KR102209937B1 - Manufacturing method of exosomes for increasing their productivity and bioactivity and its application - Google Patents

Abstract

The present invention provides a method of producing exosomes for enhancing productivity and physiological activity, wherein animal-derived cells are cultured in a serum-free medium added with platelet lysates, a culture medium is collected, and then exosomes are separated from the collected culture medium, in a cell culture and culture medium recovery step for exosome production, not in a step of proliferating cells in a growth medium.

Description

Production method of exosomes for increasing their productivity and bioactivity and its application {Manufacturing method of exosomes for increasing their productivity and bioactivity and its application}

The present invention relates to a method for producing exosomes, and to a method for producing exosomes having enhanced physiological activity and/or capable of improving the productivity of exosomes.

In addition, the present invention relates to a method for improving the productivity of exosomes, a method for enhancing physiological activity, and a serum-free medium for producing exosomes.

Additionally, the present invention relates to the application of exosomes with enhanced physiological activity obtained by the production method as described above.

Recently, studies have been reported that a variety of bioactive factors that regulate cell behavior are included in the cell secretome, and in particular, exosomes or extracellular secretions that have intercellular signaling functions Since it contains extracellular vesicles, studies on its components and functions are actively underway.

Cells release vesicles of various membrane types into the extracellular environment, and these vesicles are usually called extracellular vesicles (EVs). Extracellular vesicles are sometimes called cell membrane-derived vesicles, ectosomes, shedding vesicles, microparticles, exosomes, etc., and in some cases, they are used separately from exosomes.

Exosomes are vesicles having a size of tens to hundreds of nanometers consisting of a double phospholipid membrane identical to the structure of a cell membrane, and contain proteins and nucleic acids (mRNA, miRNA, etc.) called exosomes cargo (cargo). Exosomal cargo contains a wide range of signaling factors, and these signaling factors are known to be specific to the cell type and are regulated differently depending on the environment of the secretory cell. Exosomes are intercellular signaling mediators secreted by cells, and various cellular signals transmitted through them regulate cellular behavior including activation, growth, migration, differentiation, dedifferentiation, apoptosis, and necrosis of target cells. Is known. Exosomes contain specific genetic material and bioactive factors depending on the nature and state of the derived cell. In the case of proliferating stem cell-derived exosomes, cell behaviors such as cell migration, proliferation and differentiation are regulated, and the characteristics of stem cells related to tissue regeneration are reflected (Nature Review Immunology 2002 (2) 569-579).

In other words, exosomes, which are called cell avatars, contain bioactive factors such as growth factors, similar to cells, and serve as a carrier that carries bioactive factors between cells and cells, that is, the communication between cells and cells. Exosomes are known to be released not only from animal cells such as stem cells, immune cells, fibroblasts, and cancer cells, but also from cells of various organisms such as plants, bacteria, fungi, and algae. .

Conventional techniques for separating such exosomes include ultracentrifugation, density gradient centrifugation, ultrafiltration, tangential flow filtration (TFF), and size exclusion chromatography. (size exclusion chromatography), ion exchange chromatography, immunoaffinity capture, microfluidics-based isolation, exosome precipitation, total exosome extraction kit (total) exosome isolation kit), or polymer based precipitation.

However, although various methods of separating exosomes have been proposed as described above, continuous improvement can be made in the technical field related to the production of exosomes, like other technical fields. For example, there is a need to provide a new exosome production technology capable of improving the production of exosomes per cell culture solution (or unit cell) and improving the efficacy related to physiologically active factors of exosomes.

On the other hand, it should be understood that the matters described as background art are only for improving understanding of the background of the present invention, and are not cited as approval that it can be used as "prior art" of the present invention.

Republic of Korea Patent Publication No. 10-1985941 (2019.06.04)

It is an object of the present invention to provide a method for producing exosomes that can improve the productivity of exosomes and/or have enhanced physiological activity.

Another object of the present invention is to provide a method for enhancing the productivity of exosomes and a method for enhancing physiological activity.

Another object of the present invention is to provide a serum-free medium for producing exosomes.

Another object of the present invention is to provide various applications of exosomes with enhanced physiological activity obtained by the production method as described above.

However, the subject of the present invention as described above is exemplary, and the scope of the present invention is not limited thereby. Further, other objects and advantages of the present invention will become more apparent by the following detailed description, claims, and drawings.

The inventors of the present invention have been intensively researching the production method of exosomes that can improve the productivity of exosomes and have enhanced efficacy, while culturing cells and recovering the culture medium for the production of exosomes rather than the step of proliferating cells in the growth medium. In the step (so-called "conditioning" step), when culturing animal-derived cells in a serum-free medium to which platelet lysate is added, collecting conditioned media, and separating exosomes from the collected culture, the unit culture solution (or The present invention was completed by confirming that the production of exosomes per unit cell) was increased and the physiological activity of the produced exosomes was improved.

As used herein, the term "extracellular vesicles (EVs)" generally encompasses membrane-derived vesicles (membrane vesicles), ectosomes, shedding vesicles, microparticles, or equivalents thereof. It is used in the sense of Depending on the separation environment, conditions, and methods, extracellular vesicles may have the same meaning as exosomes, and may have the same or similar size as exosomes, but may have a meaning including nanovesicles that do not have the composition of exosomes. Meanwhile, the term exosomes used in relation to productivity in the present specification is used in the sense of encompassing the extracellular vesicles.

