WO2020032186A1

WO2020032186A1 - Topical composition containing extracellular vesicles produced by buccal epithelial cells

Info

Publication number: WO2020032186A1
Application number: PCT/JP2019/031429
Authority: WO
Inventors: セバスチャンショークビスト; 信雄金井; 隆紀岩田
Original assignee: 学校法人東京女子医科大学
Priority date: 2018-08-08
Filing date: 2019-08-08
Publication date: 2020-02-13
Also published as: JPWO2020032186A1

Abstract

The present invention provides a novel topical composition that can be easily manufactured and is capable of improving the state of bodily tissue, especially epithelial tissue such as the skin or mucosae. In particular, the present invention provides a topical composition containing extracellular vesicles derived from buccal epithelial cells.

Description

Topical composition comprising extracellular vesicles produced by oral epithelial cells

The present invention relates to a composition for topical application to epithelium such as skin, which contains extracellular vesicles derived from oral epithelial cells, particularly exosomes.

In recent years, research and development of products such as regenerative medicine using cells and tissues have been promoted to solve unmet medical needs. For example, epithelial cells are known to promote wound healing by transplanting to epithelial defect sites, and products such as regenerative medicine using the same have been put on the market. In addition, cultures of oral epithelial cells have been used for applications such as corneal regeneration and improved esophageal mucosal healing.

Studies are also being conducted on the effect of promoting extracellular vesicles (EVs) such as exosomes secreted from various cell types, and most of the research is on bone marrow mesenchymal stromal cells. It is performed on exosomes derived from cells (BM-MSC)-(Journal of Translational Medicine (2015), 13:49). However, few studies have been reported on extracellular vesicles derived from epithelial cells. Particularly, extracellular vesicles derived from oral epithelial cells have been reported on viral immunology (Journal of Virology, Vol.90 No.7). (2016), p. 3469-3479) only exists.

An object of the present invention is to provide a new topical composition which can be easily prepared and can improve the condition of body tissues, particularly epithelial tissues such as skin and mucous membranes.

The present inventors have found that a composition containing extracellular vesicles secreted into a medium when culturing oral epithelial cells has an effect such as promoting wound healing of epidermis or epithelial tissue such as skin or mucous membrane. And completed the invention of the present application.
That is, the present invention provides a topical composition comprising extracellular vesicles derived from oral epithelial cells. Further, the present invention provides a step of culturing oral epithelial cells in a medium, a step of separating oral epithelial cells from the culture to recover a conditioned medium, and a step of isolating extracellular vesicles of oral epithelial cells from the conditioned medium. And adding the isolated extracellular vesicles to a medium suitable for topical application.

The topical composition of the present invention can be easily prepared by isolating extracellular vesicles from a culture solution of oral epithelial cells and the like, and can heal and fibrosis wounds and ulcers on the epidermis such as skin or mucous membrane. Can be used to treat or prevent scarring by alleviation. It also has a rejuvenating effect by improving the condition of the epidermis by promoting tissue repair. Further, as an effect of reducing fibrosis, fibrosis of the heart, lungs, liver, kidney and the like can be improved.

4 shows the results of Western blot of fractions containing extracellular vesicles. The electron micrograph of the isolated extracellular vesicle is shown (scale bar: 50 nm). 4 shows the results of a fibroblast proliferation assay. Fig. 3 shows the results of a cytotoxicity assay on fibroblasts. The results of gene expression analysis of various growth factors in fibroblasts (FIG. 5a), HGF-release assay by ELISA (FIG. 5b), and expression analysis of type IV collagen gene (FIG. 5c) are shown. 3 shows the results of a proliferation test using squamous cell carcinoma cells. The observation results of an in vivo test using a rat full-thickness wound healing model (FIG. 7a) and a micrograph after staining with hematoxylin / eosin and picrosirius red (FIG. 7b) are shown. FIG. 4 shows the results of wound area measurement in an in vivo test using a rat full thickness wound healing model. The micrograph after hematoxylin and eosin staining in an in vivo test using a porcine skin wound model is shown (scale bar: 500 nm).

The topical composition of the present invention comprises extracellular vesicles derived from oral epithelial cells. Oral epithelial cells are cells that constitute the surface of the oral mucosa, and include any cells that constitute epithelial tissue. The sampling method and the instrument for the collection are well known to those skilled in the art. In addition, various cells have been developed or marketed for research, and these cells can also be used.
In the present invention, preferably, oral epithelial cells derived from mammals are used. Oral epithelial cells may be derived from a species different from the species to which the final topical composition is applied, but from the viewpoint of safety and availability in consideration of application to humans, Preferably, human oral epithelial cells are used. The human is not particularly limited and may be non-adult / adult, but preferably oral epithelial cells derived from an adult are used.

