KR20220080050A - Exosome isolated from dermal papilla precursor cells and uses thereof - Google Patents

Abstract

The present invention relates to exosomes isolated from dermal papilla progenitor cells, and provides exosomes isolated from dermal papilla progenitor cells, which are excellent in preventing, improving, and treating hair loss, as well as improving skin and wound healing, and various uses thereof can do.

Description

Exosome isolated from dermal papilla precursor cells and uses thereof

The present invention relates to exosomes and their uses, and more particularly, to exosomes isolated from dermal papilla progenitor cells and various uses thereof.

Hair loss is a phenomenon in which hair falls out of the scalp through growth, regression, and resting phases in the hair cycle. Generally, hair growth and hair loss occur periodically for about 3-5 years. However, if the growth/hair loss balance is disrupted by various factors and the number of hairs lost per day becomes more than 100, it is judged as a disease called "hair loss". The factors of such alopecia are known as internal factors such as heredity or male hormone degeneration, and external factors caused by stress in daily life and harmful substances (lipid peroxide, etc.) accumulated on the scalp. It is denaturation (modification from testosterone to dihydrotestosterone).

Common methods for treating alopecia include drug treatments such as Propecia and Avodart, which are known as representative hair loss treatment drugs, and a hair transplantation method in which another person's hair is transplanted. Propecia and Avodart, which are widely used for drug treatment, were originally known as drugs for the treatment of benign prostatic hyperplasia. On the other hand, the hair transplantation method is a method of transplanting one's own or another person's hair, so it has the best effect, but it is temporary because it does not treat the underlying cause, and there is a problem in that it causes pain during transplantation. In order to solve the above problems, a bio-derived material or component can be used as an active ingredient, and an example of the bio-derived material is an exosome. An exosome is a vesicle composed of a lipid-bilayer, and is a constituent of a substance secreted by a cell to the outside of the cell. Exosomes are known to play a role in transporting (transporting) proteins, bioactive lipids, and RNA (miRNA), which are biomolecules in cells, in order to perform a functional role that mediates cell-cell communication and cellular immunity. . These exosomes are being studied as biomarkers for neurological diseases such as Alzheimer's, and have high selective permeability enough to penetrate the blood-brain barrier (BBB) that separates cerebrospinal fluid and blood, making them a nanocarrier for specific drugs. It is also used in the development of drug delivery systems such as

As the prior art related to the treatment for hair loss, a composition for preventing hair loss or promoting hair growth (Registration Patent Publication No. 10-2112736), a composition for preventing hair loss or promoting hair growth comprising iron as an active ingredient (Patent Publication No. 10-2019-0046697) ho), etc., but they are related to chemical drug therapeutics and thus have different technical characteristics from the present invention.

On the other hand, in the case of chemical component-based therapeutics, side effects are often accompanied as well, so there is an increasing need to develop drugs that have excellent therapeutic effects for specific diseases and few side effects. Accordingly, the present inventors have studied the efficacy of exosomes, and as a result of separating the exosomes from dermal papilla progenitor cells, they attempted to complete the invention for various uses thereof.

Registered Patent Publication No. 10-2112736 Unexamined Patent Publication No. 10-2019-0046697

Gnedeva et al., PLoS One 10, e0116892, 2015

In order to solve the above problems, the present invention provides an exosome derived from dermal papilla progenitor cells with few side effects and excellent effects for preventing, improving or treating hair loss, improving skin and treating wounds, and hair loss comprising the same as an active ingredient. To provide a composition for preventing, improving, or treating, a composition for skin improvement, and a composition for treating wounds or scars.

In addition, it is intended to provide a composition for a filler comprising the exosome as an active ingredient.

In addition, it is intended to provide a method for preventing, improving or treating hair loss, a method for skin improvement, and a method for treating a wound or scar using a composition comprising the exosome.

In order to achieve the above object, the present invention provides an exosome isolated from a dermal papilla progenitor cell or a dermal papilla progenitor cell culture.

The exosome of the present invention is derived from dermal papilla precursor cells (DPPC), and the dermal papilla precursor cells may be derived from human-derived dermal papilla precursor cells, mouse-derived dermal papilla precursor cells, or cultures thereof, but are limited thereto. it is not

In addition, the dermal papilla progenitor cells are differentiated from human stem cells or mouse-derived stem cells, including embryonic stem cells, adult stem cells, induced pluripotent stem cells, hematopoietic stem cells, neural stem cells, mesenchymal stem cells, or the like. It may be isolated from dermal papilla progenitor cells or their cultures.

The culture may refer to a culture medium obtained by culturing the human or mouse-derived stem cells or dermal papilla progenitor cells differentiated or produced from them in a culture medium, or drying, filtration and/or concentration of the culture medium, and ,

The culture solution can be cultured for 1 to 7 days, preferably from 12 hours to 120 hours, more preferably from 24 hours to 96 hours, more preferably from 48 hours to 96 hours, or from 60 hours to 84 hours, more Preferably, after culturing for 72 hours, the culture medium may be collected and centrifuged, but may be a supernatant, but is not limited thereto.

