KR102368442B1 - Manufacturing method of exosome for enhancing its bioactivity and application thereof - Google Patents

Abstract

The present invention provides a method for enhancing the physiological activity of exosomes, comprising a step of culturing animal-derived cells in a medium containing ascorbic acid, an analog or a derivative thereof. The method for producing exosomes with enhanced physiological activity or the method for enhancing the physiological activity of exosomes of the present invention can produce exosomes with enhanced physiological activity commercially and/or clinically useful, and exosomes with enhanced physiological activity produced in this way can be usefully utilized as an active ingredient in a pharmaceutical composition or a cosmetic composition.

Description

Method of producing exosomes with enhanced physiological activity and application thereof {Manufacturing method of exosome for enhancing its bioactivity and application thereof}

The present invention relates to a method for producing exosomes, and to a method for producing exosomes with enhanced physiological activity.

In addition, the present invention relates to a method for enhancing the physiological activity of exosomes, and a medium for the same.

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

Recently, it has been reported that cell secretome contains various bioactive factors that control cell behavior. In particular, cell secretion contains 'exosome' or Because it contains 'extracellular vesicle', research on its components and functions is being actively conducted.

Cells release various membrane types of ERs to the extracellular environment, and these ERs are commonly referred to as extracellular vesicles (EVs). The extracellular vesicles are also called cell membrane-derived ERs, ectosomes, shedding vesicles, microparticles, exosomes, and the like, and in some cases, they are used separately from exosomes.

Exosomes are endoplasmic reticulum with a size of several tens to hundreds of nanometers made of a double phospholipid membrane identical to the structure of the cell membrane, and contain proteins, nucleic acids (mRNA, miRNA, etc.) called exosome cargo inside. Exosome cargo includes a wide range of signaling factors, and these signaling factors are known to be cell type-specific and differentially regulated according to the environment of secretory cells. Exosomes are intercellular signaling mediators secreted by cells, and various cellular signals transmitted through them regulate cell behavior, including activation, growth, migration, differentiation, dedifferentiation, apoptosis, and necrosis of target cells. is known Exosomes contain specific genetic material and bioactive factors according to the nature and condition of the cell from which they are derived. In the case of proliferating stem cell-derived exosomes, it regulates cell behavior such as cell migration, proliferation and differentiation, and reflects the characteristics of stem cells related to tissue regeneration (Nature Review Immunology 2002 (2) 569-579).

That is, exosomes called avatars of cells contain bioactive factors such as growth factors similar to cells, and serve as a carrier for transporting bioactive factors between cells, that is, communication between cells. Exosomes are known not only to be released 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 exosomes include ultracentrifugation, density gradient centrifugation, ultrafiltration, tangential flow filtration (TFF), size exclusion chromatography (size exclusion chromatography, ion exchange chromatography, immunoaffinity capture, microfluidics-based isolation, exosome precipitation, total exosome isolation kit), or polymer-based precipitation.

However, as described above, although various exosome separation methods have been proposed, continuous improvement can be made in the technical field related to the production of exosomes as in other technical fields. For example, it is necessary to provide a novel exosome production technology that can improve the efficacy related to the physiologically active factors of the exosome.

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

Republic of Korea Patent Publication No. 10-1877793 (2018.07.13)

It is an object of the present invention to provide a method for producing exosomes with enhanced physiological activity.

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

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

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

However, the problems of the present invention as described above are 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 from the following detailed description of the invention, claims and drawings.

The present inventors have been repeating intensive studies on a method for producing exosomes with enhanced physiological activity, and when cells are cultured in a medium containing ascorbic acid, an analog or derivative thereof, the physiological activity of the exosomes isolated from the culture medium is improved It was confirmed that the present invention was completed.

As used herein, the term "extracellular vesicles (EVs)" generally encompasses membrane vesicles, ectosomes, shedding vesicles, microparticles, or equivalents thereof. used to mean Depending on the isolation environment, conditions and methods, etc., the extracellular vesicles may have the same meaning as exosomes, and may have the same or similar size as exosomes, but may have meanings including nanovesicles that do not have the composition of exosomes. On the other hand, as used herein, the term exosome is used to encompass the extracellular vesicles.

