COMPOSITION FOR SKIN REGENERATION AND WOUND HEALING, COMPRISING INDUCED EXOSOMES

Abstract

A method for producing exosomes comprises steps of: providing ultrasound stimulation directly or indirectly to cells; culturing a mixture of the cells and a medium for a predetermined time; and isolating exosomes from the mixture, wherein providing the stimulation directly to the cells comprises applying ultrasound stimulation to the medium containing the cells, and providing the stimulation indirectly to the cells comprises applying ultrasound stimulation to the medium not containing the cells and then mixing the medium and the cells. This method for producing exosomes makes it possible to obtain exosomes having skin regeneration and wound healing effects not only from stem cells and progenitor cells that are difficult to isolate and multiply, but also from somatic cells that may be easily obtained and maintained, in high yield within a short time by ultrasound treatment that is a simple process.

Claims

1. A method for producing exosomes having skin regeneration and wound healing effects, the method comprising steps of: providing ultrasound stimulation directly or indirectly to cells; culturing a mixture of the cells and a medium for a predetermined time; and isolating exosomes from the mixture, wherein the providing the stimulation directly to the cells is applying ultrasound stimulation to a medium containing the cells, and the providing the stimulation indirectly to the cells is applying ultrasound stimulation to a medium not containing the cells and then mixing the medium and the cells.

2. The method of claim 1, wherein the step of providing ultrasound stimulation directly or indirectly to cells is performed by any one method selected from among: a method of mixing the cells and a medium and then providing ultrasound stimulation to the mixture; or a method of providing ultrasound stimulation to a medium and then mixing the medium and the cells; or a method of providing ultrasound stimulation to the cells and then mixing the cells and a medium; or a method of providing ultrasound stimulation to the cells and then mixing the cells and a medium, followed by providing ultrasound stimulation to the mixture; or a method of providing ultrasound stimulation to a medium and then mixing the medium and the cells, followed by providing ultrasound stimulation to the mixture; or a method of providing ultrasound stimulation to each of the cells and a medium and then mixing the cells and the medium; or a method of providing ultrasound stimulation to each of the cells and a medium and then mixing the cells and the medium, followed by providing ultrasound stimulation to the mixture.

3. The method of claim 1, wherein the providing the ultrasound stimulation directly to the cells is performed with an ultrasound intensity of 0.1 to 3 W/cm2 and a frequency of 20 kHz to 20 MHz for a duration of 0.1 seconds to 20 minutes, and the providing the ultrasound stimulation indirectly to the cells is performed at an ultrasound intensity of 1 to 20 W/cm2 and a frequency of 20 kHz to 20 MHz for a duration of 0.1 seconds to 20 minutes.

4. The method of claim 1, wherein the cells are selected from the group consisting of mammalian stem cells, progenitor cells, fibroblasts, keratinocytes or organ tissue cells.

5. The method of claim 1, wherein the medium is any one of epithelial stem cell medium, dermal stem cell medium, adipose stem cell medium, mesenchymal stem cell medium, or embryonic stem cell medium.

6. The method of claim 1, wherein the culturing of the mixture is performed for 1 hour to 10 days.

7. The method of claim 1, wherein the step of isolating the exosomes is performed using one or more of ultracentrifugation, density gradient separation, filtration, size exclusion chromatography, immunoaffinity separation, precipitation, and microfluidic separation.

8. The method of claim 1, wherein the step of isolating the exosomes comprises steps of: centrifuging the mixture after the culturing to obtain a supernatant; filtering the supernatant through a filter to obtain a filtrate; and concentrating the filtrate.

9. The method of claim 1, wherein the step of isolating the exosomes further comprises a step of storing a supernatant at 4° C. or below for 7 days to 1 month before filtering the supernatant through the filter.