As used herein, the term "exosomes" refers to vesicles having a size of tens to hundreds of nanometers (preferably about 30 to 200 nm) composed of a double phospholipid membrane identical to the structure of the cell membrane (however, the separation object is The particle size of the exosomes may vary depending on the cell type, separation method, and measurement method. (Vasiliy S. Chernyshev et al., "Size and shape characterization of hydrated and desiccated exosomes", Anal Bioanal Chem, (2015) DOI 10.1007/s00216-015-8535-3). Exosomes contain proteins and nucleic acids (mRNA, miRNA, etc.) called exosome cargo (cargo). Exosomal cargo contains a wide range of signaling factors, and these signaling factors are known to be specific to the cell type and are regulated differently depending on the environment of the secretory cell. Exosomes are intercellular signaling mediators secreted by cells, and various cellular signals transmitted through them regulate cellular behavior including activation, growth, migration, differentiation, dedifferentiation, apoptosis, and necrosis of target cells. Is known.

On the other hand, the term "exosome" as used herein has a nano-sized vesicle structure secreted from animal-derived cells and released into the extracellular space and has a composition similar to that of exosomes (eg, exosomes -It means to include all similar veggies).

In the present invention, the type of the animal-derived cell from which the exosome is derived is not limited, but as an example that does not limit the present invention, it may be a stem cell or an immune cell. The stem cells may be embryonic stem cells, induced pluripotent stem cells (iPSC), adult stem cells, mesenchymal stem cells derived from embryonic stem cells, or mesenchymal stem cells derived from induced pluripotent stem cells. The immune cells may be T cells, B cells, NK cells, cytotoxic T cells, dendritic cells or macrophages.

As an example not limiting the present invention, the adult stem cells are at least one adult selected from the group consisting of mesenchymal stem cells, human tissue-derived mesenchymal stromal cells, human tissue-derived mesenchymal stem cells, and multipotent stem cells. It may be a stem cell. The mesenchymal stem cells may be mesenchymal stem cells derived from at least one tissue selected from the group consisting of umbilical cord, cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, Wharton jelly, and placenta. Preferably, the adult stem cells may be mesenchymal stem cells, for example, stem cells derived from fat, bone marrow, umbilical cord or umbilical cord blood, and more preferably stem cells derived from fat. The type of the stem cell or the immune cell is not limited as long as there is no risk of infection by a pathogen and does not cause an immune rejection reaction, but it may be preferably a human stem cell or a human immune cell.

However, as long as it does not cause adverse effects on the human body, it is of course possible to use various animal cells that are used in the art or can be used in the future. For example, it is also possible to use HEK 293 cells or HEK 293T cells. Therefore, the fat stem cells used in the examples described below should be understood as an example of animal cells that can be used in the present invention, and the present invention is not limited thereto.

As used herein, the term "serum-free medium" or "SFM" is a culture medium in which serum such as Fetal Bovine Serum (FBS) is not added to, for example, a basal medium (optionally including antibiotics and/or antifungal agents) Means, and "EDM" is a platelet lysate, preferably a human platelet lysate, more preferably a platelet lysate-derived exosome and an extracellular vesicle-derived human platelet lysate added to the basic medium. It means a culture medium (optionally including antibiotics and/or antifungal agents). As an example not limiting the present invention, the basic medium is α-MEM, DMEM, DMEM F12, 199 medium, RPMI-1640, L-15, Hum F-10, Hum F-12, DM-160, DM-170, etc. I can.

As used herein, the term "anti-inflammatory" means preventing, suppressing, alleviating, improving or treating inflammation, and as an example of not limiting the present invention as an inflammatory disease, dermatitis, atopic dermatitis, eczema, bacterial infection, virus Inflammation from infection or fungal infection, burns, inflammation from burns, wounds, inflammation from wounds, ulcerative colitis, arthritis, rheumatoid arthritis, hepatitis, nephritis, and the like.

The term "wound" as used herein refers to a state in which a living body is damaged, and tissues that form an internal or external surface of a living body, such as skin, muscle, nerve tissue, bone, soft tissue, internal organ or vascular tissue, are It encompasses pathological conditions that have been divided or destroyed. As an example of not limiting the present invention, wounds include abrasion, laceration, cut, cut, cut, avulsion, bedsores, bulges, tissue destruction by irradiation, penetrated wounds, gunshot wounds ( gun shot wound), burns, frostbite, surgical, post-plastic surgery sutures, chemical wounds, etc., and may include damage to any part of the object.

In the present specification, the term "iontophoresis" is a method of allowing a microcurrent to flow through the skin to which an active substance is applied to change the electrical environment of the skin by applying a potential difference so that the ionized active ingredient penetrates the skin with an electrical repulsive force. Means. Iontophoresis used in one embodiment of the present invention is a method in which a current from an external power source flows into an electrode patch on the skin and a microcurrent is introduced into the skin. Including a method in which microcurrent is introduced, a method in which microcurrent is introduced into the skin through a patch equipped with a reverse electrodialysis means that generates an electric current through the difference in ion concentration between the high concentration electrolyte solution and the low concentration electrolyte solution. I can. However, the present invention is not limited thereto, and of course, various types of iontophoresis may be used.

The present invention improves the productivity of exosomes comprising the step of culturing animal-derived cells in a serum-free medium to which platelet lysate is added, collecting conditioned media, and separating exosomes from the collected culture. Provides a way. For example, the method of improving the productivity of exosomes according to an embodiment of the present invention includes the steps of (a) culturing animal-derived cells in a growth medium to proliferate cells, and (b) converting the animal-derived cells into a platelet lysate (platelet lysate). lysate) is added to the serum-free medium, collecting the conditioned media containing exosomes secreted or released from the animal-derived cells, and separating the exosomes from the collected culture. .

In the method for improving the productivity of exosomes according to an embodiment of the present invention, a platelet lysate is not added to the growth medium in step (a).

In the method for improving the productivity of exosomes according to an embodiment of the present invention, the serum-free medium may contain about 0.01% to 20% by volume of the platelet lysate, and for example, about 0.25% by volume. .

