Method for mass-producing, with high-purity and high-efficiency, extracellular vesicles through novel cell bank construction strategy, and extracellular vesicles produced therefrom

Abstract

The present invention relates to a method for mass-producing, with high-purity and high-efficiency, extracellular vesicles by using a novel cell bank construction strategy, and extracellular vesicles produced therefrom. According to one embodiment of the present invention, extracellular vesicles can be mass-produced with high purity and high efficiency while the characteristics of genetically modified or unmodified cells are stably maintained through the construction of a novel cell bank, and the produced extracellular vesicles can be effectively used as a pharmaceutical composition for preventing or treating diseases and a functional cosmetic composition.

Description

A method for mass-producing high-purity, high-efficiency extracellular vesicles through a novel cell bank construction strategy and extracellular vesicles produced thereby

[0001] The present invention relates to a method for mass-producing high-purity, high-efficiency extracellular vesicles using a novel cell bank construction strategy, and to extracellular vesicles produced thereby.

[0002]

[0003] Extracellular vesicles (EVs) are spherical particles with a lipid bilayer structure naturally secreted by various cells in the body. They possess excellent characteristics such as high biocompatibility and low immunogenicity. They are present in most body fluids, including blood and urine, and contain physiologically active substances such as proteins, mRNA, and miRNA, playing a crucial role as intercellular signaling mediators. In particular, EVs possess the ability to deliver payloads through fusion with the target cell membrane and endocytosis, protecting endogenous substances from various immune components and enzymes in plasma, enabling stable delivery within the body.

[0004] Research is actively being conducted to utilize these extracellular vesicles in the treatment of various diseases, and accordingly, interest in processes for mass-producing extracellular vesicles with high purity and high efficiency is increasing, making research on this topic urgent.

[0005]

[0006] The present inventors completed the present invention by confirming that extracellular vesicles can be produced with high purity and high efficiency from genetically modified stem cells according to a production method according to one embodiment of the present invention.

[0007]

[0008] One object of the present invention is to provide a method for producing cells for producing extracellular vesicles, which comprises a step of passage of cells derived from a master cell bank (MCB) after constructing a master cell bank (MCB) of cells, excluding a step of constructing a working cell bank (WCB).

[0009] Another object of the present invention is to provide a method for producing extracellular vesicles using cells produced by the above production method, comprising the following steps.

[0010] a) a step of culturing the cells in three dimensions to obtain a culture; and

[0011] b) A step of purifying extracellular vesicles from the above culture.

[0012] Another object of the present invention is to provide extracellular vesicles manufactured by the above manufacturing method.

[0013] Another object of the present invention is to provide a pharmaceutical composition comprising the extracellular vesicles.

[0014] Another object of the present invention is to include a cosmetic composition comprising the extracellular vesicles.

[0015] The technical problem to be achieved according to the technical idea of ​​the invention disclosed in this specification is not limited to the problem to solve the above-mentioned problem, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

[0016]

[0017] According to one embodiment of the present invention, a novel cell bank construction strategy can be used to mass-produce high-purity, high-efficiency extracellular vesicles from genetically modified or non-modified cells while maintaining the original characteristics of the cells, and the extracellular vesicles thus produced can be effectively utilized not only as pharmaceutical compositions for the prevention or treatment of diseases but also as functional cosmetic compositions.

[0018]

[0019] Figure 1 illustrates two cellular storage systems for extracellular vesicle production.

[0020] Figure 2 shows the time-dependent metabolite concentrations of SIRPα-MSCs manufactured using the one-step system and the two-step system, respectively.

[0021] Figure 3 shows the results of confirming the cell proliferation rate and extracellular vesicle production amount of SIRPα-introduced mesenchymal stem cells (SIRPα-MSC) manufactured using the one-step system and the two-step system, respectively.

[0022] Figure 4 shows the results confirming the differentiation ability of SIRPα-MSCs into adipocytes, osteocytes, and chondrocytes.

[0023] Figure 5 shows the results of surface marker expression analysis of SIRPα-MSCs and control mesenchymal stem cells (C-MSCs) without genetic modification.

[0024] Figure 6 shows the results of comparing the secretion of cytokines related to angiogenesis and cell environment regulation of SIRPα-MSC and C-MSC.

[0025] Figure 7 shows the results of an analysis of the variation between donors or within individual donors of SIRPα-MSCs.

[0026] Figure 8 illustrates two-dimensional (2D CellSTACK) and three-dimensional (Ambr250 and STR50) cell culture systems.

[0027] Figure 9 shows a time series trace of a cell suspension in a 3D cell culture medium.

[0028] Figure 10 shows the results of metabolic profile analysis by 3D (Ambr250 and STR50) cell culture.

[0029] Figure 11 visualizes cells in two-dimensional and three-dimensional culture systems.

[0030] Figure 12 shows the results of confirming the size distribution of extracellular vesicle particles and changes in concentration over time through nanoparticle tracking analysis.

[0031] Figure 13 shows the results of confirming SIRPα expression in mesenchymal stem cells before harvesting extracellular vesicles.

[0032] Figure 14 shows the results of confirming macroscopic cell growth changes after culturing SIRPα-MSCs in 250 mL (Ambr250) and 50 L (STR50) scales in a 3D culture system.

[0033] Figure 15 shows the results of metabolic profile analysis after a 3D culture system.

[0034] Figure 16 shows the results of confirming the cell and microcarrier attachment status of the produced extracellular vesicles.

[0035] Figure 17 shows the change in the production of extracellular vesicles produced by the three-dimensional culture system.

[0036] Figure 18 shows the results of confirming the size distribution of extracellular vesicle particles produced by the 3D culture system.

[0037] Figure 19 is a schematic diagram of a conventional two-dimensional culture post-purification process and a three-dimensional culture post-purification process of the present invention.

[0038] Figure 20 shows the quantitative analysis and cumulative removal rate evaluation of double-stranded DNA (dsDNA) in an extracellular vesicle sample separated by a purification process according to one embodiment of the present invention.

[0039] Figure 21 shows the results of measuring the concentration of human serum albumin in an extracellular vesicle sample separated by a purification process according to one embodiment of the present invention.

[0040] Figure 22 shows the results of analysis of the removal rate of dsDNA and human serum albumin in an extracellular vesicle sample separated by a purification process according to one embodiment of the present invention.

[0041] Figure 23 shows the total protein concentration and step-by-step removal rate evaluation at each stage of the purification process.

[0042] Figure 24 shows the results of analysis of the protein accumulation removal rate in an extracellular vesicle sample separated by a purification process according to one embodiment of the present invention.

[0043] Figure 25 shows the concentration of extracellular vesicle particles and the step-by-step yield evaluation at each process step.

[0044] Figure 26 shows an evaluation of the cumulative yield of extracellular vesicles separated by a purification process according to one embodiment of the present invention.

[0045] Figure 27 shows the change in particle number of extracellular vesicles (C-EVs) of genetically unmodified stem cells produced by a three-dimensional culture system and a two-dimensional culture system.

