WO2022177104A1 - Procédé de production d'exosomes présentant une activité biologique améliorée, et application associée - Google Patents
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- WO2022177104A1 WO2022177104A1 PCT/KR2021/017394 KR2021017394W WO2022177104A1 WO 2022177104 A1 WO2022177104 A1 WO 2022177104A1 KR 2021017394 W KR2021017394 W KR 2021017394W WO 2022177104 A1 WO2022177104 A1 WO 2022177104A1
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- exosomes
- ascorbic acid
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- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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- A—HUMAN NECESSITIES
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
Definitions
- the present invention relates to a method for producing exosomes, and to a method for producing exosomes with enhanced physiological activity.
- the present invention relates to a method for enhancing the physiological activity of exosomes, and a medium for the same.
- the present invention relates to the application of the exosomes with enhanced physiological activity obtained by the production method as described above.
- cell secretome contains various bioactive factors that regulate cell behavior.
- cell secretion contains 'exosome' or Because it contains 'extracellular vesicle', research on its components and functions is being actively conducted.
- extracellular vesicles 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 the inside contains 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.
- Exosomes contain specific genetic material and bioactive factors according to the nature and state of the cell from which they are derived. In the case of proliferating stem cell-derived exosomes, it regulates cell behaviors 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).
- 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. .
- 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.
- 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.
- extracellular vesicles generally encompasses membrane vesicles, ectosomes, shedding vesicles, microparticles, or equivalents thereof. used in the sense that Depending on the isolation environment, conditions and methods, etc., the extracellular vesicle may have the same meaning as an exosome, and may have the same or similar size as the exosome, but may also have a meaning including nanovesicles that do not have the composition of the exosome.
- the term exosome used herein is used to encompass the extracellular vesicles.
- 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
- exosome 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 vesicles).
- 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.
- the adult stem cells include one or more 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.
- 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, 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.
- adipose 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.
- ascorbic acid an analog or derivative thereof
- ascorbic acid ascorbic Acid
- ascorbic acid-2-glucoside Ascorbic acid-2-glucoside
- ascorbic acid-2 -Sulphate Ascorbic acid-2-sulfate
- AA-2S ascorbic acid-2-phosphate
- magnesium ascorbyl phosphate Magnnesium 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 (Polyethoxylated ascorbate) acid)
- Ethyl Ascorbic Acid Aminopropyl Ascorbate
- pretreatment refers to culturing animal-derived cells in a medium containing ascorbic acid, an analog or derivative thereof
- 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 that does not contain ascorbic acid, an analog or derivative thereof
- untreated exosomes, untreated control exosomes or untreated control refers to the cell culture step and exo It refers to exosomes obtained without treatment with ascorbic acid, an analog or a derivative thereof, in any step of the isolation step.
- anti-inflammatory means preventing, inhibiting, alleviating, ameliorating or treating inflammation
- 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.
- 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.
- 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.
- Iontophoresis refers to a method of allowing an ionized active ingredient to permeate the skin with an electrical repulsive force by flowing a microcurrent to the skin to which an active material is applied to change the electrical environment of the skin by giving a potential difference 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 to introduce a microcurrent to the skin, and a battery is mounted on the electrode patch itself to give 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
- 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 culturing an animal-derived cell 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.
- the concentration of the ascorbic acid, an analog or derivative thereof is about 1 ⁇ g/mL to 300 ⁇ g/mL, preferably about 10 ⁇ g/mL to 100 ⁇ g/mL mL.
- the enhancement of the physiological activity may be anti-inflammatory, wound treatment or wound treatment promoting efficacy enhancement.
- 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 improvement 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.
- 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 improvement enhancement enhancement, or skin regeneration enhancement enhancement.
- the concentration of the ascorbic acid, an analog or derivative thereof is about 1 ⁇ g/mL to 300 ⁇ g/mL, preferably about 10 ⁇ g/mL mL to 100 ⁇ g/mL.
- the present invention provides a pharmaceutical composition
- 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.
- the exosomes with enhanced physiological activity may be exosomes with enhanced anti-inflammatory, wound treatment or wound treatment promoting efficacy
- 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.
- 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 conditioned media containing the exosomes.
- the ascorbic acid, analog or derivative thereof is present in the medium at a concentration of about 1 ⁇ g/mL to 300 ⁇ g/mL, preferably about 10 ⁇ g/mL to 100 ⁇ g/mL. concentration may be included.
