or treatment of lipid metabolism disorders.

[0002] Lipid metabolism is an essential physiological process for maintaining the body's energy homeostasis and configuring cell membranes, requiring precise regulation of lipid synthesis, transport, storage, and breakdown. However, when this balance of lipid metabolism is disrupted by dietary habits, genetic factors, aging, or metabolic abnormalities, blood cholesterol and triglyceride levels increase abnormally, leading to various metabolic diseases. Abnormalities in lipid metabolism are represented by hyperlipidemia, hypercholesterolemia, and dyslipidemia, and these pathological conditions can lead to cardiovascular diseases, atherosclerosis, coronary artery disease, and non-alcoholic fatty liver disease. In particular, the accumulation of low-density lipoprotein cholesterol (LDL-cholesterol) is known to be a major cause of atherosclerosis; as it oxidizes in the vascular endothelium, it induces the formation of foam cells by macrophages, which in turn promotes inflammatory responses and plaque formation. Currently, drugs such as statins and PCSK9 inhibitors are used in clinical practice to lower blood cholesterol, but these drugs have limitations, including reported side effects such as myalgia and hepatotoxicity, as well as drug tolerance or compensatory LDL elevation when taken long-term. Accordingly, there is an increasing demand for new cholesterol-lowering technologies that have fewer side effects and excellent biocompatibility.

[0003] Against this backdrop, mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) are attracting attention as an alternative to cell-based therapies. MSC-EVs possess the advantage of maintaining the therapeutic characteristics of progenitor cells while avoiding potential risks associated with cell therapy, such as immunogenicity or tumorigenesis, and have been reported to exhibit effects in regulating inflammation, tissue protection, and improving lipid metabolism in various animal models. However, EVs produced using conventional two-dimensional (2D) culture methods have faced limitations in clinical application due to low production yields and functional heterogeneity between batches. To overcome these limitations, various EV production technologies have been proposed; among them, a three-dimensional (3D) spheroidal cell aggregate culture system using micropatterned wells has been reported to increase EV production and enhance the expression of therapeutic factors such as miRNAs and proteins.

[0004] Accordingly, the inventors completed the present invention by confirming the therapeutic effect of WJ-MSC-derived extracellular vesicles produced using a three-dimensional static culture system on lipid metabolism disorders.

[0005] Therefore, the object of the present invention is to provide a composition comprising extracellular vesicles derived from three-dimensional spheroid-shaped cell aggregates produced by the method of the present invention.

[0006] Another objective of the present invention is to provide a method for treating lipid metabolism disorders comprising the step of administering extracellular vesicles derived from three-dimensional spheroid-shaped cell aggregates produced by the method of the present invention to an individual in need thereof.

[0007] Another objective of the present invention is to provide a method for lowering blood cholesterol, comprising the step of administering an extracellular vesicle derived from a three-dimensional spheroid-shaped cell aggregate produced by the method of the present invention to an individual in need of it.

[0008] To achieve the above objective, the present invention provides a pharmaceutical composition for the prevention or treatment of lipid metabolism disorders 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 cells and vesicles from the three-dimensional spheroid-shaped cell aggregates.

[0009] In addition, the present invention provides a pharmaceutical composition for lowering blood cholesterol 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 cells and vesicles from the three-dimensional spheroid-shaped cell aggregates.

[0010] It was confirmed that the three-dimensional spheroid-shaped cell aggregate-derived extracellular vesicles according to the present invention increase the expression of cholesterol-excreting proteins and improve lipid metabolism in disease models. This implies that the three-dimensional spheroid-shaped cell aggregate-derived extracellular vesicles can alleviate the progression of atherosclerosis by inhibiting lipid accumulation within macrophages and promoting cholesterol removal. Therefore, the three-dimensional spheroid-shaped cell aggregate-derived extracellular vesicles of the present invention can be utilized in various ways in the field of prevention or treatment of lipid metabolism disorders, including arteriosclerosis.

[0011] Figure 1a is a figure showing the results of measuring the particle diameter of a 3D-static-spheroid EV according to the present invention.

[0012] FIG. 1b is a figure showing the shape of a 3D-static-spheroid EV according to the present invention observed with an electron microscope.

[0013] FIG. 1c is a figure showing the results of analyzing the surface antigens of a 3D-static-spheroid EV according to the present invention using a multi-bead-based flow cytometry method.

[0014] Figure 1d is a figure showing the particle per protein ratio and the detection results of organelle protein markers of a 3D-static-spheroid EV according to the present invention.

[0015] Figure 2a is a figure showing the results of analyzing changes in lipid accumulation in RAW264.7-derived foam cells by Oil Red O staining following treatment with 3D-static-spheroid EV according to the present invention.

[0016] FIG. 2b is a figure showing changes in intracellular neutral lipid content and Dil-ox-LDL uptake following treatment with 3D-static-spheroid EV according to the present invention.

[0017] FIG. 2c is a figure showing the change in total cholesterol (TC) levels according to 3D-static-spheroid EV treatment according to the present invention.

[0018] FIG. 2d is a figure showing the change in CD36 protein expression according to 3D-static-spheroid EV treatment according to the present invention.

[0019] Figure 3a is a figure showing the effect of increasing cholesterol excretion in RAW264.7-derived foam cells following treatment with 3D-static-spheroid EV according to the present invention.

[0020] Figure 3b is a figure showing the change in ABCA1 protein expression following treatment with a 3D-static-spheroid EV according to the present invention.

[0021] Figure 3c is a figure showing the change in ABCG1 protein expression following treatment with a 3D-static-spheroid EV according to the present invention.

[0022] Figure 3d is a figure showing the change in LXR-α expression according to the treatment of a 3D-static-spheroid EV according to the present invention.

[0023] Figure 3e is a figure showing the change in PPAR-γ expression following treatment with a 3D-static-spheroid EV according to the present invention.

[0024] FIG. 3f is a figure illustrating the foam cell formation mitigation mechanism of a 3D-static-spheroid EV according to the present invention.