[0046] Figure 28 shows the particle size distribution based on nanoparticle (C-EV) tracking analysis of non-genetically modified stem cells measured by culture system.

[0047] Figure 29 shows the concentration of extracellular vesicle (C-EV) particles of non-genetically modified stem cells at each stage of the purification process.

[0048] Figure 30 shows the cumulative yield of extracellular vesicles (C-EVs) from non-genetically modified stem cells obtained during the purification process.

[0049] Figure 31 shows the total protein removal rate of extracellular vesicles (C-EVs) from non-genetically modified stem cells obtained during the purification process.

[0050] Figure 32 shows the results of confirming the dsDNA removal rate of extracellular vesicles (C-EV) from non-genetically modified stem cells obtained through the purification process.

[0051] Figure 33 shows the results of confirming the human serum albumin removal rate of extracellular vesicles (C-EVs) from non-genetically modified stem cells obtained through the purification process.

[0052] Figure 34 shows the results of RNA profile analysis of SIRP-MSCs and SIRP-EVs cultured in a 3D culture system.

[0053] Figure 35 shows the results of RNA quantitative analysis of C-EVs and SIRP-EVs cultured in a two-dimensional or three-dimensional culture system.

[0054] Figure 36 shows the results of analyzing the number of particles per 1 mg of protein of extracellular vesicles produced by a purification process according to one embodiment of the present invention.

[0055] Figure 37 shows the results of protein identification consistency and reproducibility analysis of SIRPα-EV and C-EV.

[0056] Figure 38 shows the results of protein profile similarity analysis of SIRPα-EV and C-EV.

[0057] Figure 39 shows the results of confirming overexpression of SIRPα in SIRPα-EV.

[0058] Figure 40 shows the results of analyzing the expression of extracellular vesicle markers and non-extracellular vesicle markers in SIRPα-EV and C-EV manufactured by a manufacturing method according to one embodiment of the present invention.

[0059] Figure 41 shows the results of Cryo-TEM (cryogenic transmission electron microscopy) analysis of SIRPα-EV and C-EV manufactured by a manufacturing method according to one embodiment of the present invention.

[0060] Figure 42 shows the results of DLS (Dynamic Light Scattering) analysis of SIRPα-EV and C-EV manufactured by a manufacturing method according to one embodiment of the present invention.

[0061] Figure 43 shows the results of zeta potential and MemGlow staining analysis of SIRPα-EV and C-EV manufactured by a manufacturing method according to one embodiment of the present invention.

[0062] Figure 44 shows the results of a biological function analysis of the protein cargo of SIRPα-EV manufactured by a manufacturing method according to one embodiment of the present invention.

[0063] Figure 45 shows the results of quantitative analysis of cell binding ability of SIRPα-EV and C-EV manufactured by a manufacturing method according to one embodiment of the present invention.

[0064]

[0065] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention can also be applied to each other description and embodiment. In other words, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the scope of the present invention should not be considered limited by the specific descriptions described below.

[0066] Furthermore, those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments of the present invention described herein. Furthermore, it should be understood that such equivalents are encompassed by the present invention.

[0067] Additionally, numerous papers and patents are referenced and cited throughout this specification. The disclosures of these cited papers and patents are incorporated herein by reference in their entirety, thereby providing a clearer understanding of the technical field to which this application pertains and the content of this application.

[0068]

[0069] One embodiment of the present invention provides a method for producing cells for producing extracellular vesicles, comprising a step of passage cells derived from a Master Cell Bank (MCB) after constructing a Master Cell Bank (MCB) of cells, excluding a step of constructing a Working Cell Bank (WCB).

[0070] Conventional cell storage methods typically utilize a two-tier system, establishing a master cell bank and then creating a working cell bank based on that master bank. However, cell characteristics can change with increasing passages. Primary cells, such as stem cells, are particularly sensitive and have limited passages. Therefore, the existing two-tier system has limitations in stably maintaining the original cell characteristics.

[0071] Therefore, the present invention applies a 1-tier system (+1 Tier system) that omits the step of constructing a working cell bank after constructing a master cell bank of cells for producing extracellular vesicles and performs a subculture step before culturing in a cell incubator, thereby enabling efficient cell storage and mass production of extracellular vesicles while maintaining the original characteristics of the cells.

[0072] In one embodiment of the present invention, the cell may be, but is not limited to, a genetically modified cell or a genetically unmodified cell.

[0073] In one embodiment of the present invention, the cell may be selected from the group consisting of a cell line and a primary cell, but is not limited thereto.

[0074] The term "cell line" of the present invention refers to a cultured cell of the same characteristics, which is immortalized and can proliferate indefinitely.

[0075] The term "primary cell" of the present invention refers to a cell obtained by directly separating and extracting from living biological tissue, has the characteristics of a cell, and has a limited number of divisions, so that it ages and dies after a certain number of divisions.

[0076] In one embodiment of the present invention, the cell line may be at least one selected from the group consisting of HEK293 cells, HEK293T cells, Expi293 cells, Expi293F cells, ExpiCHO cells, CHO cells, HeLa cells, and BHK-21 cells.

[0077] In one embodiment of the present invention, the primary cell may be at least one selected from the group consisting of NK cells, T cells, B cells, dendritic cells, macrophages, monocytes, fibroblasts, epithelial cells, endothelial cells, and stem cells.

[0078] In one embodiment of the present invention, the stem cell may be at least one selected from the group consisting of mesenchymal stem cells, pluripotent stem cells, induced pluripotent stem cells, adult stem cells, and embryonic stem cells, but is not limited thereto.

[0079] In one embodiment of the present invention, the mesenchymal stem cells may be derived from bone marrow, umbilical cord, umbilical cord blood, fat, muscle, nerve, skin, amniotic membrane, or placenta.

[0080] In one embodiment of the present invention, the subculture may be performed once and then subcultured again.

[0081] The above additional subculture may be performed 1 to 5 times, and specifically, may be performed 1 to 4 times, 1 to 3 times, or 1 to 2 times, but is not limited thereto.

[0082] One aspect according to one embodiment of the present invention provides a method for producing extracellular vesicles using cells produced by the above production method.

[0083] a) a step of culturing the cells in three dimensions to obtain a culture; and

[0084] b) A step of purifying extracellular vesicles from the above culture.

[0085] The term extracellular vesicle of the present invention refers to a natural nanoparticle (or extracellular particle) composed of a lipid bilayer membrane derived from a cell.

[0086] In one embodiment of the present invention, the three-dimensional culture is performed through a three-dimensional cell culture device, and the three-dimensional cell culture device may be at least one selected from the group consisting of a rotary cell culture system (RCCS), a spinner flask, a shaking flask, a hydrogel-based culture device, a natural or synthetic scaffold-based culture device, a microcarrier-based culture device, a hollow fiber-based culture device, a microfluidic-based culture device, an organ-on-a-chip, a packed-bed bioreactor, a fluidized-bed bioreactor, a perfusion bioreactor, an air-lift bioreactor, a wave-mixed bioreactor, and a magnetic levitation culture device, but is not limited thereto.