In the supernatant, size exclusion chromatography, ion exchange chromatography, density gradient centrifugation, differential centrifugation, ultrafiltration, precipitation ( exosome precipitation, total exosome isolation kit, immuno-absorbent capture, affinity method, e.g., affinity capture, affinity purification ( affinity purification), immunoassay, microfluidic separation, or a combination thereof can be performed to extract exosomes, and in addition to the above method, in the method commonly used in the art for isolating exosomes It can also be separated by

More preferably, the culture medium is centrifuged at 200-400 x g for 5 to 20 minutes to remove the remaining cells and cell residues, and the supernatant is taken and centrifuged at 9,000-12,000 x g for 60-80 minutes, followed by high-speed centrifugation, again Taking the supernatant, centrifuging at 90,000-120,000 x g for 80-100 minutes, and removing the supernatant, exosomes remaining in the lower layer can be obtained.

According to a specific embodiment of the present invention, the culture medium is collected and centrifuged at 300 x g for 10 minutes to remove the remaining cells and cell residues, the supernatant is taken and filtered using a 0.22 μm filter, and then a high-speed centrifuge ( Centrifuge at 10,000 x g, 4℃ for 70 minutes using a high speed centrifuge. The centrifuged supernatant is taken again and centrifuged at 100,000 x g, 4° C. for 90 minutes using an ultracentrifuge to remove the supernatant, thereby separating the exosomes remaining in the lower layer. Exosomes can be extracted by methods commonly used to isolate exosomes in the art in addition to the above method.

The size of the exosomes isolated from the dermal papilla progenitor cells of the present invention may be 30 to 200 nm, preferably 30 to 180 nm, more preferably 50 to 150 nm, but is not limited thereto.

In addition, the exosome of the present invention may include 1x10 ¹⁶ /cell to 1x10 ¹⁷ /cell, preferably 2x10 ¹⁶ /cell to 5x10 ¹⁶ /cell per cell, but is not limited thereto.

In addition, the exosome of the present invention may express any one or more markers selected from CD63, CD9, and CD81.

The expression rate of the marker is not limited, but preferably, the expression rate of CD63 among the markers may be 80% or more, and more preferably 90% or more.

In addition, the amount of the exosomes separated from the exosomes of the present invention may vary depending on the culture method of dermal papilla progenitor cells. Compared to the two-dimensional culture, which is a general cell culture method, the number of separated exosomes may vary depending on the three-dimensional culture method, the hypoxic culture, or the mixed culture with other cells.

Specifically, the three-dimensional culture method refers to a method in which cells are cultured in a suspended state in a liquid medium without adhering to a culture dish, and preferably, 10 μg/ml FGF2, 100 units/ml penicillin, 100 μg of dermal papilla progenitor cells. Cells cultured in 10% FBS (Fetal bovine serum) DMEM/F12 + glutamax (1:1) containing /ml streptomycin were used. For dermal papilla progenitor cells, 2~3 cells were recovered by trypsin, and then 10 ⁴ cells per well in an ultra-low cluster, 96-well culture dish in 10% DMEM/F12 + glutamax (1:1) medium. Incubate in a cell incubator for 24 hours. After culture, DMEM/F12 + glutamax (1:1) may be changed to serum-free medium and cultured under culture conditions for 48 hours.

In the case of hypoxic culture, the amount of oxygen in the atmosphere may be 0.1 to 10%, and more preferably, it may be cultured under 1 to 2% oxygen condition.

Mixed culture with other cells may be cultured ⁱⁿ the same culture dish or cell culture insert (Transwell, etc.) It can be cultured in a cell incubator in DMEM/F12 + glutamax (1:1) medium for 24 hours.

After the culture, the culture medium is removed, and 10 ⁴ keratinocytes per well are put and cultured in a cell incubator. After 24 hours, it can be washed using Epilife medium without a supplement and cultured by putting the medium, and the incubation time is 24 hours to 72 hours, preferably 48 hours, but is not limited thereto.

Cells that are mixed cultured are cells related to skin and hair, and may be dermal papilla progenitor cells, dermal papilla cells, keratinocytes, lateral hair root sheath cells, melanocytes, fibroblasts, and the like, but are not limited thereto.

The present invention may provide a composition comprising the exosome as an active ingredient, and the composition may be a composition selected from pharmaceutical compositions, cosmetic compositions, and food compositions.

The pharmaceutical composition in the present invention may be a formulation selected from the group including tablets, capsules, injections, creams, gels, patches, sprays, ointments, warning agents, lotions, liniment agents, pasta agents and cataplasmases, etc. However, the present invention is not limited thereto.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, lubricant, wetting agent, sweetening agent, flavoring agent, emulsifying agent, suspending agent, preservative and the like commonly used in formulation.

The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, mucosal administration and eye drop administration, topical administration, etc. It can be administered, but is not limited thereto.

A suitable dosage of the pharmaceutical composition of the present invention may vary depending on factors such as formulation method, administration method, age, weight, sex, pathology, food intake, administration time, administration route, excretion rate and reaction sensitivity of the patient. can be prescribed. Preferably, the dosage of the pharmaceutical composition of the present invention may be 0.0001-100 mg/kg (body weight) based on an adult, but is not limited thereto.