As used herein, the term "exosomes" refers to endoplasmic reticulum with a size of several tens to hundreds of nanometers (preferably about 30 to 200 nm) consisting of a double phospholipid membrane identical to the structure of the cell membrane (provided that the separation target is The particle size of exosomes may vary depending on the type of cell to be used, 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, nucleic acids (mRNA, miRNA, etc.) called exosome cargo. Exosome cargo includes a wide range of signaling factors, and these signaling factors are known to be cell type-specific and differentially regulated according to the environment of secretory cells. Exosomes are intercellular signaling mediators secreted by cells, and various cellular signals transmitted through them regulate cell behavior, including activation, growth, migration, differentiation, dedifferentiation, apoptosis, and necrosis of target cells. is known

On the other hand, the term "exosome" used herein has a nano-sized vesicle structure that is secreted from animal-derived cells and released into the extracellular space and has a composition similar to that of exosomes (eg, exosomes). -Similar vesicles) are included.

In the present invention, the type of animal-derived cells from which the exosomes are derived is not limited, but may be stem cells or immune cells as an example not limiting the present invention. The stem cells may be embryonic stem cells, induced pluripotent stem cells (iPSCs), adult stem cells, embryonic stem cell-derived mesenchymal stem cells, or induced pluripotent stem cell-derived mesenchymal 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 one or more types of adult stem cells selected from the group consisting of mesenchymal stem cells, human tissue-derived mesenchymal stromal cells, human tissue-derived mesenchymal stem cells, and pluripotent stem cells. It may be a stem cell. The mesenchymal stem cells may be mesenchymal stem cells derived from one or more tissues selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, Wharton's 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 may be adipose-derived stem cells. 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 immune rejection, but preferably a human stem cell or human-derived immune cell.

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

As used herein, the term "ascorbic acid, an analog or derivative thereof" is, for example, ascorbic acid (Ascorbic Acid), ascorbic acid-2-glucoside (Ascorbic acid-2-glucoside; AA-2G), ascorbic acid-2 -Sulphate (Ascorbic acid-2-sulfate; AA-2S), ascorbic acid-2-phosphate (AA-2P), magnesium ascorbyl phosphate (Magnesium ascorbyl phosphate), ascorbyl palmitate (Ascorbyl palmitate), Retinyl Ascorbate (Retinyl Ascorbate), Tetrahexyldecyl Ascorbate (Tetrahexyldecyl Ascorbate), Sodium ascorbate (Sodium ascorbate), Calcium ascorbate (Calcium ascorbate), Polyethoxylated ascorbate (Polyethoxylated ascorbate) acid), Ethyl Ascorbic Acid, Aminopropyl Ascorbyl Phosphate, and/or 3-Ascorbyl Carbonyl Dipeptide-17. . However, the above-described ascorbic acid, analogs or derivatives thereof should be understood as examples of ascorbic acid, analogs or derivatives thereof that can be used in the present invention, and the present invention is not limited thereto.

As used herein, the term "pretreatment" refers to culturing animal-derived cells in a medium containing ascorbic acid, an analog or derivative thereof, and "pretreatment exosome or ascorbic acid-treated exosome" refers to ascorbic acid, an analog or derivative thereof. It refers to an exosome obtained by culturing animal-derived cells in a containing medium. "Untreated" means culturing animal-derived cells in a medium not containing ascorbic acid, an analog or derivative thereof, and "untreated exosomes, untreated control exosomes, or untreated control" refers to the cell culture step and exo It refers to an exosome obtained without treatment with ascorbic acid, an analog or a derivative thereof, in any step of the separation step.

As used herein, the term "anti-inflammatory" means preventing, suppressing, alleviating, ameliorating or treating inflammation, and inflammatory diseases are examples that are not limited to the present invention, and include dermatitis, atopic dermatitis, eczema, bacterial infection, virus Inflammation due to infection or fungal infection, burns, inflammation due to burns, wounds, inflammation due to wounds, ulcerative colitis, arthritis, rheumatoid arthritis, hepatitis, nephritis, and the like.

As used herein, the term “wound” refers to a state in which a living body is damaged, and a tissue constituting the internal or external surface of the living body, for example, skin, muscle, nerve tissue, bone, soft tissue, internal organ or vascular tissue It encompasses segmented or disrupted pathological conditions. As an example not limiting the present invention, the wound is abrasion, laceration, cut, cut, avulsion, pressure sore, pit wound, tissue destruction by radiation, penetrated wound, gunshot wound ( gun shot wound), burns, frostbite, surgical wounds, sutures after plastic surgery, chemical wounds, etc., and may include damage to any part of an object.