[0025] FIG. 4a is a figure showing the experimental design of an atherosclerosis model mouse of a 3D-static-spheroid EV according to the present invention.

[0026] FIG. 4b is a figure comparing the degree of gross plaque deposition on the aortic arch after 3D-static-spheroid EV treatment according to the present invention.

[0027] Figure 4c is a figure showing the en face Oil Red O staining result of the aorta after 3D-static-spheroid EV treatment according to the present invention.

[0028] FIG. 4d is a figure showing the change in plaque formation in the aortic cross-section after 3D-static-spheroid EV treatment according to the present invention.

[0029] Figure 4e shows the Picro Sirius Red staining results of the aortic sinus after 3D-static-spheroid EV treatment according to the present invention.

[0030] FIG. 4f shows M1 macrophages (F4 / 80) in an aortic lesion after 3D-static-spheroid EV treatment according to the present invention. + CD68 + This is a diagram confirming changes in infiltration using immunofluorescence.

[0031] Figure 5a shows the results of analyzing CD36 protein expression in the aortic tissue of apolipoprotein E-deficient mice after 3D-static-spheroid EV treatment according to the present invention.

[0032] Figure 5b is a figure showing changes in ABCA1 and ABCG1 protein expression in aortic tissue of apolipoprotein E-deficient mice after 3D-static-spheroid EV treatment according to the present invention.

[0033] Figure 5c shows LXR-α expression in the aortic tissue of apolipoprotein E-deficient mice after 3D-static-spheroid EV treatment according to the present invention.

[0034] Figure 5d shows PPAR-γ expression in the aortic tissue of apolipoprotein E-deficient mice after 3D-static-spheroid EV treatment according to the present invention.

[0035] Figure 6a is a figure showing changes in blood total cholesterol, triglycerides, and LDL-C levels in apolipoprotein E-deficient mice after 3D-static-spheroid EV treatment according to the present invention.

[0036] Figure 6b is a figure showing the change in serum hs-CRP levels after 3D-static-spheroid EV treatment according to the present invention.

[0037] Figure 6c is a figure showing the change in serum IL-6 levels after 3D-static-spheroid EV treatment according to the present invention.

[0038] Figure 6d is a figure showing the change in serum PCSK9 levels after 3D-static-spheroid EV treatment according to the present invention.

[0039] Figure 6e is a figure showing the change in serum Lp(a) levels after 3D-static-spheroid EV treatment according to the present invention.

[0040] Figure 7a is a figure showing the change in body weight of apolipoprotein E-deficient mice after 3D-static-spheroid EV treatment according to the present invention.

[0041] FIG. 7b is a figure showing changes in liver weight and liver / body weight ratio after 3D-static-spheroid EV treatment according to the present invention.

[0042] FIG. 7c is a figure showing the effect of alleviating hepatic steatosis after 3D-static-spheroid EV treatment according to the present invention.

[0043] FIG. 7d is a figure showing the effect of alleviating liver fibrosis after 3D-static-spheroid EV treatment according to the present invention.

[0044] Figure 7e is a figure showing changes in LDL-R, PCSK9, and IL-6 protein expression in liver tissue after 3D-static-spheroid EV treatment according to the present invention.

[0045] The present invention will be described in detail below.

[0046] According to an embodiment of the present invention, the present invention provides a pharmaceutical composition or a health functional food composition 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 cells and vesicles from the three-dimensional spheroid-shaped cell aggregates. The pharmaceutical composition is for the prevention or treatment of lipid metabolism disorders; or for lowering blood cholesterol. Additionally, the health functional food composition is for the prevention or improvement of lipid metabolism disorders; or for lowering blood cholesterol.

[0047] In the present invention, the cell may be used without limitation as long as it is a cell capable of separating extracellular vesicles, and may be a cell isolated from a biological organism in nature. Additionally, 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.

[0048] 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, three-dimensional culture allows for growth in all directions in vitro and creates an environment that is more similar to an in vivo cell environment.

[0049] In the present invention, the three-dimensional culture of step (a) can be performed by any three-dimensional cell culture technology known in the art to which the present invention belongs, for example, by 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 dynamic culture or static culture, and more preferably, static culture. In the present invention, when the three-dimensional culture of step (a) is performed as static culture, the devices required for shaking culture are not required, making the culture easier, and enabling mass culture in a GMP (Good Manufacturing Practices) facility.

[0050] 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 particular, 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.

[0051] 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 200 to 500 cells / well.

[0052] In the present invention, the step of separating the extracellular vesicles in step (b) may be by physical separation or chemical separation. The physical separation may be the extrusion of a sample containing cells or cell aggregates, and the chemical separation may be the treatment with a chemical substance capable of separating extracellular vesicles from cells or cell aggregates. For example, the separation step of the present invention may be performed using a method selected from the group consisting of ultrasonic decomposition, cell lysis, homogenization, freeze-thaw, electroporation, chemical treatment, mechanical decomposition, and treatment of physical stimulation that applies force to the cell externally, and may be a physicochemical method of separation through binding of ions with specific biomarkers by ion or affinity chromatography, or a method of separating by destroying cells and binding only extracellular vesicles using polymers and reagents, such as Hansabiomed Life Sciences’ Immunofinity EV Capture or Creative Biolabs’ Exosome Purification Reagent, or Microgentas’ ExoCAS-2 reagent or method, and preferably may be separated by a Tangential Flow Filtration (TFF) method, but is not limited thereto.

[0053] In the present invention, the extracellular vesicle may be one that highly expresses various substances exhibiting efficacy in preventing, improving, or treating lipid metabolism disorders compared to known extracellular vesicles, for example, preferably the extracellular vesicle of the present invention may be one that highly expresses one or more selected from the group consisting of miR-146a, miR-27a, miR-132, miR-184, miR-210, and miR-301b compared to extracellular vesicles derived from spheroid-type cell aggregates cultured in three-dimensional dynamic culture of mesenchymal stem cells; or one or more selected from the group consisting of miR-27a, miR-146a, and miR-146b compared to extracellular vesicles derived from mesenchymal stem cells cultured in two dimensions, or may be one that highly expresses VEGF (Vascular endothelial growth factor), Hif-1a (Hypoxia-inducible factor 1-alpha), and FGF (Fibroblast growth factor).