- the pharmaceutical composition of one embodiment of the present invention may be administered or treated by injection, microneedling, iontophoresis, application, or a combination thereof.
- the pharmaceutical composition may be in the form of an injection, injection, spray, liquid or patch.
- 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 and mineral oil, 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.
- 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.
- 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.
- the pharmaceutical composition of one embodiment of the present invention can be administered by any device capable of transporting an active substance to a target tissue or cell.
- 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 also 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 (for example, peanut oil, sesame oil, camellia oil, etc.), monoglyceride, diglyceride, propylene glycol, camphor, benzoate estradiol, bismuth salicylate, 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.
- 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.
- 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 the pharmaceutical composition of one embodiment of the present invention is administered to an adult, it may be administered in one to several divided doses at a dose of 0.001 mg/kg to 100 mg/kg per day.
- the present invention provides a cosmetic composition
- 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.
- 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.
- the ascorbic acid, analog or derivative thereof is present in a concentration of about 1 ⁇ g/mL to 300 ⁇ g/mL in the medium, preferably about 10 ⁇ g/mL to 100 ⁇ g/mL. concentration may be included.
- 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.
- 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, colorants, water-based components, water, and various skin nutrients can be appropriately blended as needed.
- a moisturizer for example, a moisturizer, an antioxidant, an oily component, a UV absorber, an emulsifier, a surfactant, a thickener , alcohols, powder components, colorants, water-based components, water, and various skin nutrients can be appropriately blended as needed.
- 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.
- 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.
- 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.
- 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.
- 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.
- the cosmetic composition of one embodiment of the present invention includes components commonly used in the cosmetic composition, for example, it may include conventional adjuvants and carriers such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances.
- 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 treatment or wound treatment 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 commercially and/or clinically useful exosomes with enhanced physiological activity, and thus produced physiological activity This reinforced exosome can be usefully utilized as an active ingredient in a pharmaceutical composition or cosmetic composition.
- 1A is a graph showing the particle size distribution and the 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 pretreatment and culturing of stem cells in a medium not containing ascorbic acid, analogs or derivatives thereof. .
- Figure 2 is an anti-inflammatory of the exosomes 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 exosomes”) 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").
- FIG. 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.
- FIG. 4 is a comparative graph showing that the cell proliferation promoting effect of the ascorbic acid-treated exosomes of the present invention is significantly increased compared to the untreated control exosomes.
- Human adipose-derived stem cells were suspended in DMEM culture medium containing 10% fetal bovine serum (FBS), 100 Units/mL penicillin, and 100 ⁇ g/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 ⁇ g/mL of ascorbic acid was added to the culture medium.
- FBS fetal bovine serum
- penicillin 100 Units/mL
- streptomycin 100 ⁇ g/mL
- 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.
- PBS phosphate-buffered saline
- SFM serum-free media
- 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.
- culture solution hereinafter, culture solution
- RAW 264.7 a mouse macrophage cell line
- DMEM purchased from Thermo Scientific
- fetal bovine serum purchased from Thermo Scientific
- antibiotics-antimycotics purchased from Thermo Scientific
- 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.
- the culture solution recovered in Example 1 was filtered with a 0.22 ⁇ m filter to remove impurities such as cell debris, wastes, and large particles.
- the filtered culture solution was separated from the exosomes through a separation process using tangential flow filtration (TFF).
- TFF tangential flow filtration
- a cartridge filter aka hollow fiber filter; purchased from GE Healthcare or Spectrum Labs
- a cassette filter purchased from Pall or Sartorius or Merck Millipore
- TFF filters can be selected by various molecular weight cutoff (MWCO). 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.
- 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 ⁇ m 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.
- 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.
- the exosomes isolated in Example 2 were measured for particle size and concentration by nanoparticle tracking analysis ( NTA; purchased from Malvern).
- NTA nanoparticle tracking analysis
- the NTA analysis results of the exosomes isolated according to the separation method of one embodiment of the present invention are shown in FIGS. 1A and 1B.
- the exosomes obtained after pretreatment 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.
- 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.
- 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 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 in RAW 264.7 cells.
- 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
- the production amount of IL-6 (inflammatory cytokine) in the treated group was confirmed.