10. The method of claim 1, wherein the isolated exosomes have a diameter of 50 to 200 nm.

11. Exosomes having skin regeneration and wound healing effects, which are produced by the method according to claim 1.

12. The exosomes of claim 11, wherein the exosomes have increased RNA of a gene that promotes skin regeneration and wound healing, compared to exosomes secreted from the cells before being subjected to the method of claim 1, wherein the gene that promotes skin regeneration and wound healing is any one or more of EGFR, FN1, MMP9, MMP2, MAPK1, FGF2, CTGF, AKT1, ALB, TGFB1, MAPK3, VEGFA, FGF7, HGF, IL6, EGF, miR-210, miR-31, Col1α1, ELN, PCNA, N-cadherin and cyclin-D1.

13. The exosomes of claim 11, wherein the exosomes, when applied to a skin, further increase RNA expression of a gene that promotes skin regeneration and wound healing in the skin, compared to exosome secreted from the cells before being subjected to the method of claim 1, wherein the gene that promotes skin regeneration and wound healing is any one or more of Col1α1, Col3α1, ELN, PCNA, N-cadherin and cyclin-D1.

14. A composition containing the exosomes having skin regeneration and wound healing effects according to claim 11.

15. The composition of claim 14, containing the exosomes at a concentration of 106 exosomes/ml or more.

Description

DESCRIPTION OF DRAWINGS

[0028] FIG. 1 shows data related to the production of exosomes having skin regeneration and wound healing effects according to Example 1 of the present invention, and depicts immunofluorescence staining images showing induction of the exosome marker CD63.

[0029] FIG. 2 shows the data of gene expression in the exosomes having skin regeneration and wound healing effects according to Example 1 of the present invention, and shows the results of gene ontology analysis of genes having 12 or more read counts in the exosomes (iExo) by RNA-seq.

[0030] FIG. 3 shows the data of gene expression in the exosomes having skin regeneration and wound healing effects according to Example 1 of the present invention, and shows the results of RNA-seq of wound healing related genes.

[0031] FIG. 4 shows the data of gene expression in the exosomes having skin regeneration and wound healing effects according to Example 1 of the present invention, and shows the results of RNA-seq related to angiogenesis, cell growth, cell migration, cell proliferation, inflammatory response, regeneration, secretion, tissue regeneration, and tissue remodeling.

[0032] FIG. 5 shows the data of gene expression in the exosomes having skin regeneration and wound healing effects according to Example 1 of the present invention, and shows qRT-PCR data obtained by analyzing the RNAs of skin regeneration- and wound healing-related genes in the exosomes.

[0033] FIG. 6A and FIG. 6B show data related to the in vitro effects of the exosomes having skin regeneration and wound healing effects according to Example 1 of the present invention, and shows data obtained by analyzing the proliferation of HDF cells treated with various concentrations of the exosomes.

[0034] FIG. 7A and FIG. 7B show data related to the in vitro effects of the exosomes having skin regeneration and wound healing effects according to Example 1 of the present invention, and shows the results of measuring the migration rate of HDFs, treated with various concentrations of the exosomes, by scratch assay.

[0035] FIG. 8A, FIG. 8B and FIG. 8C show data related to the in vitro effects of the exosomes having skin regeneration and wound healing effects according to Example 1 of the present invention, and shows data obtained by analyzing tube formation of HUVECs treated with various concentrations of the exosomes.

[0036] FIG. 9 shows data related to the in vitro effects of the exosomes having skin regeneration and wound healing effects according to Example 1 of the present invention, and shows qRT-PCR data obtained by analyzing the RNAs of skin regeneration- and wound healing-related genes in cells treated with the exosomes.

[0037] FIG. 10 shows data related to the in vivo effects of the exosomes having skin regeneration and wound healing effects according to Example 1 of the present invention, and depicts showing the time-dependent wound healing process in mice treated with various concentrations of the exosomes.