[0054] 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.

[0055] In the present invention, extracellular vesicles derived from three-dimensional spheroid-shaped cell aggregates exhibiting preventive, improving, or therapeutic effects for lipid metabolism disorders can be used interchangeably with “3D-static-spheroid-EVs.” Additionally, in comparison, extracellular vesicles derived from three-dimensionally dynamically cultured spheroid-shaped cell aggregates can be used interchangeably with “3D-dynamic-PEG-spheroid-EVs.”

[0056] 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).

[0057] In the present invention, the three-dimensional spheroid-shaped cell aggregate may have an average diameter of 74.43 ± 7.756 μm, preferably having a size range of 55 to 95.0 μm, and the average diameter may be 74.43 μm and the coefficient of variation (CV) may be 9.59% as a result of measuring the size distribution of 155 of the three-dimensional spheroid-shaped cell aggregates. The kurtosis of the size distribution of the three-dimensional spheroid-shaped cell aggregate of the present invention may be higher compared to the “spheroid-shaped cell aggregate obtained by three-dimensional dynamic culture of mesenchymal stem cells.” Accordingly, the size of the three-dimensional spheroid-shaped cell aggregate may be smaller and have a relatively uniform size distribution compared to the “spheroid-shaped cell aggregate obtained by three-dimensional dynamic culture of mesenchymal stem cells.”

[0058] 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.

[0059] The microwell of the present invention may have a diameter of 200 to 800 μm, preferably 300 to 800 μm, and more preferably 400 to 800 μm.

[0060] 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.

[0061] 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.

[0062] When extracellular vesicles are manufactured using the “method for manufacturing extracellular vesicles derived from three-dimensional spheroid-shaped cell aggregates” of the present invention, the extracellular vesicles can be mass-produced rapidly and efficiently, along with the manufacturing advantages associated with static culture. In particular, the method for manufacturing extracellular vesicles of the present invention is characterized by being suitable for GMP application.

[0063] In the present invention, the lipid metabolism disorder refers to a disease caused by an imbalance in the lipid synthesis, breakdown, transport, or excretion processes of the human body. Such a disease can be caused by various factors such as genetics, dietary habits, obesity, and insulin resistance, and in particular, it can develop due to an abnormal increase in blood cholesterol.

[0064] In a specific embodiment of the present invention, the lipid metabolism disorder may be a disease caused by an increase in blood cholesterol.

[0065] In a specific embodiment of the present invention, the lipid metabolism disorder may be one or more diseases selected from the group consisting of arteriosclerosis, atherosclerosis, hyperlipidemia, hypercholesterolemia, dyslipidemia, coronary artery disease, peripheral vascular disease, ischemic heart disease, myocardial infarction, non-alcoholic fatty liver, non-alcoholic fatty liver hepatitis, fatty liver, liver fibrosis, metabolic syndrome, obesity, type 2 diabetes, insulin resistance disease, vasculitis and vascular endothelial dysfunction, and preferably may be arteriosclerosis or atherosclerosis, but the scope of the present invention is not limited thereto.

[0066] In a specific embodiment of the present invention, the lipid metabolism disorder may be a cardiovascular disease caused by lipid metabolism disorder, and preferably, it may be one or more selected from the group consisting of arteriosclerosis, atherosclerosis, hyperlipidemia, hypercholesterolemia, dyslipidemia, coronary artery disease, peripheral vascular disease, ischemic heart disease, and myocardial infarction.

[0067] In a specific embodiment of the present invention, it is preferable that the extracellular vesicle derived from the three-dimensional spheroidal cell aggregate inhibits LDL cholesterol excretion; or inhibits the expression of lipid-associated biomarkers.

[0068] The pharmaceutical composition of the present invention may further comprise, in addition to the active ingredient, a suitable carrier, excipient, and diluent conventionally used in the preparation of the pharmaceutical composition. The pharmaceutical composition of the present invention may further comprise other pharmaceutical active ingredients or active mixtures.

[0069] The pharmaceutical composition of the present invention may be formulated and used in the form of oral formulations such as patches, coatings, 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, the product is prepared using diluents or excipients such as commonly used fillers, fillers, binders, humectants, 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. Liquid dosage forms for oral administration include suspensions, liquid formulations, emulsions, and syrups, and may include various excipients, for example, humectants, sweeteners, flavorings, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. As non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used.Witepsol, Macrogol, Tween 61, cacao oil, laurin oil, glycerozelatin, etc. can be used as bases for suppositories.

[0070] 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.

[0071] 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, orally, transdermally, rectally, or intravenously, intramuscularly, subcutaneously, or by injection.

[0072] 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.

[0073] The composition of the present invention may be a health functional food composition for the prevention or improvement of metabolic disorders; or for lowering blood cholesterol.

[0074] In the present invention, a health functional food refers to a group of foods to which added value is imparted so that the function of the food acts or manifests for a specific purpose using physical, biochemical, or biotechnological methods, or to a food processed by designing it to sufficiently express in the body the in vivo regulatory functions regarding the regulation of biological defense rhythms, disease prevention, and recovery possessed by the food composition. For the purposes of the present invention, the health functional food is intended to enhance blood cholesterol-lowering activity.

[0075] In the present invention, the food composition can be prepared by methods commonly used in the art, and the composition can be prepared by adding raw materials and ingredients commonly added in the art. In addition, the formulation of the food composition can also be prepared without limitation as long as it is a formulation recognized as a food composition.