[0087] In one embodiment of the present invention, the three-dimensional culture may be performed at 30°C to 40°C, and specifically, may be performed at 30°C to 38°C, 32°C to 38°C, 34°C to 38°C, or 35°C to 38°C, but is not limited thereto.

[0088] In one embodiment of the present invention, the three-dimensional culture may be performed at a dissolved oxygen (DO) level of 50% to 100%, but is not limited thereto.

[0089] In one embodiment of the present invention, step a) may further include a step of replacing the medium with a protein-free medium.

[0090] In the present invention, the protein-free medium is for collecting extracellular vesicles, and any medium that is commercially available or can be manufactured directly for that purpose can be used without limitation.

[0091] In one embodiment of the present invention, the purification may include depth filtration, first tangential flow filtration (TFF), and second tangential flow filtration steps.

[0092] Specifically, in the embodiment of the present invention, it was confirmed that the extracellular vesicles obtained from the culture through the purification step have very high purity, and the total particle recovery rate reaches 50%, indicating excellent process efficiency.

[0093] For example, the filter used for the depth filtration filtering may have a pore size of 0.1 μm to 5 μm, specifically, but not limited to, 0.1 μm to 3 μm, 0.1 μm to 1.2 μm, or 0.1 μm to 0.45 μm.

[0094] In one embodiment of the present invention, a guard filtration and chromatography step may be further included between the first parallel flow filtration step and the second parallel flow filtration step.

[0095] For example, the filter used for the guard filtering may have a pore size of 0.1 μm to 0.5 μm, specifically, but not limited to, 0.1 μm to 0.45 μm, 0.1 μm to 0.3 μm, or 0.1 μm to 0.2 μm.

[0096] In one embodiment of the present invention, a filtering step may be further included after the secondary parallel flow filtration.

[0097] For example, the filter used in the filtering step after the secondary parallel flow filtration may have a pore size of 0.1 μm to 0.5 μm, specifically, but not limited to, 0.1 μm to 0.45 μm, 0.1 μm to 0.3 μm, or 0.1 μm to 0.2 μm.

[0098] In one embodiment of the present invention, the step of replacing the secondary parallel flow filtration process with a sucrose-containing salt buffer may be included.

[0099] By controlling the osmotic pressure with the above sucrose-containing salt buffer, dehydration of the manufactured extracellular vesicles can be prevented, structural damage to the extracellular vesicles due to freezing can be reduced, and the formation of ice crystals during freezing of the extracellular vesicles can be prevented, thereby preventing mechanical damage inside and outside the extracellular vesicles.

[0100] In one embodiment of the present invention, the total particle recovery rate of extracellular vesicles manufactured by the above manufacturing method may be 40% or more.

[0101] In one embodiment of the present invention, the cell may be, but is not limited to, a genetically modified cell.

[0102] In one embodiment of the present invention, extracellular vesicles manufactured by the above manufacturing method may exhibit a higher level of expression of a target protein compared to extracellular vesicles derived from genetically unmodified cells.

[0103] In one specific example, the target protein may be, but is not limited to, SIRPα, HIF1α, CAR peptide, Apelin, T7 peptide, or ESM.

[0104] Another aspect according to one embodiment of the present invention provides an extracellular vesicle manufactured by the above manufacturing method.

[0105] Another aspect according to one embodiment of the present invention provides a pharmaceutical composition comprising the extracellular vesicle. The pharmaceutical composition of the present invention may further comprise one or more pharmaceutically acceptable carriers in addition to the active ingredient for administration. Pharmaceutically acceptable carriers may include saline solution, sterile water, Ringer's solution, buffered saline, Plasma-Rite A, dextrose solution, maltodextrin solution, glycerol, ethanol, propylene glycol, polyethylene glycol, mannitol, sorbitol, sucrose, trehalose, dextran, albumin, and a mixture of one or more of these components. If necessary, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition into an injectable formulation such as an aqueous solution, suspension, or emulsion, or into pills, capsules, granules, or tablets. Accordingly, the pharmaceutical composition of the present invention may be a patch, a liquid, a pill, a capsule, a granule, a tablet, a suppository, etc. These preparations may be prepared by a conventional method used for formulation in the art or by a method disclosed in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton PA, and may be formulated into various preparations depending on each disease or ingredient.

[0106] The composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and not causing side effects. The effective dosage level may be determined based on factors including the patient's health condition, the type and severity of the disease, the activity and sensitivity of the drug, the method of administration, the time of administration, the route and excretion rate, the duration of treatment, drugs used in combination or simultaneously, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. It is important to administer an amount that achieves the maximum effect with the minimum amount without side effects by taking all of the above factors into consideration, and this can be easily determined by those skilled in the art. The daily dosage of the compound of chemical formula 1 of the present invention is about 0.01 to 1000 mg / kg, preferably 0.1 to 100 mg / kg, and can be administered once or several times a day.

[0107] The term "administration" in the present invention means introducing a predetermined substance into a patient by an appropriate method, and the administration route of the composition may be administered through any common route as long as it can reach the target tissue. In addition, the pharmaceutical composition of the present invention may be administered by any device that allows the active substance to move to the target tissue. For example, it may be administered by transdermal administration, oral administration, intrathecal administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, topical administration, intranasal administration, intrapulmonary administration, rectal administration, inner ear administration, intrauterine epidural administration, sublingual administration, and intracerebrovascular injection, but is not limited thereto.

[0108] In the present invention, the pharmaceutical composition may appropriately contain, if necessary, a suspending agent, a solubilizing agent, a stabilizer, an isotonic agent, a preservative, an adsorption inhibitor, a surfactant, a diluent, an excipient, a pH adjuster, a soothing agent, a buffer, a reducing agent, an antioxidant, etc. depending on the administration method or formulation. Pharmaceutically acceptable carriers and formulations suitable for the present invention, including those exemplified above, are described in detail in the literature [Remington's Pharmaceutical Sciences, 19th ed., 1995]. The pharmaceutical composition may be manufactured in a unit dose form or may be manufactured by inserting it into a multi-dose container by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily performed by a person skilled in the art to which the present invention pertains. At this time, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or in the form of a powder, granules, tablets, or capsules.

[0109] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are formulated by mixing at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc., with the above composition. In addition to simple excipients, lubricants such as magnesium stearate and talc may be used.

[0110] Oral liquid preparations include suspensions, solutions, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, flavoring agents, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin.

[0111] Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include withepsol, macrogol, Tween 61, cacao butter, laurin, and glycerogelatin. Meanwhile, injections can include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives.

[0112] The route of administration of the pharmaceutical composition of the present invention may be through any general route as long as it can reach the target tissue, but may be through subcutaneous injection using an osmotic pump, intradermal injection, intravenous injection, intraperitoneal injection, intravitreal injection, oral administration, etc.

[0113] The term “prevention” used in the present invention means any act of suppressing a disease or delaying its onset by administering the composition.

[0114] In the present invention, “treatment” means any act in which the symptoms of a disease are improved or beneficially changed by administering the composition.