In the method for improving the productivity of exosomes of one embodiment of the present invention, the platelet lysate may preferably be a human platelet lysate, more preferably platelet lysate-derived exosomes and extracellular vesicles. It may be a human platelet lysate removed.

In the method for improving the productivity of exosomes of one embodiment of the present invention, the culture in step (b) may be performed for about 24 hours to 72 hours, for example, it may be performed for 24 hours.

In the method for improving the productivity of exosomes of one embodiment of the present invention, the productivity may be defined as the number of exosome particles or exosome protein amount per mL of the culture medium.

The present invention is physiological activity of exosomes comprising the step of culturing animal-derived cells in a serum-free medium to which platelet lysate is added, collecting conditioned media, and separating exosomes from the collected culture. Provides a method of strengthening. For example, the method for enhancing physiological activity of exosomes according to an embodiment of the present invention includes the steps of (a) culturing animal-derived cells in a growth medium to proliferate cells, and (b) converting the animal-derived cells into a platelet lysate ( platelet lysate) is added to the serum-free medium, collecting conditioned media containing exosomes secreted or released from the animal-derived cells, and separating exosomes from the collected culture. have.

In the method for enhancing the physiological activity of exosomes according to an embodiment of the present invention, a platelet lysate is not added to the growth medium in step (a).

In the method for enhancing the physiological activity of exosomes according to an embodiment of the present invention, the serum-free medium may contain about 0.01% to 20% by volume of the platelet lysate, for example, about 0.25% by volume. have.

In the method for enhancing the physiological activity of exosomes of one embodiment of the present invention, the platelet lysate may preferably be a human platelet lysate, more preferably platelet lysate-derived exosomes and extracellular vesicles It may be a human platelet lysate from which is removed.

In the method for enhancing the physiological activity of exosomes according to an embodiment of the present invention, the culturing in step (b) may be performed for about 24 to 72 hours, for example, for 24 hours.

In the method for enhancing the physiological activity of exosomes according to an embodiment of the present invention, the strengthening of the physiological activity may be enhanced anti-inflammatory, wound treatment, or wound treatment promoting efficacy. In addition, the physiological activity enhancement may be an increase in collagen synthesis, for example, may be at least one of enhancing skin elasticity or wrinkle improvement.

The present invention is a serum-free medium used in the step of culturing cells and collecting culture medium for producing exosomes, not in the step of proliferating cells, and a serum-free medium for producing exosomes derived from animal cells, characterized in that platelet lysates are added. Provide a medium.

The serum-free medium for producing exosomes according to an embodiment of the present invention may be a serum-free medium for enhancing the productivity of exosomes derived from animal cells or a serum-free medium for enhancing physiological activity of exosomes derived from animal cells. The strengthening of the physiological activity may be an anti-inflammatory, wound treatment, or enhanced efficacy of promoting wound treatment. In addition, the physiological activity enhancement may be an increase in collagen synthesis, for example, may be at least one of enhancing skin elasticity or wrinkle improvement.

The serum-free medium for the production of exosomes derived from animal cells according to an embodiment of the present invention may contain about 0.01% to 20% by volume of the platelet lysate, and for example, about 0.25% by volume.

In the serum-free medium for the production of exosomes derived from animal cells according to an embodiment of the present invention, the platelet lysate may preferably be a human platelet lysate, more preferably platelet lysate-derived exo It may be a human platelet lysate from which moths and extracellular vesicles have been removed.

In addition, the present invention is separated from the culture medium collected after culturing animal-derived cells in a serum-free medium to which platelet lysate has been added, and collecting conditioned media, anti-inflammatory, wound treatment or wound treatment promoting efficacy It provides a composition for promoting anti-inflammatory, wound treatment or wound treatment, comprising the enhanced exosomes as an active ingredient.

For example, the composition of one embodiment of the present invention comprises the steps of (a) culturing animal-derived cells in a growth medium to proliferate cells, and (b) adding platelet lysate to the animal-derived cells. It can be obtained by culturing in a serum-free medium, collecting a conditioned media containing exosomes secreted or released from the animal-derived cells, and then separating exosomes from the collected culture.

In the composition of an embodiment of the present invention, in the step (a), platelet lysate is not added to the growth medium.

In the composition of one embodiment of the present invention, the serum-free medium may contain about 0.01% to 20% by volume of the platelet lysate, and, for example, about 0.25% by volume.

In the composition of one embodiment of the present invention, the platelet lysate may preferably be a human platelet lysate, and more preferably, a platelet lysate derived from exosomes and extracellular vesicles are removed. Can be

In the composition of one embodiment of the present invention, the culturing in step (b) may be performed for about 24 to 72 hours, for example, for 24 hours.

As an example not limiting the present invention, the composition of one embodiment of the present invention may be administered or treated by injection, microneedling, iontophoresis, application, or a combination thereof.

The composition of one embodiment of the present invention may be a pharmaceutical composition, a quasi-drug, an external preparation for skin or a functional cosmetic composition. For example, the pharmaceutical composition may be an injection form, an injection form, a spray form, a liquid form, or a patch form, and the quasi-drug or the skin external preparation may be a liquid, ointment, cream, spray, patch, gel, or aerosol. have. In addition, the functional cosmetic composition may be, for example, a lotion or cream.

When the composition of one embodiment of the present invention is used as a pharmaceutical composition, it may include a pharmaceutically acceptable carrier, excipient, or diluent. The carrier, excipient and diluent include lactose, dextrose, trehalose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium carbonate, calcium silicate, cellulose , Methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, etc., but are not limited thereto. .