As used herein, the term "iontophoresis" refers to a method of allowing an ionized active ingredient to penetrate the skin with an electrical repulsive force by flowing a microcurrent to the skin to which an active material is applied, giving a potential difference, and changing the electrical environment of the skin 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, and a battery is installed in the electrode patch itself to protect the skin. A method in which microcurrent is introduced, a method in which microcurrent is introduced into the skin through a patch equipped with a reversed electrodialysis means that generates an electric current through a difference in ion concentration between a high-concentration electrolyte solution and a low-concentration electrolyte solution, etc. can However, the present invention is not limited thereto, and it goes without saying that various types of iontophoresis may be used.

The present invention provides a method for enhancing the physiological activity of exosomes, comprising the step of culturing animal-derived cells in a medium containing ascorbic acid, an analog or derivative thereof.

The method for enhancing the physiological activity of an exosome of one embodiment of the present invention may include further culturing the animal-derived cells in a serum-free medium containing ascorbic acid, an analog or a derivative thereof.

In the method of enhancing the physiological activity of exosomes of one embodiment of the present invention, the concentration of ascorbic acid, an analog or derivative thereof is about 1 μg/mL to 300 μg/mL, preferably about 10 μg/mL to 100 μg/mL mL.

In the method of enhancing the physiological activity of the exosome of one embodiment of the present invention, the enhancement of the physiological activity may be anti-inflammatory, wound treatment or wound treatment promoting efficacy enhancement. In addition, the enhancement of the physiological activity may be enhancement of the effect of increasing collagen synthesis, for example, may be at least one of skin elasticity enhancement enhancement, wrinkle enhancement enhancement enhancement, or skin regeneration enhancement enhancement.

The present invention provides a medium for enhancing the physiological activity of exosomes derived from animal cells, comprising ascorbic acid, an analog or derivative thereof. The enhancement of the physiological activity may be anti-inflammatory, wound treatment or wound treatment promoting efficacy enhancement. In addition, the enhancement of the physiological activity may be enhancement of the effect of increasing collagen synthesis, for example, may be at least one of skin elasticity enhancement enhancement, wrinkle enhancement enhancement enhancement, or skin regeneration enhancement enhancement.

In the medium for enhancing the physiological activity of animal cell-derived exosomes of one embodiment of the present invention, the concentration of the ascorbic acid, its analog or derivative is about 1 μg/mL to 300 μg/mL, preferably about 10 μg/ mL to 100 μg/m.

In addition, the present invention provides a pharmaceutical composition comprising exosomes with enhanced physiological activity obtained by culturing animal-derived cells in a medium containing ascorbic acid, an analog or derivative thereof. For example, the exosomes with enhanced physiological activity may be exosomes with enhanced anti-inflammatory, wound treatment or wound treatment promoting efficacy, and the pharmaceutical composition includes the exosomes with enhanced physiological activity as an active ingredient, anti-inflammatory , it may be a pharmaceutical composition for treating wounds or promoting wound healing.

For example, the pharmaceutical composition of one embodiment of the present invention comprises the steps of (a) culturing animal-derived cells in a medium containing ascorbic acid, an analog or derivative thereof, and (b) secreted or released from the animal-derived cells. It includes exosomes with enhanced anti-inflammatory, wound treatment or wound healing promoting efficacy, obtained by performing the step of separating the exosomes from the collected culture medium after collecting the culture medium containing the exosomes (conditioned media).

In the pharmaceutical composition of one embodiment of the present invention, the ascorbic acid, an analog or derivative thereof, is present in a concentration of about 1 μg/mL to 300 μg/mL in the medium, preferably about 10 μg/mL to 100 μg/m. concentration may be included.

By way of example and not limitation of the present invention, the pharmaceutical composition of one embodiment of the present invention may be administered or treated by injection, microneedling, iontophoresis, application, or a combination thereof. For example, the pharmaceutical composition may be in the form of injections, injections, sprays, liquids, or patches.

The pharmaceutical composition of one embodiment of the present invention 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, acacia gum, alginate, gelatin, calcium phosphate, calcium carbonate, calcium silicate, cellulose , methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like, 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 suitable 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 mentioned However, it is not limited thereto and does not exclude various administration methods known in the art. In addition, the pharmaceutical composition of one embodiment of the present invention may be administered by any device capable of transporting an active substance to a target tissue or cell. In addition, the effective amount of the pharmaceutical composition of one embodiment of the present invention means an amount required for administration in order to expect anti-inflammatory, wound healing, and/or wound healing promoting effects.