[0115] The term "subject" in the present invention refers to any animal that has developed or is likely to develop a disease, and typically refers to an animal that can exhibit a beneficial effect from treatment using the composition of the present invention, but includes, without limitation, any subject that has symptoms of the disease or is likely to have such symptoms. As described above, by administering the pharmaceutical composition of the present invention to a subject, the disease can be effectively prevented or treated. The pharmaceutical composition of the present invention can be administered as an individual therapeutic agent, or in combination with existing disease therapeutic agents, and can be administered sequentially or simultaneously with existing therapeutic agents.

[0116] In addition, another aspect according to one embodiment of the present invention provides a treatment method comprising a step of administering the pharmaceutical composition.

[0117] In addition, another aspect according to one embodiment of the present invention provides a cosmetic composition comprising the extracellular vesicle.

[0118] In the present invention, the composition can be formulated in various forms by adding various components as auxiliary components for delivery and stabilization, etc.

[0119] In the present invention, the cosmetic composition may have a formulation such as a mist, serum, nourishing toner, emulsifying toner, emulsion, suspension, skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nourishing lotion, massage cream, nourishing cream, moisture cream, hand cream, foundation, powder, makeup base, essence, nourishing essence, pack, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion, body cleanser, facial cleanser, treatment, beauty liquid, beauty pack, ointment, gel, liniment, liquid, patch, spray, bath agent, sunscreen, sun oil, and hair product. The scope of the formulation is not limited thereto, and the formulation of the cosmetic composition may be manufactured into any formulation commonly manufactured in the art.

[0120] In the present invention, the cosmetic composition may further comprise a cosmetically acceptable carrier. The type of the cosmetically acceptable carrier of the present invention is not particularly limited, as long as it does not inhibit the activity and properties of the cosmetic composition of the present invention, and any cosmetically acceptable carrier commonly used in the art may be used. Non-limiting examples of the cosmetically acceptable carrier include saline solution, sterile water, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, and ethanol. These may be used alone or in combination of two or more.

[0121] In the present invention, the cosmetically acceptable carrier varies depending on the formulation of the cosmetic composition.

[0122] When the above formulation is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component.

[0123] When the above formulation is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in particular, when it is a spray, it may additionally include a propellant such as chlorofluorohydrocarbon, propane / butane or dimethyl ether.

[0124] When the above formulation is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

[0125] When the above formulation is a suspension, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth may be used as a carrier component.

[0126] When the formulation of the above cosmetic composition is soap, alkali metal salts of fatty acids, fatty acid hemiester salts, fatty acid protein hydrolysates, isethionates, lanolin derivatives, fatty alcohols, vegetable oils, glycerol, sugars, etc. may be used as carrier components, but are not limited thereto.

[0127] When the formulation of the above cosmetic composition is a pack, it includes all forms of a peel-off pack containing polyvinyl alcohol or the like, a wash-off pack containing pigments such as kaolin, talc, zinc oxide, or titanium dioxide in a general emulsified cosmetic, or a mask sheet pack, but is not particularly limited thereto.

[0128] The ingredients included in the above cosmetic composition may include, in addition to the composition containing extracellular vesicles as an active ingredient, ingredients commonly used in cosmetic compositions, and may include, for example, conventional auxiliary agents and carriers such as stabilizers, solubilizers, preservatives, moisturizers, pigments, bactericides, antioxidants, surfactants, vitamins, pigments, and fragrances. In addition, the cosmetic composition may further include a skin absorption promoter to enhance its effectiveness.

[0129] In the present invention, the composition may further include one or more active ingredients exhibiting the same or similar efficacy.

[0130] In the present invention, the composition including the extracellular vesicle is in an amount of 0.000000001 wt% to 90 wt%, for example, 0.000000001 wt% to 80 wt%, 0.000000001 wt% to 60 wt%, 0.000000001 wt% to 30 wt%, 0.000000001 wt% to 20 wt%, 0.000000001 wt% to 10 wt%, 0.000000001 wt% to 5 wt%, 0.000000001 wt% to 0.001 wt%, 0.000000001 wt% to 0.0001 wt%, 0.000000001 wt% to 0.00001 wt%, % by weight, 0.0001 wt% to 90 wt%, 0.0001 wt% to 80 wt%, 0.0001 wt% to 60 wt%, 0.0001 wt% to 30 wt%, 0.0001 wt% to 20 wt%, 0.0001 wt% to 10 wt%, 0.0001 wt% to 5 wt%, 0.001 wt% to 90 wt%, 0.001 wt% to 80 wt%, 0.001 wt% to 60 wt%, 0.001 wt% to 30 wt%, 0.001 wt% to 20 wt%, 0.001 wt% to 10 wt%, 0.001 wt% to 5 wt%, 0.01 wt% to 90 wt%, 0.01 wt% to 80 wt%, It may be included in an amount of 0.01 wt% to 60 wt%, 0.01 wt% to 30 wt%, 0.01 wt% to 20 wt%, 0.01 wt% to 10 wt%, 0.01 wt% to 5 wt%, 0.1 wt% to 90 wt%, 0.1 wt% to 80 wt%, 0.1 wt% to 60 wt%, 0.1 wt% to 30 wt%, 0.1 wt% to 20 wt%, 0.1 wt% to 10 wt%, or 0.1 wt% to 5 wt%. When included in an amount of less than 0.000000001 wt%, the effect, etc. is minimal, and 90.If it exceeds 0 wt%, it is not economical as the efficiency is low compared to the amount of material input.

[0131] In one specific example, the cosmetic composition comprises 10 compositions comprising extracellular vesicles. 5 10 to 100 pcs / ml 14 dog / ml, for example, 10 5 10 to 100 pcs / ml 13 dog / ml, 10 5 10 to 100 pcs / ml 12 dog / ml, 10 5 10 to 100 pcs / ml 11 dog / ml, 10 5 10 to 100 pcs / ml 10 dog / ml, 10 5 10 to 100 pcs / ml 9 dog / ml, 10 5 10 to 100 pcs / ml 8 dog / ml, 10 5 10 to 100 pcs / ml 7 dog / ml, 10 5 10 to 100 pcs / ml 6 dog / ml, 10 6 10 to 100 pcs / ml 10 dog / ml, 10 6 10 to 100 pcs / ml 9 dog / ml, 10 6 10 to 100 pcs / ml 8 dog / ml, 10 6 10 to 100 pcs / ml 7 dog / ml, 10 7 10 to 100 pcs / ml 10 dog / ml, 10 7 10 to 100 pcs / ml 9 dog / ml, 10 7 10 to 100 pcs / ml 8 dog / ml, 10 8 10 to 100 pcs / ml 10 dog / ml, 10 8 10 to 100 pcs / ml 9 dog / ml or 10 9 10 to 100 pcs / ml 10 It may be included in a concentration of 100 mg / ml.

[0132]

[0133] Hereinafter, the present invention will be described in more detail through examples. These examples are intended to explain the present invention more specifically, and the scope of the present invention is not limited by these examples.