Administration of the pharmaceutical composition of one embodiment of the present invention means introducing a predetermined substance to the patient by any appropriate method, and the administration route of the pharmaceutical composition is administered through any general route as long as the drug can reach the target tissue. Can be. For example, the pharmaceutical composition of one embodiment of the present invention may be administered orally or parenterally, and parenteral administration includes intra-articular administration, intra-articular administration, intrasynovial administration, intrasternal administration, intrathecal administration. ) Administration, intralesional and intracranial administration, transdermal administration, intraperitoneal administration, intravenous administration, intraarterial administration, intralymphatic administration, intramuscular administration, subcutaneous administration, intradermal administration, topical administration, rectal administration, etc. Can be discussed. However, it is not limited thereto and does not exclude various methods of administration known in the art. In addition, the pharmaceutical composition of one embodiment of the present invention can be administered by any device capable of moving the active substance to a target tissue or cell. In addition, the effective amount of the pharmaceutical composition of one embodiment of the present invention refers to an amount required for administration in order to expect anti-inflammatory, wound healing, and/or wound healing promoting effects.

The preparation for parenteral administration of the pharmaceutical composition of one embodiment of the present invention may be a sterilized aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized preparation, or a suppository. The formulation for parenteral administration of the pharmaceutical composition of one embodiment of the present invention may be prepared as an injection. The injection of one embodiment of the present invention may be an aqueous injection, a non-aqueous injection, an aqueous suspension injection, a non-aqueous suspension injection, or a solid injection used by dissolving or suspending, but is not limited thereto. According to the type of injection, distilled water for injection, vegetable oil (e.g., peanut oil, sesame oil, camellia oil, etc.), monoglyceride, diglyceride, propylene glycol, camphor, benzoate estradiol, bismuth subsalicylate, etc. , Arcenobenzol sodium, or streptomycin sulfate may contain at least one, and may optionally contain a stabilizer or preservative.

The blending ratio of the pharmaceutical composition according to an embodiment of the present invention may be appropriately selected according to the type, amount, and form of additional ingredients as described above. For example, with respect to the total amount of the injection, the pharmaceutical composition of the present invention may be included in about 0.1 to 99% by weight, preferably about 10 to 90% by weight. In addition, the appropriate dosage of the pharmaceutical composition of one embodiment of the present invention may be adjusted according to the severity of the disease, the type of the formulation, the formulation method, the patient's age, sex, weight, health condition, diet, excretion rate, administration time, and administration method. I can. For example, when administering the pharmaceutical composition of an embodiment of the present invention to an adult, it may be administered in a dose of 0.001 mg/kg to 100 mg/kg per day in 1 to several times.

In addition, the present invention has the efficacy of increasing collagen synthesis obtained by culturing animal-derived cells in a serum-free medium to which platelet lysate is added, collecting conditioned media, and separating exosomes from the collected culture. It provides a composition comprising the enhanced exosomes as an active ingredient. The composition may be, for example, a composition for improving skin elasticity or wrinkles.

For example, the composition of one embodiment of the present invention comprises the steps of (a) culturing animal-derived cells in a growth medium to proliferate cells, and (b) adding platelet lysate to the animal-derived cells. It can be obtained by culturing in a serum-free medium, collecting a conditioned media containing exosomes secreted or released from the animal-derived cells, and then separating exosomes from the collected culture.

In the composition of an embodiment of the present invention, in the step (a), platelet lysate is not added to the growth medium.

In the composition of one embodiment of the present invention, the serum-free medium may contain about 0.01% to 20% by volume of the platelet lysate, and, for example, about 0.25% by volume.

In the composition of one embodiment of the present invention, the platelet lysate may preferably be a human platelet lysate, and more preferably, a platelet lysate derived from exosomes and extracellular vesicles are removed. Can be

In the composition of one embodiment of the present invention, the culturing in step (b) may be performed for about 24 to 72 hours, for example, for 24 hours.

The composition of one embodiment of the present invention may be a quasi-drug, an external preparation for skin, or a functional cosmetic composition. The quasi-drug or the external preparation for skin may be a liquid, ointment, cream, spray, patch, gel, or aerosol. In addition, the cosmetic composition may be, for example, a lotion or cream.

On the other hand, when the anti-inflammatory, wound treatment or wound treatment promotion composition, or skin elasticity improvement or wrinkle improvement composition of one embodiment of the present invention is prepared as a quasi-drug, skin external preparation and/or cosmetic composition, the effect of the present invention is impaired. Ingredients commonly used in quasi-drugs, external skin preparations and/or functional cosmetic compositions, such as moisturizers, antioxidants, oily ingredients, ultraviolet absorbers, emulsifiers, surfactants, thickeners, alcohols, powder ingredients, colorants, Water-based ingredients, water, and various skin nutrients can be appropriately blended as needed.

In addition, the quasi-drug, skin external preparation, and/or functional cosmetic composition of one embodiment of the present invention, in addition to exosomes with enhanced physiological activity, have their actions (e.g., anti-inflammatory, wound treatment, wound treatment promotion, skin elasticity improvement and/or Anti-inflammatory agents, anti-inflammatory agents, wound treatments, skin elasticity improving agents, anti-wrinkle agents, and/or moisturizing agents, which have been conventionally used, can be mixed together to the extent that they do not damage wrinkles, etc.). For example, the composition comprising the exosomes with enhanced physiological activity of the present invention as an active ingredient may be applied to at least one of a hydrogel, hyaluronic acid, hyaluronic acid salt (eg, sodium hyaluronate), or hyaluronic acid gel. It can be supported or mixed. In the quasi-drug, skin external preparation and/or cosmetic composition of one embodiment of the present invention, the type of the hydrogel is not limited, but it may be preferably a hydrogel obtained by dispersing a gelling polymer in a polyhydric alcohol. The gelling polymer is at least one selected from the group consisting of pluronic, purified agar, agarose, gellan gum, alginic acid, carrageenan, cassia gum, xanthan gum, galactomannan, glucomannan, pectin, cellulose, guar gum, and locust bean gum. It may be a species, and the polyhydric alcohol may be at least one selected from the group consisting of ethylene glycol, propylene glycol, 1,3-butylene glycol, isobutylene glycol, dipropylene glycol, sorbitol, xylitol, and glycerin.