[0038] FIG. 11 shows data related to the in vivo effects of the exosomes having skin regeneration and wound healing effects according to Example 1 of the present invention, and depicts H&E staining images of wound site tissue sections in mice treated with various concentrations of the exosomes.

[0039] FIG. 12 shows data related to the in vivo effects of the exosomes having skin regeneration and wound healing effects according to Example 1 of the present invention, and shows data obtained by analyzing a cell proliferation marker in wound sites in mice treated with various concentrations of the exosomes.

[0040] FIG. 13 shows data related to the in vivo effects of the exosomes having skin regeneration and wound healing effects according to Example 1 of the present invention, and shows data obtained by analyzing skin penetration of the exosomes.

[0041] FIG. 14 shows data related to the in vivo effects of the exosomes having skin regeneration and wound healing effects according to Example 1 of the present invention, and shows the results of RT-PCR for genes in wound sites in mice treated with various concentrations of the exosomes.

[0042] FIG. 15 shows data related to the in vivo effects of the exosomes having skin regeneration and wound healing effects according to Example 1 of the present invention, and shows images comparing the development of subcutaneous blood vessels in the wound site 3 weeks after wound formation in mice treated with the exosomes.

BEST MODE

[0043] Hereinafter, the present invention will be described in more detail. However, the present invention may be embodied in various different forms and is not limited by the embodiments described herein, and the scope of the present invention should be defined only by the appended claims.

[0044] The terminology used herein is only for the purpose of describing particular embodiments and is not intended to be limiting of the present invention. Singular expressions include plural expressions unless otherwise specified in the context thereof. Throughout the present specification, it is to be understood that when any part is referred to as “comprising” any component, it does not exclude other components, but may further comprise other components, unless otherwise specified.

[0045] A method for producing exosomes having skin regeneration and wound healing effects according to one aspect of the present invention comprises steps of: providing ultrasound stimulation directly or indirectly to cells; culturing a mixture of the cells and a medium for a predetermined time; and isolating exosomes from the mixture, wherein providing the stimulation directly to the cells comprises applying ultrasound stimulation to the medium containing the cells, and providing the stimulation indirectly to the cells comprises applying ultrasound stimulation to the medium not containing the cells and then mixing the medium and the cells.

[0046] Here, the step of providing ultrasound stimulation directly or indirectly to cells may be performed by any one method selected from among: a method of mixing the cells and the medium and then providing ultrasound stimulation to the mixture; or a method of providing ultrasound stimulation to the medium and then mixing the medium and the cells; or a method of providing ultrasound stimulation to the cells and then mixing the cells and the medium; or a method of providing ultrasound stimulation to the cells and then mixing the cells and the medium, followed by providing ultrasound stimulation to the mixture; or a method of providing ultrasound stimulation to the medium and then mixing the medium and the cells, followed by providing ultrasound stimulation to the mixture; or a method of providing ultrasound stimulation to each of the cells and the medium and then mixing the cells and the medium; or a method of providing ultrasound stimulation to each of the cells and the medium and then mixing the cells and the medium, followed by providing ultrasound stimulation to the mixture. The ultrasound stimulation may be provided directly or indirectly to the cells one or more times or may be provided using a combination of one or more selected from the above methods, and as the number of times increases, the skin regeneration and wound healing effects of exosomes may increase proportionally. When the ultrasound stimulation is provided one or more times as described above, it is preferable to provide a time interval between the provisions so that the cells can recover, and the time interval may be at least 1 day, more preferably at least 2 days.

[0047] Providing the ultrasound stimulation directly to the cells may be performed at an ultrasound intensity of 0.1 to 3 W/cm.sup.2 and a frequency of 20 kHz to 20 MHz for a duration of 0.1 seconds to 20 minutes, and Providing the ultrasound stimulation indirectly to the cells may be performed at an ultrasound intensity of 1 to 20 W/cm.sup.2 and a frequency of 20 kHz to 20 MHz for a duration of 0.1 seconds to 20 minutes. More preferably, providing the ultrasound stimulation directly may be performed at an ultrasound intensity of 0.5 to 2 W/cm.sup.2 and a frequency of 20 kHz to 2 MHz for a duration of 0.1 seconds to 10 minutes, and providing the ultrasound stimulation indirectly to the cells may be performed at an ultrasound intensity of 2 to 10 W/cm.sup.2 and a frequency of 20 kHz to 2 MHz for a duration of 1 second to 15 minutes.