[0134]

[0135] Example 1: Production of extracellular vesicles derived from genetically modified stem cells

[0136] 1-1. Cell culture

[0137] 293FT cells (derived from HEK293 cells, ThermoFisher Scientific, R70007) were cultured in high glucose DMEM medium (Cytiva, SH30243.01) supplemented with 10% fetal bovine serum (FBS, Gibco, 12483-020) and 1% antibiotic-antimycotic (AA, Gibco, 15240-062).

[0138] To generate a stable cell line, Plat-E cells were used to produce a retrovirus containing the DNA sequence of interest, including the puromycin resistance gene. When 293FT cells reached 80-90% confluency, transduction with Plat-E-derived retrovirus was performed. Plat-E and 293FT cells were cultured in DMEM containing 10% FBS and 1% antibiotic-antimycotic, respectively. The virus was harvested from the Plat-E cell culture medium and treated with 293FT cells. The virus-transduced 293FT cells were grown in the same 293FT cell medium supplemented with puromycin. Human bone marrow-derived mesenchymal stem cells (hBM-MSCs, RoosterBio) were cultured in Rooster Nourish™ CellBIND® Polystyrene CellSTACK® (Corning) for 2D cell culture. TM(RoosterBio). CD47 KO cell line (Abcam, ab266324) and CD47WT cell line (Abcam, ab244449) were cultured according to the manufacturer's handling procedures.

[0139]

[0140] 1-2. Manufacturing of genetically modified human bone marrow-derived mesenchymal stem cells

[0141] Human bone marrow-derived mesenchymal stem cells (hBM-MSC) master cell bank (Passage 0, RoosterBio) were cultured in T-75 flasks (Corning) using RoosterNourish TM Cells were cultured in a medium (RoosterBio) at 37°C under 5% CO2 conditions. After 2 days, cells were harvested and counted for inoculation into T-75 flasks. To ensure that extracellular vesicles express SIRPα, a SIRPα mutant capable of binding to human and mouse CD47 with high affinity was constructed and fused to a type I transmembrane domain to enable effective expression on the surface of extracellular vesicles.

[0142] Lentiviral transfection was performed using RoosterGEM TM Transfection was performed using lentiviruses encoding the SIRPα construct (Flash Therapeutics) using genetic engineering media (RoosterBio). The lentivirus injection dose was determined based on the viral titer and multiplicity of infection (MOI).

[0143] When transfected with lentivirus, culture medium is mixed with lentivirus and RoosterGEM TM The transfection medium was replaced with the one containing this, and the control mesenchymal stem cells were treated with RoosterGEM without lentivirus. TM Only RoosterGEM cells were used. TM After 24 hours of incubation, the medium was replaced with RoosterNourish TMwas replaced. Intermediate washing was omitted during this process. After maintaining for 24 hours, the cells were re-inoculated into appropriate culture vessels and cultured for 4–5 days, expanding until cell coverage reached 80%.

[0144] After harvesting the cells, they were suspended in CryoStor 5 (Biolife Solutions), filled into 2 mL cryovials (Corning), and cryopreserved in a controlled-speed freezer in the second subculture (passage 2) state, and stored in a liquid nitrogen vaporizer.

[0145] The SIRPα-introduced mesenchymal stem cells (SIRPα-MSCs) manufactured above were used as the starting point for all subsequent seed train expansion experiments.

[0146]

[0147] 1-3. Isolation of extracellular vesicles from genetically modified human bone marrow-derived mesenchymal stem cells

[0148] Dissolve SIRPα-MSC (Master Cell Bank) and CellBIND ® After two passages in a Polystyrene CellSTACK® Chamber (Corning), the cells were cultured in a 3D culture incubator. To form seed trains, each passage was performed for 4 or 5 days to reach 80-95% confluence. Key process parameters for mass production were established through previous optimization studies. SIRPα-MSCs and microcarriers in the seed train were cultured in Ambr ® 250 (Sartorius) or STR ® 50 (Sartorius) cell culture was inoculated. Ambr ® After inoculation into a 250-cell culture vessel, the conditions were set to 37°C, 100% dissolved oxygen (DO), 24 mL / min air flow, and 5% CO2. STR ®For the 50-cell culture, the conditions were set to 37°C, 50% dissolved oxygen (DO), 1.5 L / min air flow, and 5% CO2. RoosterNourish according to the manufacturer's instructions. TM RoosterReplenish on badge TM Media additives (RoosterBio) were added on the third day of culture. After expansion of the cells for 5 days, the media was removed at the end of the culture, and the cells / microcarriers were washed twice. Afterwards, RoosterCollect, a protein-free, chemically defined media, was used to collect extracellular vesicles. TM -EV(RoosterBio) to Ambr ® 250 mL for 250, STR ® For 50, 15 L or 50 L was added. To check the cell health, the cell count and metabolite concentration were measured daily during the culture period. The particle count (particels / mL) was measured regularly, and the condition of cells / microcarriers was confirmed through macroscopic and microscopic images. After the collection of extracellular vesicles was completed, the impeller of the cell culture medium was stopped to allow the microcarriers to settle to the bottom. After that, the medium containing extracellular vesicles was transferred to Ambr ® For 250, by pipetting, STR ® For 50, the particles were collected by pumping through a deep tube. The particle number (particels / mL), particle size and distribution, and SIRPα protein content were analyzed.

[0149] As a control, SIRPα-MSCs were treated with CellBIND ® The cells were inoculated into flasks and cultured for 5 days at 37°C and 5% CO2. After culture, the medium was removed, the attached cells were washed, and the cells were replaced with RoosterCollect-EV medium to collect extracellular vesicles. TrypLE was performed on the flask or microcarriers as needed. TM (Thermo Fisher) was used.

[0150]

[0151] Example 2: Purification of extracellular vesicles derived from genetically modified stem cells

[0152] In Example 1, extracellular vesicles (hereinafter referred to as SIRPα-EVs) collected from SIRPα-MSCs were purified. First, AgentV (RoosterBio) was added to the medium according to the manufacturer's instructions, and then filtered through a depth filter (Sartorius) to remove cell debris. The pre-filter pressure was monitored in real time and maintained within the manufacturer's recommended range.

[0153] The filtered medium was concentrated up to 10-fold using tangential flow filtration (TFF, Repligen). Continuous filtration was then performed to replace the buffer, followed by guard filtration to prepare a concentrated extracellular vesicle solution suitable for chromatography. The concentrated solution was further purified using an AKTA Avant150 chromatography system (Cytiva) and a multimodal chromatography resin (Cytiva). System flow rate, pressure, conductivity, and column UV were continuously controlled and monitored throughout the process. SIRPα-EVs were collected from the effluent fraction and showed consistent results across various scales. Purified SIRPα-EVs were formed in a sucrose-containing salt buffer through a second TFF diafiltration step and sterile filtered through a sterile filtration filter (Satorius). Finally, the purified extracellular vesicles (SIRPα-EVs) were filled into particle-free vials and stored at -80°C.

