JPWO2020032186A1 - Topically applied composition containing extracellular vesicles produced by oral epithelial cells - Google Patents

Abstract

INDUSTRIAL APPLICABILITY The present invention provides a novel topically applied composition that can be easily prepared and can improve the condition of body tissues, particularly epithelial tissues such as skin and mucous membranes. In particular, the present invention provides topically applied compositions comprising extracellular vesicles derived from oral epithelial cells.

Description

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

In recent years, in order to solve unmet medical needs, research and development of products such as regenerative medicine using cells and tissues have been promoted. For example, it is known that epithelial cells promote wound healing by transplanting them into epithelial defect sites, and products such as regenerative medicine using these cells have been put on the market. In addition, cultures of oral epithelial cells have been used for applications such as corneal regeneration and improvement of healing of the esophageal mucosa.

In addition, studies on the regeneration-promoting effect of extracellular vesicles (EVs) such as exosomes secreted from various cell types are underway, and most of the studies are on the bone marrow mesenchymal stroma. It has been performed on cell (BM-MSC) -derived exosomes (Journal of Translational Medicine (2015), 13:49). However, few studies have been reported on extracellular vesicles derived from epithelial cells, and especially for extracellular vesicles derived from oral epithelial cells, a report 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 novel topical composition that can be easily prepared and can improve the condition of body tissues, particularly epithelial tissues such as skin and mucous membranes.

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

The locally applied composition of the present invention can be easily prepared by isolating extracellular vesicles from a culture solution of oral epithelial cells, and can be used for wound healing and fibrosis in epidermal burns and ulcers such as skin or mucous membranes. It can be used to treat or prevent scarring due to relief. It also has a rejuvenating effect by improving the condition of the epidermis by promoting tissue repair. Furthermore, as an effect of reducing fibrosis, fibrosis of the heart, lungs, liver, kidneys, etc. can be improved.

The results of Western blotting of the fraction containing extracellular vesicles are shown. An electron micrograph of the isolated extracellular vesicle is shown (scale bar: 50 nm). The results of the fibroblast proliferation assay are shown. The results of the cytotoxicity assay for fibroblasts are shown. 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. The results of a proliferation test using squamous cell carcinoma cells are shown. The observation results of the in vivo test using the rat full-thickness wound healing model (Fig. 7a) and the microscopic view after staining with hematoxylin eosin and picrosirius red (Fig. 7b) are shown. The results of wound area measurement in an in vivo test using a rat full-thickness wound healing model are shown. A microscopic view after hematoxylin / eosin staining in an in vivo test using a porcine skin wound model is shown (scale bar: 500 nm).

Topically applied compositions of the present invention include extracellular vesicles derived from oral epithelial cells. Oral epithelial cells are cells that make up the surface of the oral mucosa and include arbitrary cells that make up epithelial tissue. Those skilled in the art are familiar with the collection method and the instruments for that purpose. In addition, various cells have been developed or marketed for research, and these cells can also be used.
In the present invention, mammalian-derived oral epithelial cells are preferably used. The oral epithelial cells may be derived from a species different from the species to which the final topically applied composition is applied, but from the viewpoint of safety and availability in consideration of human application, the oral epithelial cells may be derived from a different species. Often, human-derived oral epithelial cells are used. The above-mentioned human is not particularly limited and may be non-adult / adult, but adult-derived oral epithelial cells are preferably used.

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, the medium (conditioned medium) from which the cells and residues have been removed from the cultured culture is collected and then known. Extracellular vesicles derived from oral epithelial cells can be isolated from the conditioned medium according to the above method.

The medium and culture conditions used for culturing the cells are not particularly limited, but a liquid medium, for example, a high glucose Dulbecco modified Eagle's medium is preferably used. Other components required for culturing may be appropriately added to the medium, and may include, for example, a nutritional mixture such as F-12 Ham, corticosteroids such as Sol Cotef, antibiotics, autologous serum and the like. The culture conditions are not particularly limited and can be appropriately set by those skilled in the art according to the amount of cells to be used and the amount of extracellular vesicles required, but for example, 33 to 40 ° C., preferably 35 to 38 ° C. , 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 exchanged during the culture, and the media can be used together thereafter. After performing the above culture, the conditioned medium is recovered by separating the cells from the culture by centrifugation, filtration, or the like. The collected conditioned medium may be further centrifuged or filtered to further remove impurities such as cell residues. The conditions for centrifugation and filtration can be appropriately determined by those skilled in the art. For example, the culture is first centrifuged at 200xg to 400xg, preferably 250xg to 350xg for about 10 minutes to separate the cells, and then the cells are separated. Fine impurities can be removed by centrifuging again at 2500xg to 3500xg or by, for example, filtering 0.22 μm.