[0048] The cells may be selected from among mammalian cells excluding mammalian germ cells. Preferably, the cells may be selected from the group consisting of mammalian stem cells, progenitor cells, fibroblasts, keratinocytes, or organ tissue cells, and more preferably, may be any one type of mammalian fibroblasts or organ tissue cells. This is because the method for producing exosomes having skin regeneration and wound healing effects according to one aspect of the present invention makes it possible to obtain the exosomes using any type of cells, and thus cells that are easy to obtain, maintain and multiply are easier and more efficient than stem cells or progenitor cells, which are difficult to obtain and difficult to multiply. The cells may be either autologous, allogeneic or heterologous depending on the future use of the exosomes in vivo as described below. When the cells are heterologous, the cells may be of mammalian origin. Since there is a possibility for an immune rejection reaction, preferably the cells may be allogeneic, most preferably autologous. In order to reduce the likelihood of immune rejection, the cells are preferably allogeneic cells, most preferably autologous cells.

[0049] The step of providing ultrasound stimulation to the cells may be performed either by directly treating the cells with ultrasound or in a state in which only a minimum amount of the cells are contained in an initial culture medium so that the cells barely covers the initial culture medium. Here, the initial culture medium is a common medium used to maintain the cells in a healthy state, and may be a medium suitable for normal culture of the cells. For example, the cells may be fibroblasts, and the initial culture medium may be DMEM medium containing antibiotics and serum.

[0050] The culture medium (a medium that is treated with ultrasound without cells) may be selected from among epithelial stem cell medium, dermal stem cell medium, adipose stem cell medium, mesenchymal stem cell medium, or embryonic stem cell medium, but is not limited thereto. For example, the culture medium may be any one of skin stem cell medium, bone marrow stem cell medium, vascular endothelial progenitor cell medium, vascular progenitor cell medium, mesenchymal progenitor cell medium, epithelial stem cell medium, dermal stem cell medium, hematopoietic stem cell medium, adipose stem cell medium, mesenchymal stem cell medium, embryonic stem cell medium, and various adult stem cell media. The invention of Korean Patent Application No. 10-2018-0060317 filed in the name of present inventor discloses that, when exosomes are obtained by culturing a mixture of an ultrasound-treated second cell culture medium (a medium capable of culturing second cells or inducing differentiation into second cells) and ultrasound-treated first cells and other cells are treated with the exosomes, the cells may be reprogrammed into the second cells, and that stem cells or extracts or conditioned media thereof all exhibit an effect on skin regeneration and wound healing.

[0051] The culturing of the mixture may be performed for 1 to 10 days, preferably 1 to 6 days, most preferably 1 to 2 days. This is because exosomes are secreted in the largest amount on day 1 after ultrasound treatment, and their secretion decreases over time, and this decrease in secretion indicates a possibility that there is a change in the components.

[0052] The step of isolating the exosomes may be performed using one or more of ultracentrifugation, density gradient separation, filtration, size exclusion chromatography, immunoaffinity separation, precipitation, and microfluidic separation.