The formulation for parenteral administration of the pharmaceutical composition of one embodiment of the present invention may be a sterile aqueous solution, a non-aqueous solution, a suspension, an emulsion, a lyophilized formulation, 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. The injection of one embodiment of the present invention is distilled water for injection, vegetable oil (eg, peanut oil, sesame oil, camellia oil, etc.), monoglyceride, diglyceride, propylene glycol, camphor, benzoate estradiol, bismuth hyposalicylate, depending on the type , Arsenobenzol sodium, or at least one of streptomycin sulfate, and may optionally include a stabilizer or a preservative.

The blending ratio of the pharmaceutical composition of one embodiment of the present invention can be appropriately selected according to the type, amount, form, etc. of the additional ingredients as described above. For example, based on 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, a suitable dosage of the pharmaceutical composition of one embodiment of the present invention may be adjusted depending on the severity of the disease, the type of formulation, the formulation method, the patient's age, sex, weight, health status, diet, excretion rate, administration time and administration method. can For example, when administering the pharmaceutical composition of one embodiment of the present invention to an adult, it may be administered at a dose of 0.001 mg/kg to 100 mg/kg per day, divided into 1 to several times.

In addition, the present invention provides a cosmetic composition comprising exosomes with enhanced collagen synthesis increasing efficacy obtained by culturing animal-derived cells in a medium containing ascorbic acid, an analog or derivative thereof. The cosmetic composition may be, for example, a cosmetic composition for skin elasticity improvement, wrinkle improvement and/or skin regeneration.

For example, the cosmetic composition of one embodiment of the present invention comprises the steps of (a) culturing animal-derived cells in a medium containing ascorbic acid, an analog or derivative thereof, and (b) secreted or released from the animal-derived cells. After collecting the culture medium containing the exosomes (conditioned media), it includes exosomes with enhanced collagen synthesis effect obtained by performing the step of separating the exosomes from the collected culture medium.

In the cosmetic composition of one embodiment of the present invention, the ascorbic acid, an analog or derivative thereof, is present in a concentration of about 1 μg/mL to 300 μg/mL in the medium, preferably about 10 μg/mL to 100 μg/m concentration may be included.

For example, the cosmetic composition may include shampoo, soap, rinse, surfactant-containing cleansing, cream, lotion, ointment, tonic, treatment, conditioner, suspension, emulsion, paste, gel, oil, wax, spray, aerosol, It may be a mist or powder, and preferably a lotion or cream.

On the other hand, the cosmetic composition of one embodiment of the present invention is a component commonly used in a cosmetic composition within a range that does not impair the effects of the present invention, for example, a moisturizer, an antioxidant, an oily component, a UV absorber, an emulsifier, a surfactant, a thickener , alcohols, powder components, color materials, water-based components, water, and various skin nutrients can be appropriately blended as needed.

In addition, the cosmetic composition of one embodiment of the present invention, in addition to exosomes with enhanced physiological activity, conventionally used skin as long as it does not impair its action (eg, skin elasticity improvement, wrinkle improvement and/or skin regeneration, etc.) An elasticity improving agent, an anti-wrinkle agent, and/or a moisturizing agent may be mixed and used together. For example, the cosmetic composition comprising the exosome with enhanced physiological activity of the present invention as an active ingredient is at least one of hydrogel, hyaluronic acid, hyaluronic acid salt (eg sodium hyaluronate, etc.), or hyaluronic acid gel may be supported or mixed. In the cosmetic composition of one embodiment of the present invention, the type of the hydrogel is not limited, but may preferably be 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. 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.

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

The cosmetic composition of one embodiment of the present invention may be used for the purpose of skin elasticity improvement, wrinkle improvement and/or skin regeneration, and the cosmetic formulation may be prepared in any formulation conventionally prepared in the art. For example, patch, mask pack, mask sheet, flexible lotion, nourishing 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 wash, hair dye, hair tonic, etc., but is not limited thereto.

The cosmetic composition of one embodiment of the present invention includes components commonly used in cosmetic compositions, for example, it may include conventional adjuvants and carriers such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances. In addition, in each formulation for the cosmetic composition, other components can be appropriately selected and formulated by those skilled in the art without difficulty depending on the type or purpose of use of the cosmetic composition.