Quasi-drugs, skin external preparations and/or functional cosmetic compositions of one embodiment of the present invention include, for example, patches, mask packs, mask sheets, creams, tonics, ointments, suspensions, emulsions, pastes, lotions, gels, oils, packs, It can be applied to various forms such as spray, aerosol, mist, foundation, powder, and oil paper. For example, the quasi-drug, skin external preparation and/or functional cosmetic composition may be applied or deposited on at least one surface of a patch, mask pack, or mask sheet.

The functional cosmetic composition of one embodiment of the present invention may be used for the purpose of anti-inflammatory, promoting wound treatment, improving wounds, improving skin elasticity and/or wrinkles, and functional cosmetic formulations can be used in any formulation commonly manufactured in the art. Can be manufactured. For example, patch, mask pack, mask sheet, softening lotion, nutrient lotion, astringent lotion, nourishing cream, massage cream, eye cream, cleansing cream, essence, eye essence, cleansing lotion, cleansing foam, cleansing water, sunscreen, lipstick , Soap, shampoo, surfactant-containing cleansing, bathing agent, body lotion, body cream, body oil, body essence, body cleaner, hair dye, hair tonic, etc., but is not limited thereto.

The quasi-drug, skin external preparation, and/or functional cosmetic composition of one embodiment of the present invention includes ingredients commonly used in quasi-drugs, skin external preparations and/or functional cosmetic compositions, such as antioxidants, stabilizers, solubilizers, vitamins, pigments And conventional adjuvants and carriers such as fragrances. In addition, in each formulation for a quasi-drug, external skin preparation and/or functional cosmetic composition, other ingredients can be appropriately selected and blended by those skilled in the art according to the type or purpose of use of the quasi-drug, external skin preparation and/or functional cosmetic composition. have.

In addition, the present invention uses a composition comprising exosomes having enhanced anti-inflammatory, wound treatment or wound treatment promoting efficacy as an active ingredient, to prevent, inhibit, alleviate, improve or treat inflammatory diseases, or treat wounds, Or, it provides a method of accelerating wound healing.

The method of one embodiment of the present invention includes administering to a mammal a therapeutically effective amount of the composition, or treating the composition to an inflamed area and/or a wound area of the mammal.

In the method of one embodiment of the present invention, the composition may be administered or treated to a mammal by injection, microneedling, iontophoresis, application, or a combination thereof.

In the method of one embodiment of the present invention, the composition is, for example, a patch, a mask pack, a mask sheet, a cream, a tonic, an ointment, a suspension, an emulsion, a paste, a lotion, a gel, an oil, a pack, a spray, an aerosol, It can be applied to various forms such as mist, foundation, powder, and oil paper. For example, the composition may be applied or deposited on at least one surface of a mask pack, mask sheet, or patch.

The method of one embodiment of the present invention may further include the step of performing iontophoresis by flowing a microcurrent to an inflammation site and/or a wound site of a mammal treated with the composition.

In the method of an embodiment of the present invention, the step of performing the iontophoresis may be performed by contacting or attaching the iontophoresis device to the inflamed area and/or the wound area of the mammal.

In the method of one embodiment of the present invention, the iontophoresis device is a group consisting of a flexible battery, a lithium ion secondary battery, an alkaline battery, a dry cell, a mercury battery, a lithium battery, a nickel-cadmium battery, and a reverse electrodialysis battery. It may include at least one type of battery selected from.

In the method of one embodiment of the present invention, the mammal may be a human, dog, cat, rodent, horse, cow, monkey, or pig.

According to the present invention, it is possible to not only improve the productivity of exosomes separated from the cell culture fluid, but also exhibit excellent effects that can enhance the anti-inflammatory, wound treatment or wound treatment promoting efficacy of exosomes, or physiological activities such as collagen synthesis. have. Therefore, the production method of exosomes of the present invention can economically and efficiently produce exosomes with enhanced physiological activity useful commercially and/or clinically at high yield, and exosomes with enhanced physiological activity thus produced It can be usefully used as an active ingredient in pharmaceutical compositions, quasi-drugs, skin external preparations, or functional cosmetic compositions.

Meanwhile, the scope of the present invention is not limited by the effects as described above.

1 is a schematic diagram showing a process of removing exosomes from a platelet lysate.
2A to 2C show exosome-specific surface marker proteins in ED-hPL (TFF permeate from which exosomes were removed from human platelet lysate) and EC-hPL (TFF concentrate of human platelet lysate) prepared in Example 1. As a graph showing the analysis results for phosphorus CD9, CD63 and CD81, it indicates that the exosome surface marker proteins CD9, CD63 and CD81 do not exist in ED-hPL.
Figure 3 is a process of culturing and proliferating cells in a growth medium containing serum, and a process of culturing the proliferated cells in SFM medium or EDM medium to obtain a culture solution containing secreted or released exosomes (exosomal production It is a schematic diagram showing the cell culture and culture solution recovery process).
Figure 4a shows the NTA results of exosomes (hereinafter referred to as "SFM exosomes") isolated from the collected culture after culturing cells in SFM medium and collecting the culture medium in the cell culture and culture medium recovery step for exosome production 4B is an exosome (hereinafter referred to as "EDM exosome") isolated from the collected culture after culturing the cells in EDM medium and collecting the culture medium in the cell culture and culture medium recovery step for exosome production. This is the NTA result. In Figures 4a and 4b, it is confirmed that the productivity of EDM exosomes is significantly higher than that of SFM exosomes.
5A is a graph showing that the productivity of EDM exosomes (the number of exosome particles per mL of culture solution) is significantly higher than that of SFM exosomes, and FIG. 5B is a graph showing that the productivity of EDM exosomes (amount of protein per mL of culture solution) is SFM. It is a graph showing that the productivity of exosomes is remarkably high.
6A to 6C are graphs showing the results of inflammatory cytokine analysis using a multiplex panel. When EDM exosomes are treated for RAW 264.7 cells, IL induced by LPS compared to the case where SFM exosomes are treated. -6, IFN-β and IL-27 are markedly reduced.
7 is a graph showing the results of analyzing the effect of increasing collagen synthesis of EDM exosomes and SFM exosomes, showing that EDM exosomes significantly increase collagen synthesis in human fibroblasts compared to SFM exosomes.