[0053] The step of isolating the exosomes may comprise steps of: centrifuging the mixture after the culturing to obtain a supernatant; filtering the supernatant through a filter to obtain a filtrate; and concentrating the filtrate. The centrifugation is performed to remove cell debris and dead cells, and may preferably be performed at 1,000 to 5,000 g for 10 to 60 minutes. The step of filtering the supernatant through the filter is performed to further remove cellular debris and obtain only particles having a specific size or less, and the filter used herein may be preferably a syringe filter. The step of concentrating the filtrate may preferably be performed using a centrifugal filter. Where the centrifugal filter is used, it is possible to remove particles having a specific size or less while concentrating the filtrate. The step of isolating the exosomes may further comprise a step of storing the supernatant at 4° C. or below for 7 days to 3 months before filtering the supernatant through the filter. The storage may preferably be done at 4° C. or less for 7 days or less, more preferably −20° C. or less for 1 month or less, most preferably −80° C. or less for 3 months or less. The active ingredients of the exosomes include mRNA and protein, and these ingredients may be more easily denatured or degraded as the temperature is higher or closer to the temperature at which the enzyme activity is high.

[0054] The isolated exosomes may have a diameter of 50 to 200 nm, preferably 100 to 150 nm.

[0055] Exosomes having skin regeneration and wound healing effects according to another aspect of the present disclosure may be produced by the above-described production method.

[0056] Here, skin regeneration and wound healing may include one or more of anti-aging, wrinkle prevention, wrinkle reduction, wound healing, scar healing, and tissue regeneration.

[0057] The exosomes may have increased RNA of a gene that promotes skin regeneration and wound healing, compared to exosomes secreted from the cells before being subjected to the above-described production method, wherein the gene that promotes skin regeneration and wound healing may be any one or more of EGFR (epidermal growth factor receptor), FN1 (fibronectin 1), MMP9 (matrix metallopeptidase 9), MMP2 (matrix metallopeptidase 2), MAPK1 (mitogen-activated protein kinase 1), FGF2 (fibroblast growth factor 2), CTGF (connective tissue growth factor), AKT1 (AKT serine/threonine kinase 1), ALB (albumin), TGFB1 (transforming growth factor beta 1), MAPK3 (mitogen-activated protein kinase 3), VEGFA (vascular endothelial growth factor A), FGF7 (fibroblast growth factor 7), HGF (hepatocyte growth factor), IL6 (interleukin 6), EGF (epidermal growth factor), miR-210 (MicroRNA 210), miR-31 (MicroRNA 31), Col1α1 (collagen 1 alpha 1), ELN (elastin), PCNA (proliferatin cell nuclear antigen), N-cadherin, and cyclin-D1.

[0058] The exosomes, when applied to the skin, may further increase RNA expression of a gene that promotes skin regeneration and wound healing in the skin, compared to exosome secreted from the cells before being subjected to the above-described production method, wherein the gene that promotes skin regeneration and wound healing may be any one or more of Col1α1, Col3α1, ELN, PCNA, N-cadherin, and cyclin-D1.

[0059] Still another aspect of the present invention provides a composition containing the exosomes having skin regeneration and wound healing effects.

[0060] Here, the composition may contain the exosomes at a concentration of at least 10.sup.6 exosomes/ml, more preferably 10.sup.6 to 10.sup.14 exosomes/ml, more preferably 10.sup.11 to 10.sup.12 exosomes/ml. This is because if the concentration of exosomes in the composition is less than 10.sup.6 exosomes/ml or more than 10.sup.14 exosomes/ml, the skin regeneration and wound healing effects may decrease, and in particular, if the concentration is excessively high, the economic efficiency may also decrease.

[0061] It is obvious that the composition may further contain a pharmaceutically acceptable carrier and/or other additives that can further enhance the uptake of active ingredients or regeneration and healing responses, in addition to the exosomes, and thus detailed description of the pharmaceutically acceptable carrier and other additives will be omitted.

MODE FOR INVENTION

[0062] Hereinafter, examples of the present invention will be described in detail so that the present invention can be easily carried out by those skilled in the art. However, the present invention may be embodied in various different forms, and is not limited to the examples described herein.

[0063] All cell culture processes in the Examples and Experimental Examples of the present invention were performed at 37° C. under 5% CO.sub.2.