The present invention exhibits an excellent effect capable of enhancing the anti-inflammatory, wound healing or wound healing promoting efficacy of exosomes or physiological activities such as collagen synthesis. Therefore, the method for producing exosomes with enhanced physiological activity or the method for enhancing the physiological activity of exosomes of the present invention can produce exosomes with enhanced physiological activity commercially and/or clinically useful, and thus produced physiological activity This reinforced exosome can be usefully utilized as an active ingredient in a pharmaceutical composition or cosmetic composition.

On the other hand, the scope of the present invention is not limited by the above-described effects.

1A is a graph showing the particle size distribution and number of particles obtained by performing NTA (nanoparticle tracking analysis) on exosomes obtained after pretreatment and culturing of stem cells in a medium containing ascorbic acid, an analog or derivative thereof.
1B is a graph showing the particle size distribution and the number of particles obtained by performing NTA (nanoparticle tracking analysis) on exosomes obtained after culturing stem cells in a medium not containing ascorbic acid, analogs or derivatives thereof.
2 is anti-inflammatory of the exosome obtained after pretreatment and culturing of stem cells in a medium containing ascorbic acid, an analog or derivative thereof according to an embodiment of the present invention (hereinafter, referred to as "ascorbic acid-treated exosome") It is a comparative graph showing that the efficacy is significantly increased compared to the exosomes obtained after culturing the stem cells in a medium not containing ascorbic acid, analogs or derivatives thereof (hereinafter referred to as "untreated control exosomes").
3 is a comparative graph showing that the collagen synthesis promoting effect of the ascorbic acid-treated exosome of the present invention is significantly increased compared to that of the untreated control exosome.
4 is a comparative graph showing that the cell proliferation promoting efficacy of the ascorbic acid-treated exosomes of the present invention significantly increased compared to the untreated control exosomes.

Hereinafter, the present invention will be described in more detail in the following examples. However, the following examples are merely illustrative of the content of the present invention and do not limit or limit the scope 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. The references cited herein 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: Cultivation of cells

Human adipose-derived stem cells were suspended in DMEM culture medium containing 10% fetal bovine serum (FBS), 100 Units/mL penicillin, and 100 μg/mL streptomycin, and then inoculated into a flask. 37° C., 5% CO ₂ Incubated in an incubator. In the case of the ascorbic acid-treated group, 30 μg/mL of ascorbic acid was added to the culture medium.

When both the ascorbic acid-treated and untreated groups reached a cell confluency of 80% or more, the cells were washed with phosphate-buffered saline (PBS, purchased from Thermo Scientific), and the culture medium was cultured at 100 units/mL. DMEM medium containing penicillin and 100 μg/mL streptomycin (hereinafter referred to as “serum-free media” or “SFM”) was replaced, and in the case of the ascorbic acid treatment group, 30 μg/mL ascorbic acid was added to the medium. did

Then, the cells were cultured for 24 hours to 72 hours, and the supernatant (hereinafter, culture solution) was collected, and the cell count was measured using a cell counter for the collected cells.

On the other hand, RAW 264.7, a mouse macrophage cell line, was purchased from the Korea Cell Line Bank and cultured. For cell culture, 5% CO in DMEM (purchased from Thermo Scientific) medium containing 10% fetal bovine serum (purchased from Thermo Scientific) and 1% antibiotics-antimycotics (purchased from Thermo Scientific). ₂ , subcultured at 37 ℃ condition. In addition, human dermal fibroblasts and a medium for culturing them were purchased from Cell Bio Co., Ltd. (Seoul, Korea) and subcultured at 5% CO ₂ , 37°C conditions.

Example 2: Isolation and purification of exosomes

The culture solution recovered in Example 1 was filtered with a 0.22 μm filter to remove impurities such as cell debris, wastes, and large particles. From the filtered culture medium, exosomes were separated through a separation process using tangential flow filtration (TFF). 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 with various molecular weight cutoffs (MWCOs). Exosomes were selectively separated and concentrated by the selected MWCO, and particles smaller than the MWCO, proteins, lipids, nucleic acids, and low molecular weight compounds were removed.

In order to separate and concentrate the 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. While the culture medium was concentrated to a volume of about 1/100 to 1/10 using the TFF method, substances smaller than MWCO were removed to separate the exosomes.