- ascorbic acid-treated exosomes obtained by pre-treating and culturing stem cells in a medium containing ascorbic acid, an analog or derivative thereof according to the present invention are anti-inflammatory, wound treatment or wound treatment promoting efficacy It can be seen that it is superior to the treatment control exosomes. Therefore, it can be seen that the method of producing exosomes with enhanced physiological activity or the method of enhancing the physiological activity of exosomes of the present invention is a technique that can be usefully utilized clinically or commercially.
- 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 of a pharmaceutical composition for anti-inflammatory, wound treatment or wound treatment promotion.
- Human skin fibroblasts purchased from Cell Bio Co., Ltd.
- 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 serum-free medium.
- 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. .
- the ascorbic acid-treated exosomes and the untreated control exosomes were adjusted to the same concentration and treated with human skin fibroblasts.
- each of the ascorbic acid-treated exosome and the untreated control exosome was treated in human skin fibroblasts at a concentration of 5 ⁇ 10 8 particles/mL, and when treated at a high concentration, ascorbic acid-treated exo
- Each of the exosomes and untreated control exosomes was treated with human skin fibroblasts at a concentration of 1.5 ⁇ 10 9 particles/mL.
- the 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 protein amount in the cell lysate was measured with a BCA protein quantification kit (purchased from ThermoFisher Scientific). The measured collagen amount was divided by the total protein amount and normalized to determine the relative collagen amount.
- ascorbic acid-treated exosomes obtained by pre-treating and culturing stem cells in a medium containing ascorbic acid, an analog or derivative thereof according to the present invention are superior to the untreated control exosomes in terms of collagen synthesis increasing efficacy.
- the method of producing exosomes with enhanced physiological activity or the method of enhancing the physiological activity of exosomes of the present invention is a technique that can be usefully utilized clinically or commercially.
- 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 human skin fibroblast proliferation efficacy of exosomes
- Human skin fibroblasts (purchased from Cell Bio 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 serum-free medium. Thereafter, each of the ascorbic acid-treated exosomes and the untreated control exosomes and Nuclight reagent (purchased from Sartorius) were diluted in serum-free medium, treated with human skin fibroblasts, and human skin fibroblasts were incubated at 37°C, 5% CO 2 incubated for 72 hours.
- 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.
- the ascorbic acid-treated exosome of the present invention enhances physiological activity related to promotion of human skin fibroblast proliferation, for example, skin elasticity improvement, wrinkle improvement, skin regeneration, wound treatment and/or wound treatment promotion, than the untreated control exosome. Efficacy is excellent. Therefore, it can be seen that the method of producing exosomes with enhanced physiological activity or the method of enhancing the physiological activity of exosomes of the present invention is a technique that can be usefully utilized clinically or commercially.
- 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 wound treatment promotion.
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Abstract
La présente invention concerne un procédé pour améliorer l'activité biologique des exosomes, comprenant une étape de culture de cellules d'origine animale dans un milieu contenant de l'acide ascorbique, ou un analogue ou un dérivé de ce dernier. Le procédé de production d'exosomes présentant une activité biologique améliorée ou le procédé d'amélioration de l'activité biologique des exosomes, de la présente invention, permet la production d'exosomes commercialement et/ou cliniquement utiles présentant une activité biologique améliorée, et les exosomes présentant une activité biologique améliorée, ainsi produits, peuvent être efficacement utilisés comme un principe actif d'une composition pharmaceutique ou d'une composition cosmétique.
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| KR101877793B1 (ko) | 2016-07-15 | 2018-07-13 | 주식회사 엔바이오텍 | 줄기세포 배양용 무혈청 배지 조성물 및 이를 이용한 줄기세포의 배양 방법 |
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| JP2020528085A (ja) * | 2017-07-24 | 2020-09-17 | 株式会社エキソステムテックＥｘｏｓｔｅｍｔｅｃｈ Ｃｏ．，Ｌｔｄ． | 幹細胞から抽出されたエキソソームを有効成分として含む骨粗鬆症の予防または治療用の組成物 |
| KR20200113225A (ko) * | 2018-01-23 | 2020-10-06 | 노바셀라 가부시키가이샤 | 피부 질환 치료용 조성물 |
| KR102159915B1 (ko) * | 2019-09-18 | 2020-09-24 | 주식회사 엑소코바이오 | 엑소좀 및/또는 세포외 소포체의 생성 촉진 방법 |
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| KR102368442B1 (ko) | 2022-02-28 |
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