Example 1. Production of Exosomes Having Skin Regeneration and Wound Healing Effects

[0064] To obtain exosomes having skin regeneration and wound healing effects, ultrasound stimulation with 20 KHz and 1.0 W/cm.sup.2 was applied directly to 1×10.sup.6 human dermal fibroblasts (HDFs) for 5 seconds by means of UltraRepro 1001 (STEMON Inc., Seoul, Republic of Korea). 2×10.sup.5 UHDFs (the HDFs to which ultrasound stimulation was applied) were cultured with an ultrasound-treated (at 20 KHz and 5.0 W/cm.sup.2 for 10 min) embryonic stem cell medium (DMEM/F12, 15% FBS, 2 mM GlutaMAX, 0.1% NEAA, 0.1% penicillin/streptomycin, 0.1 mM β-mercaptoethanol, 1000 units/ml leukemia inhibitory factor (LIF)) in a 35-mm Petri dish for one day. Exosomes were isolated from the conditioned medium of the ultrasound-treated HDFs (UHDFs) as follows: The conditioned medium was centrifuged at 3,000×g for 20 minutes to remove cell debris and dead cells, and then the supernatant was passed through a 0.22-mm filter (Minisart® Syringe Filter, Sartorius, Goettingen, Germany). The medium that passed through the filter was placed in Amicon® Ultra-15 100,000 kDa device (Millipore, Billerica, Mass., USA) and centrifuged at 14,000×g for 20 minutes to concentrate exosomes (iExo).

Experimental Example 1. Experiment for Analysis of Exosomes Having Skin Regeneration and Wound Healing Effects

[0065] The UHDFs cultured according to the method of Example 1 for inducing exosomes having skin regeneration and wound healing effects were analyzed through immunofluorescence staining for the CD63 exosome marker (at this time, counter staining was performed using the nuclear staining dye DAPI). As a result, it could be confirmed that the CD63 signal intensity in the UHDFs was significantly higher than that in the control group, suggesting that a large amount of exosomes were produced in the UHDFs (FIG. 1). As a result of RNA-Seq analysis for the produced exosomes, it was shown that expression of particularly genes related to wound healing, angiogenesis, cell growth, cell migration, cell proliferation, inflammatory response, regeneration, secretion, tissue regeneration, and tissue remodeling, was significantly higher in the exosomes (iExo) produced according to Example 1 than in exosomes (nExo) isolated from cells cultured without ultrasound stimulation (FIGS. 2 to 4), the genes typically including genes encoding proteins such as EGFR, FN1, MMP9, MMP2, MAPK1, FGF2, CTGF, AKT1, ALB, TGFB1, MAPK3, VEGFA, FGF7, HGF, IL6, and EGF, and microRNAs such as miR-210 and miR-31 (FIG. 3).

[0066] In addition, RNA was extracted from the exosomes (iExo) isolated from the conditioned medium of the cells and was analyzed by qRT-PCR. As a result, it could be confirmed that expression of Col1α1, Col3α1 and ELN, which are extracellular matrix-related genes involved in tissue regeneration and wound healing, and PCNA, N-cadherin and cyclin-D1, which are cell proliferation-related genes, was higher in the exosomes (iExo) than in the exosomes (nExo) isolated from the control group (FIG. 5).

Experimental Example 2. Experiment for Analysis of Phenotypes of Cells Treated with Exosomes Having Skin Regeneration and Wound Healing Effects

[0067] The exosomes produced according to the method of Example 1 for producing exosomes having skin regeneration and wound healing effects were added to medium, and HDFs were cultured in the medium. Then, concentration-dependent effects of the endosomes were analyzed. First, as a result of analyzing the cell proliferation effect of the exosomes through immunofluorescence staining for Ki67, a related marker, it could be confirmed that expression of the marker was higher in the experimental group than in the control group, and particularly when the cells were treated with the exosomes at concentrations of 5×10.sup.11 exosomes/ml and 10×10.sup.11 exosomes/ml, the effect of the exosomes was better and the number of the cells increased more (FIGS. 6A and 6B). The HDFs were cultured to fill the plate, and then wound healing assay was performed by creating lines by a 20 μl yellow tip to increase the spacing between the cells, and then treating the cells with the exosomes, followed by culture. As a result, it was confirmed that the empty space in the exosome-treated group was filled relatively quickly (FIGS. 7A and 7B), suggesting that the migration rate of the cells in the exosome-treated group was higher.