[0154]

[0155] Comparative Example: Production and Purification of Extracellular Vesicles by Two-Dimensional Culture

[0156] To confirm the difference in productivity according to the 3D culture and culture method performed in Examples 1-3, 2D culture was performed using 293FT cells and MSCs introduced with SIRPα to produce extracellular vesicles.

[0157] Specifically, when 293FT cells were cultured in the same manner as in Example 1-1 and the coverage reached 90%, the medium was replaced with 1% GLUTAMAX TM (Gibco, 35050061), DMEM or RoosterCollect containing 1% amino acids TM -The medium was replaced with serum-free medium such as EV (RoosterBio). After 48 hours, the supernatant was collected and centrifuged successively at 300 g for 10 minutes, 2,000 g for 10 minutes, and 10,000 g for 30 minutes. The supernatant was filtered through a KrosFlo equipped with a rubber filter (TFF, Repligen, Spectrum Labs). ® The samples were filtered and purified using a KR2i Tangential Flow filter system. The samples were concentrated and diluted with BPS, and sterilized after parallel flow filtration. The extracellular vesicles were then pelleted by centrifugation at 150,000 g for 1.5 h. The extracellular vesicle pellet was resuspended in phosphate-buffered saline (PBS) containing a protease inhibitor cocktail (Sigma-Aldrich, 535140).

[0158]

[0159] Example 3: Experiments to evaluate the characteristics of extracellular vesicles derived from genetically modified stem cells.

[0160] 3-1. Total RNA analysis

[0161] Total RNA from cells and extracellular vesicles was extracted using the miRNeasymicrokit (Qiagen, 217084). RNA electrophoresis was performed using two standard markers (Thermo Fisher, SM1831, SM1821) to analyze the various size ranges in the samples, and the results were confirmed using the GelDocGoImagingSystem (Bio-Rad). RNA quantification was performed on samples pretreated with RNA-free DNase using the Quant-it TM It was performed using the RiboGreen RNA assay kit (Invitrogen, R11490), and the fluorescent signal was measured using VICTOR Nivo TM Detection was performed using a multimode microplate reader (PerkinElmer, HH35000500).

[0162]

[0163] 3-2. Analysis of extracellular vesicle size and purity

[0164] The size distribution of extracellular vesicles was analyzed using dynamic light scattering (DLS) with a Zetasizer Nano S90 (Malvern Panalytical). PDI values ​​were calculated using Malvern Zetasizer software. The particle counts and zeta potentials of extracellular vesicles were measured by nanoparticle tracking analysis using a ZetaView system (ParticleMetrix), and data analysis was performed with ZetaView software version 8.05.16SP7. Prior to measuring the size distribution and ZP of extracellular vesicles, automated alignment was performed using a 100 nm polystyrene bead standard solution (WithInstrument, 700074). Before injecting the sample into the instrument, the number of detected particles was confirmed to be ≤10, and sample analysis was performed after a washing process. The sample buffer was used as a background solution for size distribution analysis, and deionized water was used as a background buffer for ZP measurement. To observe the structure of extracellular vesicles, TEM images were obtained and analyzed using a transmission electron microscope (cryo-TEM) (FEI Tecnai F20 G2). To quantify the amount of protein in extracellular vesicles, the BCA assay or Bradford assay was applied, and the results were converted to the purity of extracellular vesicles (particles / mg). To determine the lipid binding ratio, MemGlow TM Conditions were optimized for concentration by applying dye. The dyed samples were analyzed using NanoFCM equipment to calculate the lipid binding ratio.

[0165]

[0166] 3-3. Extracellular Vesicle Omics Profiling

[0167] For transcriptome analysis, the SIRPα-EV obtained in Example 2 and the extracellular vesicle samples obtained in the comparative example were analyzed by LC-MS / MS. The identified proteins and quantitative values ​​were used to compare differences between each batch. A total of 7,391 proteins were analyzed in the extracellular vesicles of SIRPα-EV and the comparative example. To dissolve the samples, 4 volumes of cold acetone were added to the extracellular vesicle samples and incubated at -80°C for 90 minutes. The reaction mixture was centrifuged, the supernatant was removed, and the mixture was dried. The dried samples were resuspended in 8 M urea in 100 mM ammonium bicarbonate and sonicated for 5 minutes to dissolve them. After centrifugation at 16,000 rpm for 5 minutes, the supernatant was transferred to a new tube, and the protein concentration was measured using a BCA assay kit. The quantitation was adjusted to ensure the same protein amount for analysis. For protein reduction and alkylation, dithiothreitol (final concentration 10 mM) was added to the extracellular vesicle lysate and incubated at 450 rpm for 30 minutes at 37°C. Then, iodoacetamide (final concentration 25 mM) was added and reacted in the dark at room temperature for 30 minutes to induce alkylation. For protein digestion, the alkylated protein sample was diluted with 100 mM ABC (ammonium bicarbonate) to adjust the urea concentration to less than 1 M. Trypsin digestion was performed at an enzyme:protein ratio of 1:25 at 37°C for 16 hours, and the trypsin reaction was stopped by adding trifluoroacetic acid (final concentration 1%). The prepared sample was labeled with Tandem Mass Tag, and 100 μ of the labeled sample was divided into 12 fractions and analyzed using a nano LC-MS / MS system (Thermo Dionex Ultimate 3000, Thermo Orbitrap Exploris 480).Mass spectral data measured by the Data Dependent Analysis (DDA) method were matched to human protein sequences in the UniProt database using SAGE software. Database search parameters were limited to semi-tryptic peptides with a length of 7 to 50 amino acids. Uncleavage was allowed up to 1, and cysteine ​​alkylation and methionine oxidation were included in the peptide modifications. The false discovery rate (FDR) was set to ≤1% at the spectrum, peptide, and protein levels, and the precursor isolation purity was set to ≥0.7. Gene Ontology Biological Processes (GOBPs) analysis was performed on the top 506 SIRPα-EV proteins, and the results were generated using FunRich software v. 3.1.3. The mass spectrometry proteome data are registered in ProteomeXchange.

[0168]

[0169] Example 4. Evaluation of cell storage by the 1-step system (+1 tear system)

[0170] In order to produce extracellular vesicles at high yields while maintaining the characteristics unique to stem cells even when stem cells, which are primary cells, are genetically modified, a cell storage system was established using a one-step system, such as Example 1-3, rather than a cell storage system using a conventional two-step system.

[0171] Specifically, unlike the conventional method (2-tier system) in which a Master Cell Bank (MCB) is established for stem cells, a Working Cell Bank (WCB) is created based on the Master Cell Bank (MCB), and the WCB is thawed and applied to experiments, Examples 1-3 omit the WCB step and apply a method in which the MCB is subcultured in the MCB step and then directly introduced into a cell culture medium (1-tier system, +1 Tier system). Figure 1 illustrates the two cell storage systems.

[0172] The step 1 system and the step 2 system were applied to the SIRPα-introduced mesenchymal stem cells (SIRPα-MSC) manufactured in Example 1-2 to create cell banks, and cells collected from each cell bank were injected into a cell culture medium to compare the metabolites, cell numbers, and extracellular vesicle production.