Hereinafter, the present invention will be described in more detail in the following examples. However, the following examples are not intended to limit or limit the scope of the present invention only to illustrate the content of the present invention. What can be easily inferred by those skilled in the art from the detailed description and examples of the present invention is construed as belonging to the scope of the present invention. References cited in the present invention are incorporated herein by reference.

Throughout the specification, when a part "includes" a certain component, it means that other components may be further included rather than excluding other components unless otherwise stated.

Example

Example 1: Preparation of platelet lysate from which exosomes were removed

In order to remove extracellular vesicles and exosomes from human platelet lysate (hPL), tangential flow filtration (TFF) was used (see FIG. 1). The TFF filter can be selected by various molecular weight cutoffs (MWCO), and a TFF filter of 100 kDa to 750 kDa MWCO was used to obtain a human platelet lysate from which extracellular vesicles and exosomes were removed. As shown in Figure 1, the concentrate that did not pass through the TFF filter is named EC-hPL (Exosome-concentrated hPL), and the permeate that has passed through the filter is ED-hPL (Exosome-depleted hPL). It was named as.

Meanwhile, as a method of removing extracellular vesicles and exosomes from human platelet lysates, in addition to the above-described methods, various methods known in the art may be used. For example, for the removal of extracellular vesicles and exosomes, ultrafiltration, ultracentrifuagation, density gradient centrifugation, size exclusion chromatography, ion Ion exchange chromatography, immunoaffinity capture, microfluidics-based isolation, exosome precipitation, total exosome isolation kit, or polymer Known separation methods such as polymer based precipitation may be used. However, the method of removing extracellular vesicles and exosomes is not limited to the above-described methods, and of course, various separation methods that are used in the art or may be used in the future can be adopted.

Example 2: Verification of exosome removal in ED-hPL

In order to confirm that exosomes were removed from ED-hPL, the quantitative values of ED-hPL and EC-hPL for CD9, CD63 and CD81, which are exosome-specific markers, were compared and evaluated. For the quantification of CD9, CD63 and CD81 present in EC-hPL or ED-hPL, EC-hPL or ED-hPL and exosome-human CD9/CD63/CD81 flow detection reagent (Exosome-Human CD9/CD63/CD81 Flow Detection Reagent) (Invitrogen™) was mixed overnight and then PE mouse anti-human CD9/CD63/CD81 (Mouse anti-Human CD9/CD63/CD81) corresponding to each flow detection reagent (BD Pharmigen™) Each reaction with the antibody was performed and PE mean fluorescence intensity (MFI) was measured using flow cytometry. An IgG isotype was selected as a control to be compared for the quantitative values of CD9, CD63 and CD81, and a PE mouse isotype control antibody was used for the analysis of PE mean fluorescence intensity for the IgG isotype control. CD9, CD63 and CD81 contained in EC-hPL or ED-hPL, respectively, were quantified by comparing the MFI of CD9, CD63 and CD81 measured as described above with the MFI of the control IgG isotype.

As a result, it was confirmed that EC-hPL contained CD9, CD63 and CD81, whereas in ED-hPL, the average PE intensity of CD9, CD63 and CD81 was almost the same as that of the IgG isotype control group. It was confirmed that the moth was completely removed (see FIGS. 2A, 2B and 2C). Therefore, according to the separation method of Example 1, exosomes can be efficiently removed from human platelet lysates (hPL).

Example 3: Culture of cells and production of culture medium

Human adipose-derived stem cells were suspended in α-MEM culture medium containing 10% Fetal bovine serum (FBS), 100 units/mL penicillin, and 100 μg/mL streptomycin, and then cell factory (Cell Factory) The flask was inoculated at a density of 6,000 cells/cm ² and cultured in an incubator at 37° C. and 5% CO ₂ (see “1. Cell Proliferation Step” in FIG. 3). When the cell density (confluency) of 80% or more is reached, the culture medium is α-MEM medium containing 100 units/mL penicillin and 100 μg/mL streptomycin (hereinafter referred to as "Serum-free media" or "SFM") , Or ED-hPL (0.25% by volume) prepared in Example 1, α-MEM medium containing 100 units/mL penicillin and 100 μg/mL streptomycin (hereinafter referred to as “Exosome depleted media” or “EDM”) Replaced with. After replacing the SFM medium or EDM medium, the culture medium was cultured for 24 hours, and the medium was collected (see "2. Culture and culture solution recovery for exosome production" in FIG. 3). The number of cells for which the medium was collected was measured using a cell counter, and it was confirmed that there was no change in the number of cells by the addition of ED-hPL to the basic medium. The recovered culture medium was filtered through a 0.2 μm filter to remove cells and cell debris.

Example 4: Isolation and purification of exosomes

In Example 3, a TFF (Tangential Flow Filtration) method was used for separating, concentrating, desalting, and diafiltration from the culture medium filtered with a 0.2 μm filter. As a filter for the TFF method, a cartridge filter (aka hollow fiber filter; purchased from GE Healthcare or Spectrum Labs) or a cassette filter (purchased from Pall or Sartorius or Merck Millipore) was used. TFF filters can be selected by various molecular weight cutoffs (MWCO). The exosomes were selectively separated and concentrated by the selected MWCO, and particles, proteins, lipids, nucleic acids, and small molecular compounds smaller than the MWCO were removed.