外 The isolation of extracellular vesicles from oral epithelial cells can be performed by those skilled in the art by any method. For example, since extracellular vesicles are secreted into the medium by culturing oral epithelial cells in the medium, a medium (conditioned medium) obtained by removing cells and residues from the culture after culture is collected and The extracellular vesicles derived from oral epithelial cells can be isolated from the conditioned medium by the technique described in (1).

培地 The medium used for culturing the cells and the culture conditions are not particularly limited, but a liquid medium, for example, a high glucose Dulbecco's modified Eagle medium is preferably used. Other components necessary for the culture may be appropriately added to the medium, and may include, for example, a nutrient mixture such as F-12 @ Ham, a corticosteroid such as Sol Cortev, an antibiotic, an autologous serum, and the like. Culture conditions are not particularly limited, and can be appropriately set by those skilled in the art according to the cells to be used and the amount of extracellular vesicles required. For example, 33 to 40 ° C, preferably 35 to 38 ° C And more preferably at a temperature of 37 ° C. for 12 hours to 25 days, preferably 1 to 20 days, more preferably 3 to 18 days. If necessary, the medium may be changed during the culturing, and these mediums can be used collectively thereafter. After the above culture, the conditioned medium is recovered by separating the cells from the culture by centrifugation or filtration. The collected conditioned medium may be further subjected to centrifugation or filtration to further remove impurities such as cell residues. The conditions of centrifugation and filtration can be appropriately determined by those skilled in the art.For example, first, the culture is centrifuged at 200 × g to 400 × g, preferably at 250 × g to 350 × g for about 10 minutes, and the cells are separated by centrifugation. Fine impurities can be removed by centrifugation again at 2500 × g to 3500 × g or by filtration at, for example, 0.22 μm.

As described above, the method for isolating extracellular vesicles from a conditioned medium is well known and is not particularly limited, and those skilled in the art can isolate them by any method. If necessary, after the medium is concentrated by ultrafiltration or the like, for example, size exclusion chromatography, ultracentrifugation, microfiltration, density gradient centrifugation, or the like can be performed alone or in an appropriate combination. Preferably, extracellular vesicles are isolated by performing size exclusion chromatography on the concentrated medium after ultrafiltration. For ultrafiltration, for example, Amicon (100 kDa, 1000 kDa, etc.) from Merck Millipore can be used according to the manufacturer's instructions.

外 The isolated extracellular vesicle preferably contains exosomes. In general, the distinction between types of extracellular vesicles (exosomes, microvesicles, etc.) is not always scientifically clarified, and it is technically necessary to selectively isolate specific types of extracellular vesicles. Because it is not easy, the extracellular endoplasmic reticulum will usually contain exosomes regardless of which technique is used to isolate it. Therefore, in the practice of the present invention, the modes and conditions for isolating extracellular vesicles are not limited to those that target the specific one by strictly distinguishing the type of extracellular vesicle. Absent.

However, if the separation accuracy of the individual isolation technique to be performed is acceptable, the isolation operation may be performed under conditions such that exosomes are concentrated among extracellular vesicles, and exosomes may be concentrated. May be additionally performed. For example, in the case of isolating extracellular vesicles by size exclusion chromatography, the fraction containing exosomes having a size of 30 to 200 nm, preferably 40 to 150 nm, and more preferably 80 to 130 mm, is collected to obtain a ratio of exosomes. To increase the concentration of exosomes.

After isolating the extracellular vesicle as described above, the topical composition of the present invention can be obtained by adding the extracellular vesicle to a medium suitable for topical application. The type of medium can be appropriately selected by those skilled in the art from liquids, solids, semi-solids, and the like depending on the mode of application to the body.For example, any medium for forming a solution, gel, lotion, cream, ointment, plaster Is mentioned. In addition, the topical composition of the present invention may further include any adjuvants, additives, and the like that are acceptable for the above embodiments. The concentration of extracellular vesicles in the final composition can be appropriately set based on the intended use of the composition, the desired effect, etc., so as to be an amount effective for producing a therapeutic effect or a desired effect. 0.2μg ~ 2mg / cm ² according to the area of the site, preferably 2.0 ~ 200μg / cm ^2, more preferably 10 ~ 100μg / cm ^2, particularly preferably the composition at a concentration of 20 ~ 40μg / cm ² It is preferable to add it to the product.