[0173] As a result, SIRPα-MSCs manufactured using the one-step and two-step systems, respectively, exhibited similar metabolite concentrations, including glucose and lactate, confirming that metabolite levels were normally regulated (Fig. 2). However, cell proliferation rate and extracellular vesicle production in the culture medium were significantly superior when the one-step system was applied compared to the two-step system (Fig. 3).

[0174] These results suggest that a one-step system is more effective than conventional cell storage methods when genetically engineering primary cells, such as stem cells, to produce extracellular vesicles.

[0175]

[0176] Example 5. Analysis of changes in characteristics of mesenchymal stem cells depending on the presence or absence of genetic modification.

[0177] In addition, the characteristics of SIRPα-introduced mesenchymal stem cells (SIRPα-MSC) manufactured in Example 1-2 and control mesenchymal stem cells without genetic modification (C-MSC) were compared.

[0178] As a result, it was confirmed that SIRPα-MSCs maintained the ability to differentiate into adipocytes (lipid droplet formation), osteocytes (mineral matrix formation), and chondrocytes (cartilage matrix formation) similar to the control group (Fig. 4).

[0179] In addition, it was confirmed that the expression of CD73, CD90, CD105, and CD166, which exhibit the same characteristics of mesenchymal stem cells, was maintained in SIRPα-MSCs and C-MSCs, while CD14, CD34, and CD45, which are hematopoietic and endothelial cell markers, were not expressed (Fig. 5).

[0180] In addition, a comparison of the secretion of cytokines (bFGF, HGF, IL-8, TIMP-1, TIMP-2, VEGF) related to angiogenesis and cell environment regulation revealed similar secretion patterns between SIRPα-MSCs and C-MSCs (Fig. 6).

[0181] In the analysis of SIRPα-MSC inter-donor or individual donor variations, the cell proliferation rate (Fold Expansion) 5 days after thawing, the nanoparticle concentration after the final harvest, and the percentage of SIRPα staining-positive cells were analyzed, and high reproducibility was confirmed (Fig. 7).

[0182]

[0183] Example 6. Evaluation of extracellular vesicle productivity according to culture method

[0184] In Examples 1-3, extracellular vesicles were produced from genetically modified human bone marrow-derived mesenchymal stem cells by three-dimensional culture, and as a control, extracellular vesicles were produced by two-dimensional culture in a comparative example, and the productivity of extracellular vesicles according to the culture method was compared.

[0185] Figure 8 illustrates the two-dimensional (2D CellSTACK) and three-dimensional (Ambr250 and STR50) cell culture systems. For the three-dimensional culture, the uniform cell suspension state in the cell culture was tracked and evaluated over time. As a result, it was confirmed that the cells and microcarriers were uniformly mixed (Figure 9). Furthermore, when cultured in a three-dimensional cell culture, the measured metabolic profiles were consistent regardless of scale, confirming that consistent process conditions were maintained (Figure 10).

[0186] In addition, when comparing the morphology of cells cultured in 2D and 3D cell culture vessels, it was confirmed that cells clearly attached and proliferated on the surface of the microcarriers for 10 days of culture (Fig. 11).

[0187] After replacing each culture medium with an extracellular vesicle collection medium, changes in the number and concentration of extracellular vesicle particles in the culture medium were measured, and it was confirmed that culture in a 3D cell culture vessel showed a significantly higher production of extracellular vesicles than culture in a 2D cell culture vessel (Fig. 12). In addition, it was confirmed that SIRPα expression in mesenchymal stem cells was stably maintained before harvesting extracellular vesicles (Fig. 13).

[0188] Through these results, it was confirmed that the 3D culture process is more advantageous for extracellular vesicle production than the 2D culture process and enables stable culture.

[0189]

[0190] Meanwhile, when SIRPα-MSCs were cultured in 250 mL (Ambr250) and 50 L (STR50) scales in a 3D culture system, the macroscopic cell growth changes (Fig. 14), metabolic profiles (Fig. 15), cell and microcarrier attachment status (Fig. 16), extracellular vesicle production (Fig. 17), and particle size distribution (Fig. 18) were consistently maintained, confirming that 200-fold expansion (scale-up) was successfully implemented in a 3D cell culture device.

[0191]

[0192] Example 7. Quality evaluation of extracellular vesicles after purification process

[0193] The quality of the purified extracellular vesicles (SIRPα-EV) obtained in Example 2 was evaluated. Figure 19 schematically illustrates the conventional two-dimensional culture post-purification process and the three-dimensional culture post-purification process of the present invention.

[0194] As shown in Fig. 19, the purification process of the present invention separates extracellular vesicles through removal of impurities using depth filtration of a culture harvested from a three-dimensional cell culture incubator, concentration through tangential flow filtration (TFF), additional guard filtration before chromatography, purification using chromatography, buffer exchange through secondary TFF, and further concentration.

[0195] As a result, SIRPα-EV showed a 93% removal rate of double-stranded DNA (dsDNA) (Fig. 20) and a 99% removal rate of albumin (Fig. 21) (Fig. 22), and Fig. 23 shows the total protein concentration and step-by-step removal rate evaluation at each step of the purification process.

[0196] In addition, the SIRPα-EV isolated after the purification process showed high purity with 96% of the total protein removed from the culture medium (Fig. 24), and 8 x 10 per 1 mg of protein 11 It showed high purity including the number of particles (Fig. 25), and the total particle recovery rate reached 50%, confirming that the process efficiency was very excellent (Fig. 26).

[0197] Meanwhile, human mesenchymal stem cell-derived extracellular vesicles were produced using the same method as in Examples 2 and 3, and the quality evaluation was performed. As a result, the change in the number of extracellular vesicle particles by culture system at the same level as the SIRPα-EV (Fig. 27), the particle size distribution based on nanoparticle tracking analysis measured by each culture system (Fig. 28), the concentration of extracellular vesicle particles at each stage of the purification process (Fig. 29), the cumulative yield finally obtained in the purification process (Fig. 30), the cumulative protein removal rate (Fig. 31), the cumulative double-strand removal rate (Fig. 32), and the human serum albumin removal rate (Fig. 33) were confirmed, confirming that a high extracellular vesicle recovery rate and purity were achieved.

[0198] These results suggest that the above-described production process is not limited to specific extracellular vesicles and can be universally applied to various types of extracellular vesicles.

[0199]

[0200] Example 8. Evaluation of extracellular vesicle quality according to the presence or absence of genetic modification of mesenchymal stem cells.

[0201] Experiments were performed to compare the genetic profiles and purity of extracellular vesicles derived from genetically modified and non-genetically modified mesenchymal stem cells.

[0202] As a result of comparing the RNA content of SIRPα-EV manufactured by the same process of Examples 1 and 2 and SIRPα-MSC, which is a genetically modified stem cell, it was confirmed that the RNA level was significantly lower in SIRPα-EV (Fig. 34).