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

On the other hand, as a method of isolating the exosomes from an animal-derived cell culture medium including stem cells, various methods known in the art may be used in addition to the separation method as described above. For example, for the separation of exosomes, ultracentrifugation, ultrafiltration, 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 ( A known separation method such as polymer based precipitation) may be used. However, the separation method of the exosomes is not limited to the methods described above, and of course, various separation methods that are used in the art or that can be used in the future are acceptable.

Example 3: Characterization of isolated exosomes and evaluation of productivity

The exosomes isolated in Example 2 were measured for particle size and concentration by nanoparticle tracking analysis (　NTA; purchased from Malvern). The results of NTA analysis of the exosomes isolated according to the separation method of one embodiment of the present invention are shown in FIGS. 1A and 1B. 1A and 1B, the exosomes obtained after pre-treatment and culturing of stem cells in a medium containing ascorbic acid according to an embodiment of the present invention (hereinafter referred to as "ascorbic acid-treated exosomes") 1A) It can be seen that the particle size distribution is uniform compared to the exosomes obtained after culturing the stem cells in a medium not containing ascorbic acid (hereinafter referred to as “untreated control exosomes”; FIG. 1B). there was.

Therefore, the present invention can produce exosomes having a uniform particle size distribution by pre-treating and culturing stem cells in a medium containing ascorbic acid.

Example 4: Evaluation of anti-inflammatory efficacy of exosomes

RAW 264.7 cells were suspended in DMEM medium containing 10% FBS and aliquoted to each well of a multiwell plate to have a confluency of 80 to 90%. The next day, exosomes (each of ascorbic acid-treated exosomes and untreated control exosomes) diluted in fresh medium containing LPS were treated and cultured at an appropriate concentration for 24 hours. At this time, in consideration of the increase in the productivity of the exosomes by ascorbic acid, the ascorbic acid-treated exosomes and the untreated control exosomes were adjusted to the same concentration (1.5×10 ⁹ particles/mL) and treated with RAW 264.7 cells.

After the culture was completed, the culture supernatant was taken and the inflammatory response was confirmed by measuring the inflammatory cytokines present in the culture supernatant. The amount of inflammatory cytokines in the culture supernatant was measured using an IL-6 ELISA kit. ELISA kit (purchased from R&D system) According to the manufacturer's manual, the production amount of IL-6 (inflammatory cytokine) in the group treated only with LPS, and LPS treatment with ascorbic acid-treated exosomes and untreated control exosomes, respectively The production amount of IL-6 (inflammatory cytokine) in the treated group was confirmed.

As shown in FIG. 2, when RAW 264.7 cells, which are macrophages of mice, were treated with the ascorbic acid-treated exosomes of the present invention together with LPS, compared to the case of treatment with the untreated control exosomes with LPS, IL-6 ( production of inflammatory cytokines) was significantly inhibited.

From these results, ascorbic acid-treated exosomes obtained by pre-treating and culturing stem cells in a medium containing ascorbic acid, analogs or derivatives thereof according to the present invention are anti-inflammatory, wound treatment, or non-toxic in terms of enhancing the efficacy of promoting wound treatment It can be seen that it is superior to the treatment control exosomes. Therefore, it can be seen that the method for producing exosomes with enhanced physiological activity or the method for enhancing the physiological activity of exosomes of the present invention is a technique that can be usefully utilized clinically or commercially.

In addition, the ascorbic acid-treated exosome with enhanced anti-inflammatory, wound treatment or wound treatment promoting efficacy of the present invention may be usefully utilized as an active ingredient in a pharmaceutical composition for anti-inflammatory, wound treatment or wound treatment promotion.

Example 5: Evaluation of the effect of increasing collagen synthesis of exosomes

Human skin fibroblasts (purchased from Cellbio Co., Ltd.) dispersed in DMEM medium containing fetal bovine serum were dispensed in a multi-well plate, cultured for 24 hours, and cultured for an additional 24 hours in a serum-free medium. Thereafter, each of the ascorbic acid-treated exosomes and the untreated control exosomes were diluted in serum-free medium at different concentrations, treated with human skin fibroblasts, and cultured for 24 hours at 37° C., 5% CO ₂ in an incubator. . At this time, in consideration of the increase in the productivity of the exosomes by ascorbic acid, the ascorbic acid-treated exosomes and the untreated control exosomes were adjusted to the same concentration and treated with human skin fibroblasts. In the case of treatment with a low concentration, each of the ascorbic acid-treated exosome and the untreated control exosome was treated with human skin fibroblasts at a concentration of 5×10 ⁸ particles/mL, and when treated with a high concentration, ascorbic acid-treated exo Som and untreated control exosomes were each treated with human skin fibroblasts at a concentration of 1.5×10 ⁹ particles/mL.