[0068] Next, in order to analyze angiogenesis efficiency during tissue regeneration depending on the concentration of the exosomes produced according to Example 1, endothelial cells (HUVECs) were treated with different concentrations of the exosomes, and after 10 hours, tube formation was checked. As a result, it could be visually confirmed that the exosome-treated groups easily formed tubes compared to the control group (FIG. 8A), and the tube length and the number of branch points were also higher in the exosome-treated groups (FIGS. 8B and 8C), particularly higher in the groups treated with the exosomes at concentrations of 5×10.sup.11 exosomes/ml and 10×10.sup.11 exosomes/ml.

[0069] Finally, expression of Col1α1, Col3α1, ELN, PCNA, N-cadherin and cyclin-D1, which are tissue regeneration- and wound healing-related genes, in the exosome-treated cells, was analyzed by qRT-PCR. As a result, it was confirmed that expression of the wound healing-related genes in the exosome-treated group was higher than that in the control group (FIG. 9).

Experimental Example 3. Experiment for Analysis of In Vivo Effect of Treatment with Exosomes Having Skin Regeneration and Wound Healing Effects

[0070] In order to analyze the in vivo effect of the exosomes (iExo) produced according to the method of Example 1 for producing exosomes having skin regeneration and wound healing effects, a 6-mm wound was created on the skin of each 6-week-old C57 mouse using a punch, and iExo were diluted in PBS at concentrations of 0, 5, 10 and 20×10.sup.11/ml and applied to each wound site twice a week.

[0071] As a result, it could be confirmed that the size of the externally exposed wound (opening) in the exosome-treated mice after 1 week significantly decreased compared to that in the untreated mice (FIG. 10; in this regard, the wound in the exosome-treated mice at week 2 was completely closed and the hair grew over the wound, and thus it was not possible to take a comparative picture between the treated groups). In addition, after 1 week and 2 weeks, the tissue section of each wound site was analyzed by H & E staining. As a result, it could be confirmed that the wound size in the exosome-treated mice was smaller than that in the non-exosome-treated mice, and thus skin regeneration and wound healing occurred more quickly in the exosome-treated mice. In particular, when the wound site was treated with the exosomes at a concentration of 10 or 20×10.sup.11 exosomes/ml, the skin tissue was completely recovered at week 2, and thus the epidermis, dermal layer, fat layer and muscle layer constituting the skin tissue were regenerated so that they could be clearly distinguished from one another. In addition, in this case, hair follicles, which are skin appendages, were located in the dermal layer and the fat layer without any difference between the wound site and the surrounding area. In particular, it was confirmed that, when the wound site was treated with the exosomes at a concentration of 10 or 20×10.sup.11 exosomes/ml, even the scar disappeared at week 2 (FIG. 11). Additionally, as a result of analyzing the cell proliferation marker Ki67 in the tissue section of the site recovered from the wound through immunofluorescence staining, it could be confirmed that Ki67 was more extensively observed in the exosome-treated group than in the non-exosome-treated group (FIG. 12), indicating that cell proliferation actively occurred at the wound site. iExo were stained with Vybrant™ DiD cell-labeling solution and the wound site was treated with the stained iExo, and after 2 weeks, the iExo-treated site was analyzed under a fluorescence microscope. As a result, it could be confirmed that the iExo penetrated the entire wound site, and the above-described regeneration and healing effects could appear due to this effective penetration (FIG. 13).