In order to separate and concentrate exosomes, a TFF filter of MWCO 100,000 Da (Dalton), 300,000 Da, 500,000 Da or 750,000 Da, or a TFF filter having a size of 0.05 μm was used. The culture medium was concentrated to a volume of about 1/100 to 1/25 using the TFF method, while substances smaller than MWCO were removed to separate exosomes.

The separated and concentrated exosome solution was further subjected to desalting and buffer exchange (diafiltration) using the TFF method. At this time, desalination and buffer exchange were performed continuously (continuous diafiltration) or discontinuous diafiltration, and at least 4 times, preferably 6 times to 10 times or more, more preferably with respect to the starting volume. It was carried out using a buffer solution having a volume of at least 12 times.

Meanwhile, as a method for separating exosomes from animal-derived cell culture media including stem cells, in addition to the above-described separation method, various methods known in the art may be used. For example, for the separation of exosomes, ultrafiltration, ultracentrifugation, density gradient centrifugation, size exclusion chromatography, ion exchange chromatography ( ion exchange chromatography), immunoaffinity capture, microfluidics-based isolation, exosome precipitation, total exosome isolation kit, or polymer-based precipitation method ( polymer based precipitation) can be used. However, the method of separating exosomes is not limited to the methods as described above, and of course, various separation methods that are used in the art or may be used in the future can be adopted.

Example 5: Analysis of characteristics and productivity of isolated exosomes

The isolated exosomes were measured for particle size and concentration by nanoparticle tracking analysis (　NTA; purchased from Malvern), and total protein was also used in the Micro BCA assay kit (purchased from ThermoFisher). The amount was measured.

In the step of culturing and recovering the culture medium for the production of exosomes, the stem cells were cultured in the SFM medium according to Example 3, the culture medium was recovered, and then separated from the recovered culture medium according to the separation method of an embodiment of the present invention of Example 4. The NTA results of the exosomes (hereinafter referred to as "SFM exosomes") are shown in FIG. 4A. In addition, in the step of culturing and recovering the culture medium for the production of exosomes, according to Example 3, the stem cells were cultured in the EDM medium and the culture medium was recovered, and then from the recovered culture medium, the separation method of the present invention of Example 4 The NTA results of the separated exosomes (hereinafter referred to as "EDM exosomes") are shown in FIG. 4B. In addition, analysis of the total protein amount of SFM exosomes and EDM exosomes was also performed.

As shown in FIG. 5A, when comparing the number of particles from the NTA results, it can be seen that the productivity of EDM exosomes (that is, the number of exosome particles per mL of the culture medium) is approximately 2 times higher than that of SFM exosomes. In addition, when comparing the amount of protein as shown in FIG. 5B, it can be seen that the productivity of EDM exosomes (that is, the amount of protein per mL of the culture medium) is approximately 2 times higher than that of SFM exosomes. Therefore, in the step of culturing and recovering the culture medium for the production of exosomes, when the stem cells are cultured in EDM medium, the culture medium is recovered, and the exosomes are separated from the recovered culture medium, the exosome productivity (the number of exosome particles and protein per mL of the culture medium) Amount) can be significantly improved.

Example 6: Evaluation of anti-inflammatory efficacy of exosomes

Exosomes produced using SFM medium (hereinafter referred to as "SFM exosomes") and exosomes produced using EDM medium (hereinafter referred to as "SFM exosomes") in the culture and culture medium recovery process for exosome production (see FIG. 3) EDM exosomes") was confirmed as follows to reduce the production of inflammatory cytokines. However, both SFM exosomes and EDM exosomes were isolated according to Example 4. To this end, the experimental groups were classified as follows:

(1) negative control (control): the experimental group in which LPS was not treated in RAW 264.7 cells;

(2) LPS: an experimental group in which only LPS was treated in RAW 264.7 cells;

(3) Positive control group: an experimental group in which dexamethasone was treated before LPS was treated in RAW 264.7 cells (indicated as "DEX" in FIGS. 6A to 6C);

(4) SFM exosome treatment group: an experimental group in which SFM exosomes were treated at a concentration of 6×10 ¹⁰ particles/mL before LPS treatment in RAW 264.7 cells (indicated as “SFM” in FIGS. 6A to 6C);

(5) EDM exosome treatment group: an experimental group in which EDM exosomes were treated at a concentration of 6×10 ¹⁰ particles/mL before the LPS was treated in RAW 264.7 cells (indicated as “EDM” in FIGS. 6A to 6C).

In RAW 264.7 cells, a mouse macrophage cell line, the effect of reducing the production of inflammatory cytokines of SFM exosomes or EDM exosomes was confirmed as follows.

RAW 264.7 cells were suspended in DMEM (Dulbecco Modified Eagle Medium; purchased from ThermoFisher Scientific) medium supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin, and then 2.5×10 ⁴ cells/well in a 96-well plate. The cells were inoculated at a density of, and the RAW 264.7 cells were treated according to the conditions of each of the experimental groups (1) to (5), and then cultured in a 37°C, 5% CO ₂ incubator for 24 hours. Then, 100 ng/mL of LPS (purchased from Sigma) was treated and cultured for 24 hours in a 37°C, 5% CO ₂ incubator to induce activation of RAW 264.7 cells.

Culture supernatant of RAW 264.7 cells after culture was collected, and the production amount of IL-6, IFN-β, and IL-27 in the culture supernatant was measured in mice for LEGENDplex ^TM bead-based immnnoassay. Anti-inflammatory effects were confirmed by measuring using a mouse inflammation panel (purchased from Biolegend) and NovoCyte Flow Cytometer (purchased from AceA).