Then, the culture supernatant (culture supernatant) of human skin fibroblasts was collected and centrifuged to prepare a centrifuged culture solution. The amount of collagen synthesized from human skin fibroblasts and accumulated in the culture medium was measured using an EIA kit for procollagen type IC-peptide (PIP) (purchased from Takara). The total amount of protein in the cell lysate was measured with a BCA protein quantification kit (purchased from ThermoFisher Scientific). The measured amount of collagen was normalized by dividing by the total amount of protein to determine the relative amount of collagen.

As shown in FIG. 3 , as a result of measuring the amount of collagen after culturing for 24 hours, when the ascorbic acid-treated exosomes of the present invention were treated at low and high concentrations in human skin fibroblasts, both untreated control exosomes were treated with human skin fibers. Collagen synthesis was significantly increased compared to the case of treatment with blast cells.

From these results, ascorbic acid-treated exosomes obtained by pre-treating and culturing stem cells in a medium containing ascorbic acid, analogs or derivatives thereof according to the present invention are superior to untreated control exosomes in terms of collagen synthesis increasing efficacy. Able to know. Therefore, it can be seen that the method for producing exosomes with enhanced physiological activity or the method for enhancing the physiological activity of exosomes of the present invention is a technique that can be usefully utilized clinically or commercially.

In addition, the ascorbic acid-treated exosomes with enhanced collagen synthesis increasing efficacy of the present invention are superior to the untreated control exosomes in enhancing the physiological activity related to the increase in collagen synthesis, for example, improving skin elasticity, improving wrinkles and/or skin regeneration. And, it can be usefully used as an active ingredient in a cosmetic composition for skin elasticity improvement, wrinkle improvement and/or skin regeneration.

Example 6: Evaluation of the human skin fibroblast proliferation efficacy of exosomes

Human skin fibroblasts (purchased from Cellbio Co., Ltd.) dispersed in DMEM medium containing fetal bovine serum were dispensed in a multi-well plate, cultured for 24 hours, and cultured for an additional 24 hours in a serum-free medium. After that, each of the ascorbic acid-treated exosomes and the untreated control exosomes and Nuclight reagent (purchased from Sartorius) were diluted in serum-free medium and treated with human skin fibroblasts, and human skin fibroblasts were incubated at 37°C, 5% CO ₂ incubated for 72 hours. At this time, in consideration of the increase in the productivity of exosomes by ascorbic acid, human skin fibroblasts were treated by adjusting the ascorbic acid-treated exosomes and the untreated control exosomes to the same concentration (2×10 ⁹ particles/mL). Then, using Incucyte (purchased from Sartorius) according to the manufacturer's manual, cell proliferation was confirmed for 24 hours to 72 hours.

4, as a result of measuring the proliferative capacity of human skin fibroblasts after culturing for 72 hours, when the ascorbic acid-treated exosomes of the present invention were treated with human skin fibroblasts, the untreated control exosomes were applied to human skin fibroblasts. Compared to the treated case, the proliferation efficacy of human skin fibroblasts was significantly increased.

From these results, ascorbic acid-treated exosomes obtained by pre-treating and culturing stem cells in a medium containing ascorbic acid, analogs or derivatives thereof according to the present invention are non-treated control exosomes in terms of the proliferation promoting efficacy of human skin fibroblasts. It can be seen that the better That is, the ascorbic acid-treated exosome of the present invention enhances physiological activity related to human skin fibroblast proliferation promotion, for example, skin elasticity improvement, wrinkle improvement, skin regeneration, wound treatment and/or wound treatment promotion compared to the untreated control exosome. Efficacy is excellent. Therefore, it can be seen that the method for producing exosomes with enhanced physiological activity or the method for enhancing the physiological activity of exosomes of the present invention is a technique that can be usefully utilized clinically or commercially.

In addition, the ascorbic acid-treated exosome of the present invention can be usefully used as an active ingredient in a cosmetic composition for skin elasticity improvement, wrinkle improvement and/or skin regeneration, and a pharmaceutical composition for wound treatment or promoting wound treatment.