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

The extracellular vesicles isolated preferably contain exosomes. In general, the distinction between extracellular vesicle types (exosomes, microvesicles, etc.) is not always clarified academically, and it is technically technical to selectively isolate specific types of extracellular vesicles. Since it is not easy, no matter which method is used to isolate the extracellular endoplasmic reticulum, it usually contains exosomes. Therefore, in the practice of the present invention, the embodiments and conditions for isolating extracellular vesicles may be limited to those that strictly distinguish the types of extracellular vesicles and target specific ones. do not have.

However, if the separation accuracy allows for the individual isolation method to be performed, the isolation operation may be performed under conditions such that exosomes are concentrated in the extracellular vesicles, and exosomes are concentrated. The step to be performed may be additionally performed separately. For example, when isolating extracellular vesicles by size exclusion chromatography, the proportion of exosomes by collecting fractions containing substances sized 30-200 nm, preferably 40-150 nm, more preferably 80-130 mm. Can be increased to concentrate exosomes.

After isolating the extracellular vesicles as described above, the topical application composition of the present invention can be obtained by adding the extracellular vesicles 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 applied to the body, and any medium for making a solution, gel, lotion, cream, ointment, ointment, for example. Can be mentioned. In addition, the topically applied composition of the present invention may further contain any adjuvant, additive, etc. that are acceptable for the above aspects. The concentration of extracellular vesicles in the final composition can be appropriately set based on the use of the composition, the desired effect, etc. so as to be an effective amount for producing a therapeutic effect or a desired effect, but is applied topically. 0.2μg~2mg / cm ² according to the area of the site, the composition preferably 2.0~200μg / cm ^2, more preferably 10-100 [mu] g / cm ^2, particularly preferably at a concentration of 20~40μg / cm ² It is preferable to add it in a product.

Topical application in the present invention means application in a manner that directly acts on a part of the body intended for the effect of the composition of the present invention, and preferably means use in a manner of contacting the part. Preferably, the compositions of the invention are applied by application to the epithelium of the body surface (including contacting solid compositions). Topical application includes so-called external application, but the application site is not limited to application to the surface of the body (parts directly visible from the outside, such as skin and cornea), and is applied to mucous membranes such as the oral cavity, nasal cavity, and digestive organs. Includes applicable aspects. Furthermore, it also includes an embodiment in which it is directly applied to internal organs (heart, lung, liver, kidney, thyroid gland, parathyroid gland, pancreas, gallbladder, etc.) through surgical procedures and the like. Preferably, the composition of the present invention is preferably applied to the skin or mucous membranes, and is applied by applying to these sites.

The health of other organs with skin, mucous membranes, or epithelium (heart, lungs, liver, kidneys, thyroid, parathyroid, pancreas, gallbladder, etc.) by topically applying the compositions of the present invention to predetermined parts of the body. It is possible to restore various tissue structures or functions, and to obtain effects such as promotion of wound healing and treatment or reduction of scars. As described above, the condition of the wound or disease can be improved, for example, by promoting healing of the wound on the skin or mucous membrane. Wounds are based on any aspect and include trauma, surgical scars and even burns. This promotion of healing can relieve pain in the skin or mucous membranes and improve the appearance of the application site by treating or preventing scarring. Further, as described above, by applying to internal organs (heart, lung, liver, kidney, etc.) in which fibrosis is a problem, fibrosis of the organ can be treated or prevented.

In addition, the composition of the present invention stimulates fibroblasts as shown in Examples described later, and hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), And because it has been shown to promote the release of growth factors such as binding tissue growth factor (CTGF), application of the composition to a given site may result in any disease or disease in which the above growth factors have a therapeutic effect. Expected to have the effect of preventing or treating the condition.