[0076] Foods according to the present invention include, for example, various types of food, beverages, chewing gum, tea, vitamin complexes, functional foods, etc. In addition, foods include, but are not limited to, special nutritional foods (e.g., infant formula, baby food, etc.), processed meat products, fish products, tofu products, jelly products, noodles (e.g., ramen, noodles, etc.), bread products, health supplements, seasoning foods (e.g., soy sauce, soybean paste, red pepper paste, mixed sauce, etc.), sauces, confectionery products (e.g., snacks), candies, chocolates, chewing gum, ice cream, dairy products (e.g., fermented milk, cheese, etc.), other processed foods, kimchi, pickled foods (various types of kimchi, pickled vegetables, etc.), beverages (e.g., fruit beverages, vegetable beverages, soy milk, fermented beverages, etc.), natural seasonings (e.g., ramen soup, etc.), and food additives. The above foods, beverages, or food additives may be manufactured by conventional manufacturing methods.

[0077] When the composition of the present invention is used as an additive to a health functional food, the composition may be added as is or used together with other health functional food ingredients, and may be used appropriately according to conventional methods. The amount of the active ingredient can be appropriately determined according to the purpose of use. Generally, when manufacturing food or beverages, the composition of the present invention may be added to the raw material in an amount preferably 50 parts by weight or less, more preferably 25 parts by weight or less. However, in the case of long-term consumption for the purpose of health control and hygiene, the above amount may be less than the above range, and since there is no problem in terms of stability, the active ingredient may be used in an amount greater than the above range.

[0078] In addition to containing extracellular vesicles derived from three-dimensional spheroidal cell aggregates as an active ingredient, the food or health functional food composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients, as in conventional food compositions. Examples of the above-mentioned natural carbohydrates include monosaccharides, e.g., glucose, fructose, etc.; disaccharides, e.g., maltose, sucrose, etc.; and polysaccharides, such as conventional sugars like dextrin, cyclodextrin, etc.; and sugar alcohols such as xylitol, sorbitol, erythritol, etc. The above-mentioned flavoring agents may advantageously include natural flavoring agents (thaumatin), stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.), and synthetic flavoring agents (saccharin, aspartame, etc.).

[0079] In addition, the food composition may contain, in addition to extracellular vesicles derived from three-dimensional spheroid-shaped cell aggregates, various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. Furthermore, the food composition of the present invention may contain fruit pulp for the production of natural fruit juices, fruit juice beverages, and vegetable beverages.

[0080]

[0081] According to another aspect of the present invention, the present invention provides a method for treating lipid metabolism disorders or a method for lowering blood cholesterol, comprising the step of administering extracellular vesicles derived from three-dimensional spheroid-shaped cell aggregates to an individual in need thereof. The extracellular vesicles derived from three-dimensional spheroid-shaped cell aggregates are prepared through the following steps: (a) preparing three-dimensional spheroid-shaped cell aggregates by culturing stem cells in three dimensions (3D, 3 dimension) in microwells having a diameter of 200 to 800 μm and a depth of 100 to 1000 μm; and (b) separating extracellular vesicles from the three-dimensional spheroid-shaped cell aggregates.

[0082] In a specific embodiment of the present invention, the individual may be an individual expected to develop a lipid metabolism disorder; an individual that has developed the disorder; or an individual that has been determined to be cured, but is not limited thereto. Additionally, the individual may be an individual in which an increase in blood cholesterol has occurred; or an individual in which it is expected to occur, but the scope of the present invention is not limited thereto.

[0083]

[0084] 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.

[0085] The present invention will be described in more detail below through examples. These examples are intended solely to illustrate the present invention, and it will be obvious to those skilled in the art that the scope of the present invention is not to be interpreted as being limited by these examples.

[0086]

[0087] [Experimental Example]

[0088] Experimental Example 1. 3D Spheroid Culture of Human Umbilical Cord-Derived Mesenchymal Stem Cells and EV Production

[0089] 1-1. Preparation of Mesenchymal Stem Cells

[0090] 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.

[0091]

[0092] 1-2. Preparation of 3D Spheroidal Cell Aggregate Culture Medium

[0093] The WJ-MSC prepared in Example 1-1 above was washed with PBS, and trypsin (TrypLE TM The cells were treated with (Express, GIBCO, NY, USA) and reacted in a CO2 incubator for 5 minutes. Afterward, fresh serum-free medium was added to neutralize 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 the cell suspension was uniformly dispensed into a microarray containing microwells coated with MPC (2-Methacryloyloxyethyl Phosphorylcholine Polymer) with diameters and depths of 500 μm × 200 μm, at a density of 400 cells / well. The cells were kept static to induce spontaneous spheroid-shaped cell aggregate formation, 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).

[0094]

[0095] 1-3. Isolation of extracellular vesicles derived from 3D spheroidal cell aggregates

[0096] The 3D-static-spheroid culture medium prepared in Examples 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, and then purified once more with physiological saline to obtain high-purity extracellular vesicles derived from the 3D-static-spheroid of the present invention (hereinafter, 3D-static-spheroid EV).

[0097]

[0098] Experimental Example 2. Characterization of 3D-Static-Spheroid EVs

[0099] The characterization of 3D static spheroid EVs followed the guidelines of the International Society for Extracellular Vesicles (MISEV 2018 and 2023) and the standards of the Ministry of Food and Drug Safety of Korea. The structure and morphology of the EVs were imaged using transmission electron microscopy (TEM). Specifically, 3D static spheroid EVs were fixed for 2 hours in 1% OsO4 dissolved in 0.1 M phosphate buffer. An EM grid was adsorbed onto the extracellular vesicle droplets with the formvar side facing downward for 1 minute. Subsequently, the sample was blotted with filter paper and reacted with 2% uranyl acetate for 15 seconds. After removing excess uranyl acetate, the EM grid was observed using TEM (JEM-1011, JEOL, Japan). In addition, the size distribution, surface protein markers, purity, functional characteristics, and stability of EVs were analyzed.