[0072] Next, expression of the tissue regeneration- and wound healing-related genes Col1α1, Col3α1, ELN, N-cadherin and cyclin-D1 in the wound site treated with the exosomes was analyzed by qRT-PCR, and as a result, it was confirmed that expression of one or more of the genes in the experimental group treated with iExo was generally higher than that in the control group (FIG. 14). At this time, the gene with the greatest increase in expression was different between the exosome concentrations, and it was difficult to confirm the correlation between the concentration and the increase or decrease in expression.

[0073] Finally, at week 3 when wound healing was completed, the degree of development of subcutaneous blood vessels was analyzed. As a result, it was confirmed that blood vessels in the tissue treated with iExo more developed (FIG. 15).

[0074] Taken together, the exosomes produced according to Example 1 can improve the wound healing and regeneration ability of tissue by promoting cell proliferation and migration and blood vessel development. In addition, since the effect of the exosomes also appears in vivo, the exosomes may increase the rate of skin regeneration and wound healing, and may also induce more complete skin regeneration and wound healing so that skin appendages may be regenerated with minimized scars or without scars.

Comparative Example 1. Control for Exosomes

[0075] Non-ultrasound-treated HDFs (NHDFs) were cultured in a fibroblast medium (10% fetal bovine serum (Gibco)) containing 2 mM GlutaMAX™-I supplement (Gibco), 6 U/mL heparin (Sigma-Aldrich) and 200 μM ascorbic acid (Sigma-Aldrich), and DMEM (Gibco) containing 1% penicillin/streptomycin (Gibco)). Isolation of exosomes (nExo) from the conditioned media of the NHDFs was performed in the same manner as the exosome isolation method of Example 1.

Comparative Example 2. Control for Cells Treated with Exosomes

[0076] 1×10.sup.5 HDFs were seeded and cultured in a 35-mm Petri dish for one day, and then the medium was replaced with a medium containing the exosomes obtained according to Comparative Example 1, followed by 5 days of culture. At this time, embryonic stem cell culture medium was used as the medium containing the exosomes, and the culture medium was replaced every 2 days.

Comparative Example 3. Control for Mice in which Skin Regeneration and Wound Healing were Induced by Exosomes

[0077] The iExo dilution vehicle PBS was applied twice a week to mice with a punch wound of 6 mm in diameter.

[0078] The above description of the present invention is exemplary, and those of ordinary skill in the art will appreciate that the present invention can be easily modified into other specific forms without departing from the technical spirit or essential characteristics of the present invention. Therefore, it should be understood that the embodiments described above are exemplary in all aspects and are not restrictive. For example, each component described to be of a single type can be implemented in a distributed manner. Likewise, components described to be distributed can be implemented in a combined manner.

[0079] The scope of the present invention is defined by the appended claims, and it shall be understood that all modifications and alterations conceived from the meaning and scope of the claims and equivalents thereto are included in the scope of the present invention.

INDUSTRIAL APPLICABILITY

[0080] The exosomes having skin regeneration and wound healing effects according to the present invention and a composition containing the same contain large amounts of expression products of various factors capable of inducing skin regeneration and wound healing, particularly genes that promote skin regeneration and wound healing. In addition, the exosomes have a phospholipid-based membrane structure, and thus may easily penetrate the skin and deliver substances with high efficiency, thus effectively inducing skin regeneration and wound healing. Moreover, the exosomes do not have side effects occurring due to the use of synthetic drugs, have a longer-lasting effect, and may be used as a drug capable of inducing regeneration and healing when tissues including skin are damaged.

[0081] In addition, according to the method for producing exosomes having skin regeneration and wound healing effects according to the present invention, exosomes containing high concentrations of various active ingredients having the above-described effects may be obtained in high yield through an easy process of treating cells with ultrasound stimulation.