By blending the composition of the present invention with a pharmaceutical product, a pharmaceutical product having the above-mentioned therapeutic or preventive effect can be obtained. In particular, as shown in Examples described later, it was 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 incorporated into pharmaceuticals for various diseases or symptoms.

Furthermore, since the composition of the present invention has an epidermis rejuvenation effect by improving the condition of tissues such as skin and promoting tissue repair, it is also preferable for healthy skin by blending it in cosmetics. Can bring.
Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to the aspects of these Examples.

1. 1. Isolation of extracellular vesicles CellSeed obtained the conditioned medium used to produce clinical grade oral mucosal cell sheets from cells biopsied from the mouth of healthy adult donors. The conditioned medium provided was keratinocyte culture medium (75% high glucose Dalveco modified Eagle's medium, 25% nutritional mixture F-12 Ham, 0.475 g / L L-glutamine, 5 μg / ml Humarin regular). Regular), 0.4 μg / mL Sol Kotef, 1 nM cholera toxin, 2 nM T3, 10 ng / mL EGF, 40 μg / mL Gensumycin, 0.14 μg / mL fungizone and 5% autologous serum) The cells were cultured at 37 ° C., and 8 to 12 mL of the conditioned medium was collected during the medium exchange on the 5th, 8th, 10th, 12th, 13th, 14th, 15th and 16th days of the culture. Together, they were used to isolate extracellular vesicles.

The recovered conditioned medium was centrifuged at 300 xg for 10 minutes at 4 ° C., and then cells and cell residues were removed by ultrafiltration of 0.22 μm. To concentrate the medium with an ultrafiltration column (Amicon, Merck Millipore) 100 kDa (5,000xg, 4 ° C) and then 10 kDa (7,500xg, 4 ° C) to separate extracellular vesicles (including exosomes) from free proteins. Added to 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 measurement
Protein concentrations were measured using the Pierce BCA Protein Assay Kit (ThermoFisher Scientific). Extracellular vesicles are diluted 1: 1 with RIPA (Radioimmunoprecipitation assay) buffer and super-superimposed for 5 minutes (on / off: 15 seconds / 15 seconds, in an ice bath, high setting (Cosmo Bio / ultrasonic crusher)). Sonicated. Sonicated extracellular vesicles and cell lysates were diluted with MilliQ water (Mercipore) and a BCA (bicinchoninic acid) assay was performed according to the instructions.

3. 3. Preparation of cell lysates Cell lysates were obtained from fibroblasts (CC-2509, Lonza) and oral keratinocytes (# 2610, ScienCell). The cell suspension (approximately 4x10 ⁶ cells) was centrifuged at 300xg for 5 minutes, the supernatant was discarded, the pellet was resuspended in PBS and centrifuged again. Pellets are 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. The sample was then centrifuged at 4 ° C., 8000xg for 10 minutes, the supernatant was collected, divided into equal parts and used for subsequent analysis.
Using the extracellular vesicles and cell lysates prepared as described above, the presence and characteristics of extracellular vesicles were confirmed and measured by molecular biological techniques, electron microscopic observation, and nanoparticle tracking analysis, and further, their Function was analyzed in an in vitro study.

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 are loaded onto NuPage 4-12% 1.5x15 ThermoFisher, electrophoresed (200V, 125mA, 25 minutes; PowerStation 1000XP; Atto), then using iBlot (Invitrogen) 11-minute program. The protein was transferred onto a nitrocellulose membrane (protein iBlot gel transfer stack, Invitrogen). Subsequent blocking, antibody incubation, and washing steps were performed on a tilted shaker. The filter was blocked for 60 minutes using TBS-T (BioRad) containing 5% infant formula (CellSignaling) and exposed to the primary antibody overnight at 4 ° C. The filter was washed with TBS-T for 3x10 minutes on an orbital shaker, exposed to secondary antibody for 60 minutes at room temperature, and then again washed with TBS-T for 3x10 minutes. The buffer was removed from the filter, a detection agent (ECL Prime WB detection kit, Sigma-Aldrich) was added, and the mixture was left for 5 minutes. After that, the expression of the protein 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 are 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). The antibody was diluted in Can Get Signal solution (Toyobo). Each membrane was stripped and reused with different antibodies: 30 minutes, 2% SDS, 0.8% β-mercaptoethanol, Tris-HCl (pH 6.6), 50 ° C., then washed with TBS-T for 3x10 minutes. ..