[0100]

[0101] Experimental Example 2. EV administration in an atherosclerosis mouse model

[0102] 2-1. Design of Animal Experiments

[0103] Apolipoprotein E deficient (ApoE- / -) mice (C57BL / 6J, male, 6-8 weeks old, Jackson Laboratory, California, USA) and adult C57BL / 6N mice (Orient Bio Inc., Korea) were used. Mice were acclimatized for one week in a 12-hour light-dark cycle and temperature-controlled environment, after which they were randomly assigned to experimental groups.

[0104] The mice divided into groups as follows:

[0105] General diet (chow diet, control group),

[0106] High-fat diet (HFD, TD88137; ENVIGO, USA, fat 21%, cholesterol 0.2%).

[0107] The mice were reared for 12 weeks, and body weight and food intake were measured once a week. At week 8, mice in the high-fat diet group were randomly classified and treated with either a vehicle or a 3D-static-spheroid EV.

[0108]

[0109] 2-2. 3D-Static-Spheroid EV Administration

[0110] 3D-static spheroid EVs were administered intravenously via the tail vein at weekly intervals for 4 weeks. Specifically, the vehicle group (n=6) was injected with 100 μL of Dulbecco's phosphate-buffered saline (DPBS), and the 3D-static spheroid EV group (n=6) was 6×10⁻¹³ 3D-static spheroid EVs. 8The particles were suspended in 100 μL of DPBS and injected. The corresponding dosage and administration interval were determined based on prior preclinical studies of rodent and non-human primate stroke models. No deaths occurred in this experiment.

[0111] After the experiment, mice were sacrificed to collect aorta, liver, and serum, and histological, biochemical, and molecular analyses were performed. The effective population size (n) for some analyses varied depending on serum yield or tissue distribution.

[0112]

[0113] 2-3. Separation of the Cardiac and Arterial Systems

[0114] After anesthetizing mice with isoflurane (Hana Pharm, Korea), the thoracoabdominis were exposed by making an incision along the abdominal midline from the mandible to the pubic bone. A needle was inserted into the apex of the left ventricle to inject 20 mL of DPBS, and the fluid was drained through an incision in the right atrium. After perfusion, it was confirmed that the liver, kidneys, and lungs had faded in color. The tissues were fixed with a 4% paraformaldehyde (PFA, Sigma-Aldrich, USA) solution.

[0115]

[0116] 2-4. Serum Analysis

[0117] Blood collected from the orbital venous plexus at week 24 was placed in a 300 μL capillary tube (Drummond Scientific, USA). The blood was coagulated at room temperature for 30 minutes, and then centrifuged at 4°C, 4000 rpm, for 15 minutes to separate the serum.

[0118] Total cholesterol (TC), triglycerides (TG), glucose (Glu), C-reactive protein (CRP), and high-density lipoprotein cholesterol (HDL-C) in serum were measured using a Fuji Dri-Chem Clinical Chemistry Analyzer (Fujifilm, Japan). Low-density lipoprotein cholesterol (LDL) was calculated using the formula (TC - TG / 5) - HDL.

[0119]

[0120] 2-5. Staining of atherosclerotic plaque

[0121] The fixed arterial system was washed three times with distilled water for 1 minute each and immersed in 60% isopropyl alcohol for 5 minutes. Subsequently, it was stained in Oil Red O solution (Sigma-Aldrich, USA) at 37°C for 30 minutes. After staining, the sample was washed with 60% isopropyl alcohol and distilled water.

[0122]

[0123] Experimental Example 3. Evaluation of Foam Cell Formation and Cholesterol Metabolism in RAW264.7 Macrophages

[0124] 3-1. Cell Culture and Processing

[0125] RAW264.7 macrophages were cultured in Dulbecco's modified Eagle medium (DMEM, GIBCO, NY, USA) containing 10% (v / v) FBS (GIBCO, NY, USA) and 1% penicillin-streptomycin (GIBCO, NY, USA) at 5% CO2 and 37°C.

[0126] 3D-static-spheroid EVs were dissolved in PBS and then diluted in the culture medium for treatment. Cells were seeded into various plates and treated with oxidized low-density lipoprotein (ox-LDL, Sigma-Aldrich, USA) for 24 hours.

[0127]

[0128] 3-2. Observation of Foam Cell Formation

[0129] After treating macrophages with ox-LDL for 24 hours, they were washed three times with DPBS and fixed with 4% (w / v) paraformaldehyde for 10 minutes. Subsequently, they were stained with filtered Oil Red O solution (60°C, 30 min) and observed under a microscope (Olympus, Japan).

[0130] Absorbance was measured at 358 nm using an Infinite M1000 microplate reader (Tecan, Switzerland). The total cholesterol (TC), free cholesterol (FC), and cholesterol ester (CE) content of foam cells derived from RAW264.7 were measured using a Total Cholesterol Assay Kit (Cell Biolabs, USA) according to the manufacturer's instructions.

[0131]

[0132] 3-3. Cholesterol Absorption Analysis

[0133] RAW264.7 cells were cultured in high-concentration glucose DMEM and treated with 10 μg / mL of fluorescently labeled ox-LDL (Dil-oxLDL; Invitrogen) for 4 hours. Afterward, the cells were washed three times with DPBS, and the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen, USA). Images were acquired using a fluorescence microscope and analyzed with ImageJ (MA, USA).

[0134]

[0135] 3-4. Cholesterol Excretion Analysis

[0136] The cholesterol efflux capacity of foam cells was measured using the Cholesterol Efflux Assay Kit (ab196985; Abcam, USA). RAW264.7-derived foam cells were labeled with fluorescently labeled cholesterol for 1 hour, washed with DPBS, and cultured for 2 hours with or without 3D-static-spheroid EV treatment. After equilibration overnight with Equilibration Buffer, cholesterol acceptors were added and the cells were incubated at 37°C for 4 hours. After the reaction, the fluorescence of the supernatant (effluxed cholesterol) and the fluorescence of the cell lysate (residual cholesterol) were measured, and the efflux rate was calculated using the following formula.