局所 The topical application in the present invention means to apply in such a manner as to directly act on a site in the body where the effect of the composition of the present invention is intended, and preferably to use in such a mode as to contact the site. Preferably, the compositions of the invention are applied by application to the epithelium on the body surface (including contacting the solid composition). Topical application includes so-called topical application, but the application site is not limited to application to the body surface (a site directly visible from the outside, for example, skin, cornea), but to mucous membranes such as oral cavity, nasal cavity, and digestive organs. Includes aspects of application. Further, the present invention also includes a mode in which the method is directly applied to internal organs (heart, lung, liver, kidney, thyroid, parathyroid, pancreas, gallbladder, etc.) through a surgical procedure or the like. Preferably, the composition of the present invention is applied to the skin or mucous membrane, and is applied by applying to these sites.

By topically applying the composition of the present invention to a predetermined part of the body, the health of skin, mucous membrane, or other organs having epithelium (heart, lung, liver, kidney, thyroid, parathyroid, pancreas, gallbladder, etc.) Recovery of various tissue structures or functions, and effects such as promotion of wound healing and treatment or reduction of scarring can be obtained. In the above embodiment, for example, a wound or disease state can be improved by promoting the healing of a wound of skin or mucous membrane. Wounds include those based on any aspect, including trauma, surgical scars, and even burns. By promoting this healing, the pain of the skin or mucous membrane is alleviated, and the appearance of the application site can be improved by treating or preventing scar. Further, as described above, by applying to internal organs where fibrosis is a problem (heart, lung, liver, kidney, etc.), fibrosis of the organ can be treated or prevented.

In addition, the composition of the present invention stimulates fibroblasts, as shown in the examples below, to give hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), And has been shown to enhance the release of growth factors such as connective tissue growth factor (CTGF), so that by applying the composition to a given site, any disease or disease in which the above growth factors have a therapeutic effect The effect of preventing or treating the condition is expected.

医薬品 By blending the composition of the present invention with a drug, a drug having the above-mentioned therapeutic or preventive effect can be obtained. In particular, as shown in the Examples below, it has been confirmed that extracellular vesicles derived from oral epithelial cells do not promote tumor growth in contrast to extracellular vesicles derived from bone marrow mesenchymal stromal cells. Therefore, it can be safely compounded with pharmaceuticals for various diseases or symptoms.

Furthermore, the composition of the present invention improves the condition of tissues such as the skin and has a rejuvenating effect on the epidermis by promoting tissue repair. Can bring.
Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

1. Isolation of extracellular vesicles A conditioned medium used to produce a clinical grade oral mucosal cell sheet from cells isolated by biopsy from the mouth of a healthy adult donor was obtained from CellSeed. The provided conditioned medium was a keratinocyte culture medium (75% high glucose Dulbecco's modified Eagle medium, 25% nutrient mixture F-12 Ham, 0.475 g / L L-glutamine, 5 μg / ml Humulin Regular (Humulin Regular). Regular), 0.4 μg / mL sol Cortev, 1 nM cholera toxin, 2 nM T3, 10 ng / mL EGF, 40 μg / mL Gensumycin, 0.14 μg / mL fungizone and 5% autologous serum) When the cells were cultured at 37 ° C., 8 to 12 mL of the conditioned medium was collected when the medium was replaced on

days

5, 8, 10, 12, 13, 14, 15, and 16 of the culture. The combination was used for the isolation of extracellular vesicles.

(4) The collected conditioned medium was centrifuged at 300 × g for 10 minutes at 4 ° C., and then cells and cell debris were removed by ultrafiltration at 0.22 μm. Concentrate the medium with an ultrafiltration column (Amicon, Merck Millipore) 100kDa (5,000xg, 4 ° C) and then 10kDa (7,500xg, 4 ° C) to separate extracellular vesicles (including exosomes) from free proteins It was applied to a size exclusion chromatography column (qEV column, Izon). Fractions 7-9 (500 μl each) were collected as extracellular vesicle fractions according to the column manufacturer's instructions.

2. Total protein concentration determination Protein concentration was determined using the Pierce BCA protein assay kit (ThermoFisher Scientific). Dilute the extracellular vesicles 1: 1 with RIPA (Radioimmunoprecipitation assay) buffer, and ultrapure for 5 minutes (on / off: 15 seconds / 15 seconds, in an ice bath, high setting (Cosmo Bio / sonicator)) Sonicated. Sonicated extracellular vesicles and cell lysates were diluted in MilliQ water (Merck Millipore) and BCA (bicinchoninic acid) assay was performed according to the instructions.