5. Transmission electron microscope observation
A 10 μL extracellular vesicle suspension was placed on a copper grid for 20 minutes, followed by 2% uranyl acetate and left for 1 minute. Uranyl acetate was removed, distilled water was added, the mixture was left for 1 minute, and then removed. The grid was air-dried as it was 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. The size of the vesicles was about 125 nm (124.8 ± 4.1 nm (standard deviation)).

7. Fibroblast proliferation assay
NHDFp13 cells (CC-2509, Lonza) were seeded in 96-well plates at a concentration of 1,600 cells / well. 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 (Dojin Chemical Laboratory). It was confirmed that the sample containing extracellular vesicles suppressed the proliferation of fibroblasts in the same manner as dexamethasone, which is a corticosteroid commonly used as an anti-scarring agent (Fig. 3).

8. Fibroblast cytotoxicity and HGF-release assay
NHDFp15 cells (CC-2509, Lonza) were seeded in 24-well plates at a concentration of 50,000 cells / well. The next day, the medium was replaced with 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 metabolism of the cells was measured using the CCK kit (Dojin Chemical Laboratory). As a result, extracellular vesicles became cytotoxic to fibroblasts. It was confirmed that it was not shown (Fig. 4). Moreover, when the hepatocyte growth factor (HGF) content in the conditioned medium was measured by an ELISA kit (Abcam), it was confirmed that the HGF content was increased at the protein level (Fig. 5 (b)).

9. Gene expression analysis in fibroblasts
NHDFp14 cells (CC-2509, Lonza) were seeded in 6-well plates at a concentration of 336,000 cells / well. The next day, the medium was replaced with a serum-free medium containing 2 μg / mL extracellular vesicles (for control wells, a serum-free medium containing no 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 on various growth factors using TaqMan Fast Advanced Master Mix (ThermoFisher Scientific).
The primers used were: β-actin (4326315E-1112022, Applied Biosystem), HGF (HS00300159_m1, ThermoFischer Scientific), VEGFA (HS00900055_m1, ThermoFischer Scientific), FGF2 (HS00266645_m1, ThermoFischer Scientific), CTGF (HS01026927_g1) ). Type IV collagen analysis was performed in the same manner using NHDF cells at 5 and 17 passages and the primer Col4a1 (HS00266237_m1, ThermoFischer Scientific).

As shown in FIG. 5 (a), extracellular vesicles stimulate fibroblasts to stimulate hepatocyte growth factor (HGF), vascular endothelial growth factor A (VEGFA), fibroblast growth factor (FGF2), and Significantly increased gene expression of important growth factors such as connective tissue growth factor (CTGF). 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. Also, as shown in FIG. 5 (c), older (highly passaged) fibroblasts expressed low levels of type IV collagen-gene as compared to younger (five-passaged) fibroblasts. However, once "old" fibroblasts were exposed to extracellular vesicles, they exhibited similar expression to "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 cells / cm ² , and the next day, the medium was serum-free medium containing extracellular vesicles of various concentrations, or control medium containing no extracellular vesicles. Exchanged for. After 48 hours, the number of cells was measured using Cell Counting Kit-8 (CCK, Dojin Chemical Laboratory). As a result, it was confirmed that extracellular vesicles derived from oral epithelial cells reduce the proliferation of squamous cell carcinoma cells (TR146) in a concentration-dependent manner (Fig. 6).

11. Labeling of extracellular vesicles Labeling extracellular vesicles with fluorescent membrane dye (PKH26-GL-1KT, Sigma Aldrich), diluting extracellular vesicles with 500 μL of diluent C, and immediately immediately with 500 μL of diluent C ( (Containing 2 μL of PKH26) was added again and the incubation step was performed for 5 minutes with gentle mixing. Then 2.5 mL of PBS was added and extracellular vesicles were reconcentrated using a 10 kDa filter (Amicon). This process was performed a total of 3 times. As a control, all steps were performed similarly without extracellular vesicles.