[0137] [Mathematical Formula]

[0138] Cholesterol efflux = media fluorescence intensity / (cell fluorescence intensity + media fluorescence intensity) x 100%

[0139]

[0140] 3-5. Western blotting

[0141] Total protein was extracted from cells using RIPA lysis buffer (Invitrogen, USA), and protease and phosphatase inhibitors were added. Protein concentration was measured using a BCA protein assay kit (Invitrogen, USA).

[0142] 20 μg of protein samples were electrophoresed on 4-12% SDS-PAGE and transferred to a PVDF membrane (Millipore, Germany). The membrane was blocked with 5% skim milk for 1 hour, and the primary antibody was treated overnight at 4°C. Subsequently, the secondary antibody was incubated in a dark cow for 1 hour. Immunological reactions were detected using a ChemiDoc MP Imaging System (Bio-Rad, USA) and quantitatively analyzed using ImageJ software.

[0143]

[0144] Experimental Example 4. Statistical Analysis

[0145] Lipid accumulation in foam cells, cholesterol excretion capacity, and aortic plaque area were set as primary evaluation items, and blood lipid levels, liver protein expression, and inflammatory cytokine concentrations were set as secondary evaluation items.

[0146] All quantitative analyses were based on experiments conducted independently at least three times (n ≥ 3), and results were expressed as mean ± standard error (SEM). Differences between groups were tested using one-way analysis of variance (ANOVA, Tukey post-hoc test), and significance levels were set at p < 0.001 (***), 0.001 < p < 0.01 (**), and 0.01 < p < 0.05 (*).

[0147] The normality of the data was verified using the Shapiro-Wilk test, and non-parametric tests were applied to non-normally distributed data. Welch correction was performed when homoscedasticity was not ensured. All statistical analyses were performed using GraphPad Prism 8.0.1 (GraphPad Software, USA).

[0148]

[0149] [Example]

[0150] Example 1. Characteristics of a 3D-static-spheroid EV

[0151] In this embodiment, the characteristics of a 3D-static-spheroid EV were analyzed, and the results are shown in Fig. 1.

[0152] As shown in Fig. 1a, the particle diameter of the 3D-static-spheroid EV was an average of 130.5 ± 4.3 nm and a mode of 128.3 ± 3.1 nm.

[0153] As shown in Fig. 1b, the 3D static spheroid EV was confirmed to be a typical sphere based on electron microscopy (TEM and Cryo-EM) observations.

[0154] As shown in Figure 1c, EV surface antigens were profiled using a multi-bead-based flow cytometry method, and the presence of CD81, CD63, or CD9 was confirmed in 3D-static-spheroid EVs.

[0155] As shown in Fig. 1d, the particle-per-protein ratio of the 3D-static-spheroid EVs from three different lots was 3.45–4.56 X 10⁻⁶. 8 The particles / μg range was similar. Gentamicin and BSA, components of the 3D spheroid culture medium, and protein markers for organelle identification such as calnexin (ER marker), histone (nuclear marker), GM130 (Golgi marker), or cytochrome C (mitochondrial marker) were not detected in the 3D static spheroid EV.

[0156]

[0157] In addition, mRNA expression of 3D-static-spheroid EVs, 2D-EVs, and 3D-dynamic-spheroid EVs was analyzed.

[0158] As a result, compared to 2D-EV, 3D-static-spheroid EVs were found to have high expression of miR-27a, miR-146b, and miR-146a, which are miRNAs effective in angiogenesis / neurogenesis and immunomodulation, and high expression of integrin 1 / 2 and VEGF / R2 (Vascular endothelial growth factor / R2), which are proteins effective in angiogenesis / neurogenesis.

[0159] In addition, compared to 3D-dynamic-PEG spheroid EVs, it was confirmed that 3D-static-spheroid EVs highly express miR-146a, miR-27a, miR-132, miR-184, and miR-210

[0160]

[0161] Example 2. Inhibitory effect of 3D-static-spheroid EV on lipid accumulation in RAW264.7-derived foam cells

[0162] In this embodiment, the inhibitory effect of 3D-static-spheroid EV on lipid accumulation in RAW264.7-derived foam cells was confirmed, and the results are shown in Figure 2.

[0163] As shown in Figure 2a, in an in vitro foam cell model using RAW264.7 macrophages, Oil Red O staining results showed that intracellular lipid droplet accumulation significantly increased with ox-LDL treatment and decreased with 3D-static-spheroid EV treatment.

[0164] As shown in Figure 2b, in quantitative extraction and absorbance analysis, ox-LDL significantly increased neutral lipid content, and 3D-static-spheroid EV treatment alleviated this accumulation. Consistently, in the Dil-ox-LDL absorbance analysis, the EV group showed reduced intracellular fluorescence intensity compared to the ox-LDL alone treatment group, supporting that 3D-static-spheroid EV inhibits foam cell formation.

[0165] As shown in Figures 2c and d, in biochemical quantification, total cholesterol (TC) levels were significantly increased by ox-LDL and significantly reversed by 3D-static-spheroid EV (Figure 2c). On the other hand, no significant difference between groups was observed in the analysis of protein expression of the phagocytic receptor CD36 (Figure 2d). This suggests that lipid reduction by 3D-static-spheroid EV occurred independently of CD36 regulation.

[0166]

[0167] Example 3. Effect of 3D-Static-Spheroid EV on Increased ABCA1 / ABCG1 / LXR-α / PPAR-γ Expression and Promotion of Cholesterol Excretion in RAW264.7-Derived Foam Cells

[0168] In this example, the effect of 3D-static-spheroid EV on increasing ABCA1 / ABCG1 / LXR-α / PPAR-γ expression and promoting cholesterol excretion in RAW264.7-derived foam cells was confirmed. The results are shown in Figure 3.

[0169] As shown in Figure 3a, intracellular TC and lipid reduction were associated with increased excretion rather than decreased absorption. The 3D-static-spheroid EV group showed significantly increased cholesterol excretion compared to the negative control group.

[0170] As shown in Figures 3b and c, ox-LDL significantly inhibited ABCA1 and ABCG1 expression in RAW264.7-derived foam cells, but 3D-static-spheroid EVs restored it.