3. Preparation of cell lysates Cell lysates were obtained from fibroblasts (CC-2509, Lonza) and oral keratinocytes (# 2610, ScienCell). The cell suspension (about 4 × 10 ⁶ cells) was centrifuged at 300 × g for 5 minutes, the supernatant was discarded, and the pellet was resuspended in PBS and centrifuged again. The pellet is resuspended in RIPA (Radioimmunoprecipitation assay) buffer (Sigma) containing 1% protease / phosphatase inhibitor cocktail (Cell Signaling Technology) and placed in an ice bath for 5 minutes (15 second on, 15 second off interval) Sonicated. Next, the sample was centrifuged at 8000 × g for 10 minutes at 4 ° C., and the supernatant was collected and divided into equal amounts for use in the subsequent analysis.
Using the extracellular vesicles and cell lysates prepared as described above, confirm and measure the presence and properties of extracellular vesicles by molecular biological techniques, electron microscopy, and nanoparticle tracking analysis. Function was analyzed in in vitro studies.

4. Western blot cell lysates or extracellular vesicle isolates were diluted 1: 1 with Laemmli sample buffer (BioRad), heated at 95 ° C. for 5 minutes, and then cooled on ice. Samples were loaded onto NuPage 4-12% 1.5x15 well gels (ThermoFisher), electrophoresed (200V, 125mA, 25 minutes; Power Station 1000XP; Atto), and then used with the 11 minute program of iBlot (Invitrogen). Proteins were transferred onto a nitrocellulose membrane (Protein iBlot gel transfer stack, Invitrogen). Subsequent blocking, antibody incubation, and washing steps were performed on a tilt shaker. Filters were blocked for 60 minutes using TBS-T (BioRad) containing 5% milk powder (CellSignaling) and exposed to primary antibody overnight at 4 ° C. The filters were washed on an orbital shaker with TBS-T for 3 × 10 minutes, exposed to the secondary antibody for 60 minutes at room temperature, and then washed again with TBS-T for 3 × 10 minutes. The buffer was removed from the filter, and a detection agent (ECL Prime WB detection kit, Sigma-Aldrich) was added and left for 5 minutes. Thereafter, protein expression was confirmed using LAS 4000 mini (Fuji Film). Samples from the extracellular vesicle fraction were positive for the internationally recognized markers (CD9 and flotilin) and negative for the negative marker GRP94 (FIG. 1).
The antibodies and concentrations used were as follows: CD9 (# 13174, Cell Signaling Technologies), Flotillin (# 18634, Cell Signaling Technologies), GRP94 (# 2104, Cell Signaling Technologies), anti-rabbit IgG, HRP-linked (# 7074, Cell Signaling Technologies, 1: 5000). Antibodies were diluted in Can Get Signal solution (Toyobo). Each membrane was stripped and reused with a different antibody: 30 minutes, 2% SDS, 0.8% β-mercaptoethanol, Tris-HCl (pH 6.6), 50 ° C., then washed with TBS-T for 3 × 10 minutes .

5. Observation by transmission electron microscopy 10 μL of the extracellular vesicle suspension was placed on a copper grid for 20 minutes, and 2% uranyl acetate was added thereto for 1 minute. The uranyl acetate was removed, distilled water was added and left for 1 minute, then removed. The grid was air-dried for 15 minutes and the morphology of extracellular vesicles (exosomes) was confirmed using a transmission electron microscope (H7650, Hitachi) (FIG. 2).

6. Nanoparticle tracking analysis The size and number of extracellular vesicles were measured using the LM10 Nanoparticle Analyzer (NanoSight) according to the manufacturer's instructions. Vesicle size was approximately 125 nm (124.8 ± 4.1 nm (standard deviation)).

7. Fibroblast Proliferation Assay Cells of NHDFp13 (CC-2509, Lonza) were seeded in 96-well plates at a concentration of 1,600 cells / well. On the next day, the medium was replaced with a serum-free medium containing extracellular vesicles (2 μg / mL, 0.67 μg / mL, 0.2 μg / mL), a control medium containing no extracellular vesicles, or a medium containing dexamethasone (10 nm). . After 72 hours, cell metabolism was measured using a CCK kit (Dojindo Laboratories). It was confirmed that the sample containing extracellular vesicles suppressed the proliferation of fibroblasts in the same manner as dexamethasone, a corticosteroid generally used as an anti-scarring agent (FIG. 3).