12. Rat full-thickness wound healing model
Six-week-old male Sprague Dawley rats (n = 3) were used. Isoflurane was used to cause sensory anesthesia and the condition was maintained with a mixture of domitol (0.375 mg / kg), midazolam (2 mg / kg) and butorphanol tartrate (2.5 mg / kg). The back hair was removed and a full-thickness wound (n = 4 per animal) was created using a 5 mm biopsy trepan (Kai Medical). A 20 μl (4.44 μg) extracellular vesicle suspension or control suspension is added to the wound, held for 45 minutes and imaged using a digital camera and stereomicroscope (Olympus: MVX10), followed by The wound was covered with 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 on day 1.
Animals were anesthetized on days 3, 4, and 5 to obtain images and slaughtered on day 6. Wounds were removed using an 8 mm biopsy trepan (Kai Medical) and used for histological analysis. In a similar experiment conducted separately, rats (n = 3) were sacrificed on the 17th day. Tissue sections (8 μm) were prepared using a cryostat (MicroEdge) and stained with hematoxylin eosin and picrosirius red (Polysciences). Slides stained with picrosirius red were visualized under a polarizing microscope and images were evaluated blindly.
As shown in FIG. 7 (a), signals from fluorescently labeled extracellular vesicles were confirmed in rat wound beds up to 5 days after application. Extracellular vesicles also promoted wound healing in full-thickness skin wounds in rats, as shown by hematoxylin eosin and picrosirius red-staining, as shown in FIG. 7 (b). In addition, as shown in FIG. 8, based on a blinded assessment, the extracellular vesicle treatment group showed significantly smaller wound area compared to controls on days 6 and 17.

13. Pig skin wound healing test Pigs (Japanese farm pig) (n = 1) were sedated with domitol, midazolam and butorphanol tartrate, and anesthesia was maintained with sevoflurane. The skin on the back was shaved and sterilized with iodine. Use biopsy trepan (Kai Medical) to make a 5 mm skin wound, add 20 μL (4.75 μg) extracellular vesicles or control (PBS) suspension to the wound and hold for 45 minutes, then the wound is Tegaderm Covered with. Seven days later, the wound was dissected using an electric knife. Dissected samples were sectioned with a cryostat (MicroEdge) and stained with hematoxylin and eosin. As a result, the 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). In addition, there were no signs of side effects in the extracellular vesicle treatment group.

As shown in each of the above examples, a composition containing extracellular vesicles produced by human oral epithelial cells is directly applied to a wound model in which rat skin (epidermis, dermis) is excised to obtain physiological saline. Compared with the control group to which was applied, wound healing was promoted, scars were reduced, and a rejuvenating effect by tissue repair was observed.
In addition, as shown 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 stromal stromal cells, tumor growth. It was confirmed that it does not promote. Therefore, the compositions of the present invention have the advantage that they can be safely incorporated into pharmaceutical products for various diseases or symptoms.

Claims (15)

Topically applied composition comprising extracellular vesicles derived from oral epithelial cells. The composition according to claim 1, wherein the extracellular vesicle comprises an exosome. The composition according to claim 1 or 2, wherein the extracellular vesicles are enriched for exosomes. The composition according to any one of claims 1-3, wherein the oral epithelial cells are of human origin. The composition according to any one of claims 1-4, which is applied by application to the epithelium. The composition according to any one of claims 1-5 for restoring a healthy tissue structure or function of skin, mucous membranes, or other organs having epithelium. Claim 1 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 1-7 for treating a wound on the skin or mucous membrane. The composition according to any one of claims 1-7 for treating or preventing scarring on the skin or mucous membranes. The composition according to any one of claims 1-7 for treating or preventing fibrosis of internal organs. A drug comprising the composition according to any one of claims 1-10. A cosmetic product containing the composition according to any one of claims 1-10. The process of culturing oral epithelial cells in a medium,
The step of separating oral epithelial cells from the culture and collecting the conditioned medium,
The step of isolating extracellular vesicles of oral epithelial cells from the conditioned medium, and
The step of adding the isolated extracellular vesicles into a medium suitable for topical application,
A method for producing a topically applied composition, which comprises. 13. The method of claim 13, further comprising the step of concentrating exosomes in extracellular vesicles. 13. The method of claim 13 or 14, wherein the epithelial cells are of human origin.

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