[0171] As shown in Figure 3d, 3D-static-spheroid EV treatment induced significant upregulation of LXRα, a key transcription regulator of ABCA1 and ABCG1.

[0172] As shown in Figure 3e, PPAR-γ expression in the 3D-static-spheroid EV group increased compared to the ox-LDL control group, which suggests activation of the PPARγ / LXRα axis.

[0173] In summary, these results indicated that 3D-static-spheroid EVs can increase cholesterol excretion in RAW264.7-derived foam cells by activating the PPAR-γ / LXR-α / ABCA1 signaling pathway, thereby contributing to lipid removal and alleviating foam cell formation.

[0174]

[0175] Example 4. Inhibitory effect of 3D-static-spheroid EV on the progression of atherosclerosis in apolipoprotein E-deficient mice

[0176] In this embodiment, the inhibitory effect of 3D-static spheroid EVs on the progression of atherosclerosis in apolipoprotein E-deficient mice was confirmed. For this purpose, an in vivo atherosclerosis model was prepared by feeding 8-week-old ApoE- / - mice a high-fat diet. Subsequently, the mice were sacrificed to isolate the heart and arterial systems, and the progression of atherosclerosis was confirmed. The experimental schedule from the preparation to the sacrifice of the in vivo atherosclerosis model is shown in Fig. 4a. The results confirming the progression of atherosclerosis in the in vivo atherosclerosis model are shown in Fig. 4.

[0177]

[0178] As shown in Fig. 4b, in the gross examination of the aortic arch after sacrifice, the control group showed more distinct white plaque deposition than the 3D-static-spheroid EV group.

[0179] As shown in Figure 4c, en face Oil Red O staining of the longitudinally opened aorta showed that lipid-accumulated atherosclerotic plaques were significantly reduced in the 3D-static-spheroid EV group compared to the control group.

[0180] As shown in Figure 4d, cross-sectional analysis also confirmed that plaque formation in the 3D-static-spheroid EV group was significantly reduced.

[0181] As shown in Figure 4e, in Picro Sirius Red staining of the aortic sinus, the deposition of type I collagen was reduced, particularly in the 3D-static-spheroid EV group when observed under polarized light, confirming a reduction in fibrotic remodeling.

[0182] As shown in Fig. 4f, in the immunofluorescence analysis of the aortic lesion, the 3D-static-spheroid EV group [contained] inflammatory M1 macrophages (F4 / 80 + CD68 + The infiltration of double-positive cases decreased.

[0183] The above results indicated that 3D-static-spheroid EVs not only reduce lipid deposition and fibrous matrix accumulation but also alleviate the burden of atherosclerosis by inhibiting inflammatory cell infiltration within plaques.

[0184]

[0185] Example 5. Effect of 3D-Static-Spheroid EVs on Increased Expression of ABCA1 / ABCG1 / LXR-α / PPAR-γ and Promotion of Cholesterol Excretion in Apolipoprotein E-Deficient Mice

[0186] In this example, changes in protein expression and the effect of promoting cholesterol excretion in apolipoprotein E-deficient (ApoE- / -) mice following the administration of 3D-static-spheroid EVs were confirmed. Specifically, to verify whether the cholesterol excretion pathway activated by 3D-static-spheroid EVs in vitro (identified in Figures 2 and 3) acts identically in vivo, western blot analysis was performed on aortic tissues of ApoE- / - mice. The results of confirming the protein expression of CD36, ABCA1, ABCG1, LXR-α, and PPAR-γ are shown in Figure 5.

[0187] As shown in Fig. 5, there was no significant difference in the expression level of CD36, a phagocytic receptor mediating cholesterol absorption, between the DPBS control group and the 3D-static-spheroid EV group, which was consistent with in vitro results (Fig. 5a). On the other hand, cholesterol efflux transporters ABCA1 and ABCG1 were significantly upregulated in the aorta treated with 3D-static-spheroid EV (Fig. 5b). In addition, the expression of LXRα (Fig. 5c) and PPARγ (Fig. 5d), upstream regulators of these transporters, was significantly increased in the 3D-static-spheroid EV group.

[0188]

[0189] Example 6. Analysis of blood lipid profiles in apolipoprotein E-deficient mice by 3D-static-spheroid EV

[0190] Cardiovascular risk indicators (lipid profiles and inflammation markers) are well-established determinants of ischemic stroke risk and can serve as surrogate markers to approximate cerebrovascular outcomes, providing preclinical potential for the development of stroke prevention and treatment. Accordingly, in this example, blood lipid profiles were measured to evaluate systemic lipid metabolism in ApoE- / - mice. The results of the analysis of serum lipid profiles are shown in Figure 6.

[0191] As shown in Figure 6a, the 3D-static-spheroid EV group showed a significant decrease in total cholesterol (TC), triglycerides (TG), and LDL-C levels, while HDL-C remained unchanged.

[0192] As shown in Figures 6b and c, the 3D-static-spheroid EV group showed significantly reduced levels of serum inflammatory biomarkers IL-6 and hs-CRP.

[0193] As shown in Figures 6d and e, the 3D-static-spheroid EV group showed a significant decrease in PCSK9 and Lp(a), lipid-associated biomarkers associated with cardiovascular risk.

[0194] The above results indicate that the reduction in PCSK9 and Lp(a) levels suggests that 3D-static-spheroid EVs can target not only traditional lipid indicators but also non-LDL residual risk components.

[0195]

[0196] Example 7. Analysis of liver PCSK9 expression, LDL-R levels, and whole-body lipid profile by 3D-static-spheroid EV

[0197] In this example, liver PCSK9 expression, LDL-R levels, and whole-body lipid profiles were analyzed by 3D-static-spheroid EV, and the results are shown in Figure 7.

[0198] As shown in Figure 7a, 3D-static-spheroid EV-treated mice showed a significant decrease in body weight compared to the control group (-2.000 ± 0.6066 g, p < 0.050), confirming an improvement in systemic metabolic status.