8. Fibroblast cytotoxicity and HGF-release assay Cells of NHDFp15 (CC-2509, Lonza) were seeded at a concentration of 50,000 / well in 24-well plates. The next day, the medium was changed to a serum-free medium containing 2 μg / mL extracellular vesicles or a serum-free medium containing no extracellular vesicles. After 72 hours, the medium was collected, frozen at -80 ° C, and the cell metabolism was measured using the CCK kit (Dojindo Laboratories). As a result, extracellular vesicles showed cytotoxicity to fibroblasts. It was confirmed that it did not show (FIG. 4). Further, when the content of hepatocyte growth factor (HGF) in the conditioned medium was measured using an ELISA kit (Abcam), it was confirmed at the protein level that the HGF content was increased (FIG. 5 (b)).

9. Gene expression analysis in fibroblasts Cells of NHDFp14 (CC-2509, Lonza) were seeded at a concentration of 336,000 cells / well in a 6-well plate. The next day, the medium was replaced with serum-free medium containing 2 μg / mL extracellular vesicles (for control wells, serum-free medium without extracellular vesicles). After 72 hours, RNA was isolated using the RNeasy kit (Qiagen). CDNA was synthesized using a commercially available kit (OriGene), and quantitative PCR was performed for various growth factors using TaqMan Fast Advanced Master Mix (ThermoFisher Scientific).
The primers used are as follows: β-actin (4326315E-1112022, Applied Biosystem), HGF (HS00300159_m1, ThermoFischer Scientific), VEGFA (HS00900055_m1, ThermoFischer Scientific), FGF2 (HS00266645_m1, ThermoFischer Scientific 269gFischer 010gFisher 010GFr) ). The type IV collagen analysis was similarly performed using NHDF cells at

passages

5 and 17 and primer Col4a1 (HS00266237_m1, ThermoFischer Scientific).

As shown in FIG. 5 (a), extracellular vesicles stimulate fibroblasts, and hepatocyte growth factor (HGF), vascular endothelial growth factor A (VEGFA), fibroblast growth factor (FGF2), and Gene expression of important growth factors such as connective tissue growth factor (CTGF) was significantly increased. The results of hepatocyte growth factor (HGF) corresponded to the results of ELISA in which an increase in hepatocyte growth factor release was confirmed at the protein level in FIG. 5 (b) described above. In addition, as shown in FIG. 5 (c), the old (high passage) fibroblasts expressed the type IV collagen-gene at a lower level than the young (5 passages) fibroblasts. However, once "old" fibroblasts were exposed to extracellular vesicles, they showed similar expression as "young" cells.

10. Cancer cell proliferation test Commercially available squamous cell carcinoma cells (TR146) were cultured in DMEM containing 10% FBS and 1% antibiotics. Cells were seeded in 96-well plates at a density of 10,000 / cm ^2, the next day, serum-free medium containing the extracellular vesicles of varying concentrations of the medium, or a control medium without extracellular vesicles Was replaced. After 48 hours, the number of cells was measured using Cell Counting Kit-8 (CCK, Dojindo Laboratories). As a result, it was confirmed that extracellular vesicles derived from oral epithelial cells reduced the proliferation of squamous cell carcinoma cells (TR146) in a concentration-dependent manner (FIG. 6).

11. Labeling of extracellular vesicles The extracellular vesicles were labeled with a fluorescent membrane dye (PKH26-GL-1KT, Sigma Aldrich), the extracellular vesicles were diluted with 500 μL of diluent C, and immediately 500 μL of diluent C ( 2 μL of PKH26) was added again and an incubation step of 5 minutes was performed with gentle mixing. Thereafter, 2.5 mL of PBS was added, and the extracellular vesicles were re-concentrated using a 10 kDa filter (Amicon). This step was performed three times in total. As a control, all steps were performed without extracellular vesicles.