[0203] In addition, as a result of comparing the RNA content of SIRPα-EV and C-EV isolated by culturing in a 3D cell culture medium in the same manner as in Examples 1 and 2 and SIRPα-EV and C-EV isolated by the "2D culture post-purification process" of FIG. 19 after culturing in a 2D manner as in the comparative example as a control group, the RNA content was significantly lower than that of the control group when the purification process of the present invention was applied (FIG. 35), confirming that the purification process of the present invention can dramatically remove RNA while maintaining the high purity of the produced extracellular vesicles.

[0204] In addition, compared to the control process, it was confirmed that the number of particles per 1 mg of protein of the extracellular vesicles produced by the purification process of the present invention was more than twice as high (Fig. 36).

[0205] In addition, the characteristics of SIRPα-EV and C-EV isolated by culturing in a 3D cell culture medium in the same manner as in Examples 1 and 2 were compared.

[0206] First, we analyzed the protein cargo profile using LC / MS to identify 7,391 proteins. As a result, we confirmed that SIRPα-EVs showed consistency and reproducibility of protein identification similar to C-EVs (Fig. 37), confirmed that the protein profiles were similar (Fig. 38), and confirmed overexpression of SIRPα in SIRPα-EVs (Fig. 39). In addition, Western blot analysis confirmed the presence of extracellular vesicle markers CD81, CD9, CD63, and TSG101 and expression of SIRPα in SIRPα-EVs, while the non-extracellular vesicle marker prohibitin was not detected (Fig. 40).

[0207] In addition, cryo-TEM (cryogenic transmission electron microscopy) analysis confirmed that SIRPα-EV is a spherical nanoparticle with a lipid bilayer, similar to C-EV (Fig. 41), and DLS (dynamic light scattering) analysis confirmed that SIRPα-EV showed a uniform particle size distribution with an average diameter of approximately 200 nm, similar to C-EV, and maintained high monodispersity without aggregation (Fig. 42). In addition, Zeta potential (ZP) measurement showed that the surface charges of SIRPα-EV and C-EV were -22.56 mV and -22.30 mV, respectively, and MemGlow staining confirmed a lipidation rate of more than 95% (Fig. 43). In addition, analysis of gene ontology biological processes (GOBPs) overexpressed in SIRPα-EV protein cargo confirmed that SIRPα-EV is enriched in adhesion and binding-related proteins that promote target binding at inflammatory sites, a characteristic of stem cell-derived extracellular vesicles (Fig. 44).

[0208] To evaluate the ability of SIRPα-EV to target CD47, in vitro binding assays were performed using a CD47-deficient cell line (CD47 KO) and a wild-type CD47 expressing cell line (WT CD47). As a result, SIRPα-EV was confirmed to bind more selectively to the WT CD47 cell line than C-EV in a dose-dependent manner, while no significant binding was observed in the CD47 KO cell line (Fig. 45).

[0209]

[0210] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical spirit or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as encompassing all changes or modifications derived from the meaning and scope of the claims described below, and their equivalent concepts, rather than the detailed description above.

Claims

1. A method for producing cells for producing extracellular vesicles, comprising a step of passage of cells derived from a master cell bank (MCB) after establishing a master cell bank (MCB) of cells, excluding a step of establishing a working cell bank (WCB).

2. In paragraph 1, A cell manufacturing method for producing extracellular vesicles, characterized in that the above cells are at least one selected from the group consisting of genetically modified cells and genetically non-modified cells.

3. In paragraph 1, A method for producing cells for producing extracellular vesicles, wherein the above cells are at least one selected from the group consisting of cell lines and primary cells.

4. In paragraph 3, A method for producing cells for producing extracellular vesicles, wherein the cell line is at least one selected from the group consisting of HEK293 cells, HEK293T cells, Expi293 cells, Expi293F cells, ExpiCHO cells, CHO cells, HeLa cells, and BHK-21 cells.

5. In paragraph 3, A method for producing cells for producing extracellular vesicles, characterized in that the primary cells are at least one selected from the group consisting of natural killer cells, T cells, B cells, dendritic cells, macrophages, monocytes, fibroblasts, epithelial cells, endothelial cells, and stem cells.

6. In paragraph 5, A method for producing cells for producing extracellular vesicles, wherein the stem cells are at least one selected from the group consisting of mesenchymal stem cells, pluripotent stem cells, induced pluripotent stem cells, adult stem cells, and embryonic stem cells.

7. In paragraph 6, A method for producing cells for producing extracellular vesicles, wherein the mesenchymal stem cells are derived from bone marrow, umbilical cord, umbilical cord blood, fat, muscle, nerve, skin, amniotic membrane or placenta.

8. In paragraph 1, A method for producing cells for producing extracellular vesicles, wherein the above subculture is performed once and then subcultured again.

9. A method for producing extracellular vesicles using cells produced by the method of any one of claims 1 to 8, comprising the following steps: a) a step of culturing the cells in three dimensions to obtain a culture; and b) A step of purifying extracellular vesicles from the above culture.

10. In paragraph 9, A method for producing extracellular vesicles, wherein the above three-dimensional culture is performed at 30°C to 40°C.

11. In paragraph 9, A method for producing extracellular vesicles, wherein the above three-dimensional culture is performed at 50% to 100% dissolved oxygen (DO).

12. In paragraph 9, A method for producing extracellular vesicles, further comprising a step of replacing the medium in step a) with a protein-free medium.

13. In paragraph 9, The above purification method comprises depth filtration, first tangential flow filtration (TFF), and second tangential flow filtration steps.

14. In paragraph 13, A method for producing extracellular vesicles, wherein the filter used for the above depth filtration has a pore size of 0.1 μm to 5 μm.

15. In paragraph 13, A method for producing extracellular vesicles, further comprising a guard filtration and chromatography step between the first parallel flow filtration step and the second parallel flow filtration step.

16. In paragraph 15, A method for producing extracellular vesicles, wherein the filter used for the above guard filtering has a pore size of 0.1 μm to 0.5 μm.

17. In paragraph 13, A method for producing extracellular vesicles, further comprising a filtering step after the secondary parallel flow filtration.

18. A method for producing extracellular vesicles, characterized in that in the 17th paragraph, the filtering step is performed using a filter having a pore size of 0.1 μm to 0.5 μm.

19. In paragraph 9, A method for producing extracellular vesicles, characterized in that the total particle recovery rate of extracellular vesicles produced by the above method is 40% or more.

20. In paragraph 9, A method for producing extracellular vesicles, wherein the above cells are genetically modified cells.

21. In paragraph 20, A method for producing extracellular vesicles, characterized in that extracellular vesicles produced by the above method exhibit a higher level of expression of a target protein compared to extracellular vesicles derived from genetically unmodified cells.

22. In paragraph 21, A method for producing extracellular vesicles, wherein the target protein is SIRPα, HIF1α, CAR peptide, Apelin, T7 peptide or ESM.

23. An extracellular vesicle manufactured by any one of claims 9 to 22.

24. A pharmaceutical composition comprising the extracellular vesicle of claim 23.

25. A cosmetic composition comprising the extracellular vesicle of clause 23.

Images