[0199] As shown in Fig. 7b, consistent with the body weight loss results in Fig. 7a, the absolute liver weight of the 3D-static-spheroid EV group also decreased (-0.2398 ± 0.04735 g, p < 0.050). This implies that the pathological burden of the liver was reduced by 3D-static-spheroid EV treatment. In particular, despite the decrease in both body weight and liver weight, the liver-to-body weight ratio (%) of 3D-static-spheroid EV treated mice significantly increased compared to the control group (+0.3312 ± 0.08022%, p < 0.050), which appears to reflect a more pronounced reduction in total body weight. Furthermore, liver TC levels were significantly lower in the 3D-static-spheroid EV group compared to the control group (p < 0.050), confirming that liver lipid metabolism improved after 3D-static-spheroid EV treatment.

[0200] As shown in Figures 7c and 7d, it was confirmed that 3D-static-spheroid EV alleviated both hepatic steatosis (Figure 7c) and fibrosis (Figure 7d). This means that 3D-static-spheroid EV improves liver function and structure.

[0201] As shown in Figure 7e, LDL-R expression in the 3D-static-spheroid EV group was significantly upregulated, and PCSK9, a negative regulator of LDL-R, was significantly downregulated. In addition, the expression of IL-6, a key inflammatory cytokine, was significantly reduced after treatment with 3D-static-spheroid EVs. These results indicate that MSC-derived EVs contribute additionally to the improvement of observed lipid metabolism and liver pathology by inhibiting PCSK9, thereby restoring hepatic LDL-R levels, as well as suppressing liver inflammation.

[0202]

[0203] In summary, the inventors confirmed that the administration of 3D-static-spheroid EVs increased the expression of cholesterol-excreting proteins and improved lipid metabolism in apolipoprotein E-deficient mice. This implies that 3D-static-spheroid EVs can alleviate the progression of atherosclerosis by inhibiting lipid accumulation in macrophages and promoting cholesterol removal; thus, the 3D-static-spheroid EVs of the present invention can be utilized in various ways in the field of prevention or treatment of lipid metabolism disorders, including arteriosclerosis.

[0204]

[0205] 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 cells and vesicles from the above three-dimensional spheroid-shaped cell aggregate; comprising an extracellular vesicle derived from a three-dimensional spheroid-shaped cell aggregate prepared through this step, Pharmaceutical composition for the prevention or treatment of lipid metabolism disorders.

2. A pharmaceutical composition for the prevention or treatment of lipid metabolism disorders, wherein the stem cells in claim 1 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 lipid metabolism disorders, wherein, in claim 1, the three-dimensional culture of step (a) is a static culture.

4. A pharmaceutical composition for the prevention or treatment of lipid metabolism disorders, wherein the three-dimensional culture of step (a) is cultured by dispensing mesenchymal stem cells into microwells at a density of 100 to 1000 cells / well.

5. A pharmaceutical composition for the prevention or treatment of a lipid metabolism disorder according to claim 1, wherein the lipid metabolism disorder is a disease caused by an increase in blood cholesterol.

6. A pharmaceutical composition for the prevention or treatment of lipid metabolism disorders according to claim 1, wherein the lipid metabolism disorder is one or more diseases selected from the group consisting of arteriosclerosis, atherosclerosis, hyperlipidemia, hypercholesterolemia, dyslipidemia, coronary artery disease, peripheral vascular disease, ischemic heart disease, myocardial infarction, non-alcoholic fatty liver, non-alcoholic steatohepatitis, fatty liver, liver fibrosis, metabolic syndrome, obesity, type 2 diabetes, insulin resistance disease, vasculitis, and vascular endothelial dysfunction.

7. A pharmaceutical composition for the prevention or treatment of a lipid metabolism disorder, wherein the lipid metabolism disorder is a cardiovascular disease caused by a lipid metabolism disorder, in accordance with claim 1.

8. A pharmaceutical composition for the prevention or treatment of lipid metabolism disorders, wherein the extracellular vesicles derived from three-dimensional spheroid-shaped cell aggregates in claim 1 inhibit LDL cholesterol excretion; or inhibit the expression of lipid-associated biomarkers.

9. (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 cells and vesicles from the above three-dimensional spheroid-shaped cell aggregate; comprising an extracellular vesicle derived from a three-dimensional spheroid-shaped cell aggregate prepared through this step, Pharmaceutical composition for lowering blood cholesterol.

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 cells and vesicles from the above three-dimensional spheroid-shaped cell aggregate; comprising an extracellular vesicle derived from a three-dimensional spheroid-shaped cell aggregate prepared through this step, A health functional food composition for the prevention or improvement of lipid metabolism disorders.

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; and (b) a step of separating cells and vesicles from the above three-dimensional spheroid-shaped cell aggregate; comprising an extracellular vesicle derived from a three-dimensional spheroid-shaped cell aggregate prepared through this step, Health functional food composition for lowering blood cholesterol.

12. Includes the step of administering extracellular vesicles derived from three-dimensional spheroid-shaped cell aggregates to an individual requiring them, and The above-mentioned extracellular vesicle derived from the three-dimensional spheroid-shaped cell aggregate comprises: (a) a step of producing a three-dimensional spheroid-shaped cell aggregate by culturing stem cells three-dimensionally 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 aggregate; a method for treating lipid metabolism disorders.

13. The method includes the step of administering extracellular vesicles derived from three-dimensional spheroid-shaped cell aggregates to an individual requiring them, and The above-mentioned extracellular vesicle derived from the three-dimensional spheroid-shaped cell aggregate comprises: (a) a step of producing a three-dimensional spheroid-shaped cell aggregate by culturing stem cells three-dimensionally in a microwell having a diameter of 200 to 800 μm and a depth of 100 to 1000 μm; and (b) a method for lowering blood cholesterol, prepared by the step of separating extracellular vesicles from the above three-dimensional spheroid-shaped cell aggregate.

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