12. Rat full-thickness wound healing model Six-week-old male Sprague Dawley rats (n = 3) were used. Perceptual anesthesia was induced using isoflurane and maintained with a mixture of domitol (0.375 mg / kg), midazolam (2 mg / kg), and butorphanol tartrate (2.5 mg / kg). The hair on the back was removed, and a full-thickness wound (n = 4 per animal) was made using a 5 mm biopsy trepan (Kai Medical). 20 μl (4.44 μg) of extracellular vesicle suspension or control suspension was added to the wound, kept for 45 minutes, and an image was obtained using a digital camera and a stereomicroscope (Olympus: MVX10). The wound was covered using a Tegaderm wound dressing. The next day, the rats were anesthetized and photographed, and then 14 μL (3.1 μg) of extracellular vesicle suspension was added to the wound as in Day 1.
Animals were anesthetized on days 3, 4 and 5 and images were acquired and sacrificed on day 6. The wound was excised using an 8 mm biopsy trepan (Kai Medical) and used for histological analysis. In a similar experiment performed separately, rats (n = 3) were killed on day 17. Tissue sections (8 μm) were prepared using a cryostat (MicroEdge) and stained with hematoxylin and eosin and picrosirius red (Polysciences). Slides stained with picrosirius red were visualized under a polarizing microscope and the images were evaluated blind.
As shown in FIG. 7 (a), signals from the fluorescently labeled extracellular vesicles were observed in the wound bed of the rat until 5 days after application. Also, as shown in FIG. 7 (b), extracellular vesicles promoted wound healing in full-thickness skin wounds of rats, as indicated by hematoxylin-eosin and picrosirius red-staining. Furthermore, as shown in FIG. 8, based on the blinded evaluation, the treatment group with extracellular vesicles showed significantly smaller wound areas on

days

6 and 17 compared to the control.

13. Pig Skin Wound Healing Test Pigs (Japanese farm pig) (n = 1) were sedated with domitol, midazolam and butorphanol tartrate, and anesthesia was maintained using sevoflurane. The skin on the back was shaved and sterilized with iodine. A 5 mm skin wound is made using a biopsy trepan (Kai Medical) and 20 μL (4.75 μg) of extracellular vesicle or control (PBS) suspension is added to the wound and allowed to hold for 45 minutes, after which the wound is tegaderm Covered. Seven days later, the wound was dissected using an electric scalpel. Dissected samples were sectioned with a cryostat (MicroEdge) and stained with hematoxylin and eosin. As a result, re-epithelialization observed in the control group was limited, whereas complete re-epithelialization was confirmed in the extracellular vesicle-treated group (arrow in FIG. 9). No signs of side effects were observed in the extracellular vesicle treatment group.

As shown in each of the above Examples, physiological saline was applied by directly applying a composition containing extracellular vesicles produced by human oral epithelial cells to a wound model from which rat skin (epidermal, dermis) was excised. Compared to the control group to which was applied, wound healing was promoted, scarring was reduced, and a rejuvenating effect by tissue repair was observed.
In addition, as shown particularly in the results of the cancer cell proliferation test in FIG. 6, extracellular vesicles derived from oral epithelial cells, in contrast to extracellular vesicles derived from bone marrow mesenchymal stromal cells, exhibited tumor growth. Was not promoted. Therefore, the composition of the present invention has an advantage that it can be safely formulated into a drug for various diseases or conditions.

Claims

(4) A composition for topical application comprising extracellular vesicles derived from oral epithelial cells.
The composition of claim 1, wherein the extracellular vesicle comprises an exosome.
The composition of claim 1 or 2, wherein the extracellular vesicles are enriched for exosomes.
(4) The composition according to any one of (1) to (3), wherein the oral epithelial cells are derived from a human.
(5) The composition according to any one of (1) to (4), which is applied by application to an epithelium.
The composition according to any one of claims 1 to 5, for restoring healthy tissue structure or function of skin, mucous membrane, or another organ having epithelium.
2. For treating a disease or condition in which one or more growth factors selected from the group consisting of hepatocyte growth factor, vascular endothelial growth factor, fibroblast growth factor, and connective tissue growth factor have a therapeutic effect. The composition according to any one of -6.
The composition according to any one of claims 1 to 7, for treating skin or mucous membrane wounds.
(8) The composition according to any one of (1) to (7) for treating or preventing scarring of skin or mucous membranes.
The composition according to any one of claims 1 to 7, for treating or preventing fibrosis of internal organs.
医薬品 A pharmaceutical comprising the composition according to any one of claims 1 to 10.
化粧 Cosmetics containing the composition according to any one of claims 1 to 10.
Culturing the oral epithelial cells in a medium,
Recovering the conditioned medium by separating the oral epithelial cells from the culture,
Isolating the extracellular vesicles of the oral epithelial cells from the conditioned medium, and
Adding the isolated extracellular vesicles to a medium suitable for topical application,
A method for producing a topical composition, comprising:
14. The method of claim 13, further comprising the step of enriching exosomes in extracellular vesicles.
方法 The method according to claim 13 or 14, wherein the epithelial cells are of human origin.