6A to 6C, as a result of inflammatory cytokine analysis, the experimental group (5) in which EDM exosomes were previously treated with RAW 264.7 cells before LPS treatment on RAW 264.7 cells (5) was IL-6 induced by LPS, The production of IFN-β and IL-27 was significantly reduced. In particular, the experimental group (4) in which the SFM exosomes were previously treated on the RAW 264.7 cells before LPS treatment on the RAW 264.7 cells (4) did not decrease the production of IL-6 and IFN-β, or compared to the EDM exosome-treated experimental group (5). It was confirmed that the degree of decrease in the amount of IL-27 produced was low.

From these results, it was found that EDM exosomes produced using EDM medium in the culture and culture medium recovery process for exosome production according to the present invention are superior to SFM exosomes in terms of enhancing the anti-inflammatory, wound treatment, or wound treatment promoting efficacy. Able to know. In other words, EDM exosomes have better anti-inflammatory, wound treatment or wound treatment promotion efficacy than SFM exosomes, and can economically and efficiently produce exosomes with enhanced anti-inflammatory, wound treatment or wound treatment promotion efficacy with high yield. And/or clinically useful.

Therefore, EDM exosomes with enhanced anti-inflammatory, wound treatment or wound treatment promoting efficacy of the present invention are useful as an active ingredient of anti-inflammatory, wound treatment or wound treatment promotion pharmaceutical composition, quasi-drug, skin external preparation, or functional cosmetic composition. Can be utilized.

Example 7: Evaluation of the efficacy of increasing collagen synthesis of exosomes

Exosomes produced using SFM medium (hereinafter referred to as "SFM exosomes") and exosomes produced using EDM medium (hereinafter referred to as "SFM exosomes") in the culture and culture medium recovery process for exosome production (see FIG. 3) EDM exosomes") was confirmed as follows. However, both SFM exosomes and EDM exosomes were isolated according to Example 4. To this end, the experimental groups were classified as follows:

(1) Negative control: an experimental group in which human fibroblasts were not treated with exosomes (indicated as "Negative control" in FIG. 7);

(2) Positive control: an experimental group in which human fibroblasts were treated with 10% growth supplement (purchased from Cell Bio, Seoul, Korea) (indicated as "positive control" in FIG. 7);

(3) SFM exosome treatment group: an experimental group in which human fibroblasts were treated with SFM exosomes at a concentration of 5×10 ⁹ particles/mL (indicated as “SFM” in FIG. 7);

(4) EDM exosome treatment group: an experimental group in which EDM exosomes were treated at a concentration of 5×10 ⁹ particles/mL in human fibroblasts (indicated as “EDM” in FIG. 7).

The effect of increasing collagen synthesis of SFM exosomes or EDM exosomes in human dermal fibroblast (HDF) cells was confirmed as follows.

Human fibroblasts were suspended in HDF growth medium supplemented with 1% penicillin-streptomycin (purchased from Cell Bio Co., Ltd., Seoul, Korea), inoculated into a well plate, and cultured in a 37°C, 5% CO ₂ incubator for 24 hours I did.

Thereafter, human fibroblasts were treated according to the conditions of the experimental groups (1) to (4), and human fibroblasts were cultured in an incubator at 37° C. and 5% CO ₂ for 72 hours. After 72 hours, the culture supernatant of human fibroblasts was collected, centrifuged, and then the centrifuged culture solution was prepared. Using the EIA kit (purchased from Takara) for procollagen type I C-peptide (PIP), the amount of collagen synthesized from human skin fibroblasts and accumulated in the culture medium was measured according to the manufacturer's recommended method. The measured collagen synthesis amount was normalized by dividing by the number of cells of the final human fibroblasts measured with an MTT assay kit (purchased from Sigma).

As shown in FIG. 7, in the case of the experimental group (4) in which EDM exosomes were treated with human fibroblasts, collagen synthesis was significantly increased compared to the experimental group (3) in which SFM exosomes were treated with human fibroblasts.

From these results, it can be seen that the EDM exosomes produced using the EDM medium in the process of culturing and recovering the culture medium for the production of exosomes according to the present invention are very excellent in terms of the efficacy of increasing collagen synthesis. Therefore, EDM exosomes are more effective in enhancing physiological activity related to increased collagen synthesis than SFM exosomes, for example, skin elasticity improvement, wrinkle improvement and/or anti-aging effect, and skin elasticity improvement, wrinkle improvement and/or anti-aging effect. These more enriched exosomes can be economically and efficiently produced with high yield, and are therefore commercially and/or clinically useful.

In the above, the present invention has been described with reference to the above embodiments, but the present invention is not limited thereto. Those skilled in the art will be able to make modifications and changes without departing from the spirit and scope of the present invention, and it will be appreciated that such modifications and changes also belong to the present invention.

Claims (35)

delete delete delete (a) culturing the animal-derived cells in a growth medium to proliferate the cells,
(b) the animal-derived cells were cultured in a serum-free medium to which platelet lysate was added, and a conditioned media containing exosomes secreted or released from the animal-derived cells was collected and then collected. A method for enhancing physiological activity of exosomes, comprising the step of separating exosomes from the culture medium. The method of claim 4,
A method for enhancing physiological activity of exosomes, characterized in that platelet lysate is not added to the growth medium in step (a). The method of claim 5,
In the step (b), the culture is performed for 24 to 72 hours, a method for enhancing physiological activity of exosomes. The method according to any one of claims 4 to 6,
The strengthening of the physiological activity is anti-inflammatory, wound treatment or wound treatment promoting effect strengthening, method of enhancing the physiological activity of exosomes. The method according to any one of claims 4 to 6,
The physiological activity enhancement is a method of enhancing the physiological activity of exosomes, which is enhancing the effect of increasing collagen synthesis. delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete

Images