In the above, the present invention has been described with reference to the above examples, but the present invention is not limited thereto. Those skilled in the art can 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 (23)

A method for enhancing the efficacy of anti-inflammatory, wound treatment, wound treatment promotion or collagen synthesis increase of exosomes, comprising the step of culturing animal-derived cells in a medium containing ascorbic acid. According to claim 1,
A method for enhancing the efficacy of anti-inflammatory, wound treatment, wound treatment promotion or collagen synthesis increase of exosomes, comprising the step of further culturing the animal-derived cells in a serum-free medium containing ascorbic acid. delete delete 3. The method of claim 1 or 2,
The concentration of the ascorbic acid is 1 μg / mL to 300 μg / mL, the exosome anti-inflammatory, wound treatment, wound treatment promotion or collagen synthesis enhancing efficacy enhancement method. 6. The method of claim 5,
The concentration of the ascorbic acid is 10 μg / mL to 100 μg / m, the exosome anti-inflammatory, wound treatment, wound treatment promotion or collagen synthesis enhancing efficacy enhancement method. A medium for enhancing the efficacy of anti-inflammatory, wound treatment, wound treatment promotion or collagen synthesis increase of animal cell-derived exosomes, comprising ascorbic acid. delete delete 8. The method of claim 7,
The concentration of the ascorbic acid is 1 μg / mL to 300 μg / mL, an animal cell-derived exosome anti-inflammatory, wound treatment, wound treatment promotion or collagen synthesis enhancement medium for enhancing efficacy. 11. The method of claim 10,
The concentration of the ascorbic acid is 10 μg / mL to 100 μg / m, the anti-inflammatory, wound treatment, wound treatment promotion of animal cell-derived exosomes, or a medium for enhancing the efficacy of collagen synthesis. A cosmetic composition comprising exosomes with enhanced collagen synthesis efficacy, obtained by culturing animal-derived cells in a medium containing ascorbic acid. 13. The method of claim 12,
(a) culturing the animal-derived cells in a medium containing ascorbic acid, (b) collecting the conditioned media containing the exosomes secreted or released from the animal-derived cells, and then collecting the exo A cosmetic composition comprising an exosome having enhanced collagen synthesis increasing efficacy, obtained by performing the step of isolating the small cell. 14. The method of claim 12 or 13,
The ascorbic acid is contained in the medium at a concentration of 1 μg / mL to 300 μg / mL, the cosmetic composition. 15. The method of claim 14,
The ascorbic acid is contained in the medium at a concentration of 10 μg / mL to 100 μg / m, the cosmetic composition. 14. The method of claim 12 or 13,
A cosmetic composition that enhances at least one of skin elasticity improvement effect, wrinkle improvement effect, or skin regeneration effect. 14. The method of claim 12 or 13,
Shampoo, soap, rinse, surfactant-containing cleansing, cream, lotion, ointment, tonic, treatment, conditioner, suspension, emulsion, paste, gel, oil, wax, spray, aerosol, mist, or powder, cosmetic composition . A pharmaceutical composition for promoting anti-inflammatory, wound treatment or wound treatment, comprising exosomes having enhanced anti-inflammatory, wound treatment or wound treatment promoting efficacy, obtained by culturing animal-derived cells in a medium containing ascorbic acid. 19. The method of claim 18,
(a) culturing the animal-derived cells in a medium containing ascorbic acid, (b) collecting the conditioned media containing the exosomes secreted or released from the animal-derived cells, and then collecting the exo A pharmaceutical composition for promoting anti-inflammatory, wound treatment or wound treatment, comprising exosomes with enhanced anti-inflammatory, wound treatment or wound treatment promoting efficacy, obtained by performing the step of isolating the small. 20. The method of claim 18 or 19,
The ascorbic acid is contained in the medium in a concentration of 1 μg / mL to 300 μg / mL, anti-inflammatory, wound treatment or a pharmaceutical composition for promoting wound treatment. 21. The method of claim 20,
The ascorbic acid is contained in the medium in a concentration of 10 μg / mL to 100 μg / m, anti-inflammatory, wound treatment or a pharmaceutical composition for promoting wound treatment. 20. The method of claim 18 or 19,
A pharmaceutical composition for anti-inflammatory, wound treatment or accelerating wound healing, administered or treated by injection, microneedling, iontophoresis, application, or a combination thereof. 20. The method of claim 18 or 19,
An injection, injection, spray, liquid or patch formulation, anti-inflammatory, wound treatment or a pharmaceutical composition for promoting wound treatment.

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