The cosmetic composition in the present invention can be prepared in the form of lotion, lotion, essence, cream, etc., in addition to massage cream, body lotion, body milk, bath oil, shower gel, shower cream, sunscreen, hand lotion, hair lotion, It may be prepared in the form of soap, shampoo, soap, cleansing foam, cleansing oil, cleansing cream, scalp cleaner, etc., but is not limited thereto.

In the preparation of the cosmetic composition of the present invention, it may additionally include conventional adjuvants such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances, and carriers commonly used in the preparation of cosmetics, and also a sugar It may be prepared in a formulation conventionally prepared in the industry.

The food composition in the present invention may be prepared in the form of powder, granules, tablets, capsules or beverages, but is not limited thereto. In addition, additives, flavorings, thickeners, stabilizers, pH adjusters, etc. commonly used in the art for preparing food compositions may be further included. In addition, the method for preparing the food composition may be prepared by a method commonly used for preparing food in the art, and is not limited to a specific method.

The present invention provides a pharmaceutical composition for preventing, improving, or treating hair loss, or a cosmetic composition for preventing or improving hair loss, and a food composition for preventing or improving hair loss, comprising the exosome as an active ingredient.

The hair loss is androgenetic alopecia, telogen hair loss, chemical hair loss, mechanical hair loss, traumatic hair loss, pressure hair loss, genital hair loss, alopecia areata, syphilitic hair loss, seborrheic hair loss, symptomatic hair loss, scarring hair loss, congenital hair loss, circular hair loss hair loss, ringworm of the head, alopecia totalis, hypotrichosis, hereditary hypotrichosis simplex and generalized alopecia, but is not limited thereto.

The cosmetic composition for preventing and improving hair loss is hair tonic, hair conditioner, hair essence, hair lotion, hair nutrition lotion, hair shampoo, hair conditioner, hair treatment, hair cream, hair nutrition cream, hair moisture cream, hair massage cream , hair wax, hair aerosol, hair pack, hair nutrition pack, hair soap, hair cleansing foam, hair oil, hair dryer, hair preservative, hair dye, hair wave agent, hair bleach, hair gel, hair glaze, hair dresser, hair It may be a formulation selected from the group including lacquer, hair moisturizer, hair mousse and hair spray, but is not limited thereto.

In addition, the present invention provides a composition for improving or treating wounds or scars comprising the exosomes as an active ingredient.

The wound refers to a wound on the skin, and specifically, it may refer to damaged skin such as a diabetic foot, a pressure sore, a burn, a laceration (swept wound), or a chronic wound in which the self-healing ability is significantly lowered, but is limited thereto not.

The pharmaceutical composition for the treatment of wounds or scars means that the damaged skin or scars can be treated by promoting the cell regeneration ability of the damaged skin or scarring region to restore the same as normal skin.

The composition for improving or treating wounds or scars may be any one selected from a pharmaceutical composition, a cosmetic composition, and a food composition.

In addition, the present invention provides a pharmaceutical composition for skin improvement, a cosmetic composition or a food composition comprising the exosome as an active ingredient.

The skin improvement may be any one or more selected from skin whitening, wrinkle improvement, elasticity enhancement, skin regeneration, skin moisturizing, anti-aging, alleviating skin irritation, preventing or improving acne, preventing or improving atopy, but is not limited thereto.

In addition, the present invention provides a composition for a filler comprising the exosome as an active ingredient.

The filler composition may be used for promoting hair growth, preventing, improving or treating hair loss, improving skin, treating wounds, etc., but is not limited thereto.

The administration method of the filler composition is not limited in a particular manner, and may be administered by various administration methods known in the art, but preferably may be administered by injection administration.

It is not limited thereto and does not exclude various administration methods known in the art.

The composition for filler may be injected into the skin region including the scalp, preferably administered to the dermis. The administration may include administering to the patient at a depth of about 1 mm or less from the surface of the skin. The composition can be injected at a depth of preferably, but not limited to, about 0.8 mm or less, more preferably about 0.6 mm or less, or about 0.4 mm or less. The composition for fillers, in addition to including the exosomes of the present invention as an active ingredient, additives, fillers, anti-aggregants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, etc. commonly used in the art for manufacturing fillers are added. may include

The method for preparing the composition for filler may be prepared by a method commonly used in the art for preparing the composition for filler, and is not limited to a specific method.

The preparation form of the composition for filler may also be prepared in a form that can be conventionally prepared for preparing the composition for filler, and is not limited to a specific dosage form.

When the exosome is included as an active ingredient of a pharmaceutical composition, a cosmetic composition, a food composition, or a filler composition, it is not limited, but may be included in the number of 1.0x10 ⁶ particles to 1.0x10 ¹² particles (particles). When the content of the exosome is less than 1.0x10 ⁶ particles, the effect may be insignificant, and when the content exceeds 1.0x10 ¹² particles, it may cause toxicity to the human body.

In addition, the present invention can provide a method for preventing, improving, or treating hair loss, a method for improving skin, a method for treating a wound or a scar, and the like by using the exosomes.

The present invention relates to exosomes isolated from dermal papilla progenitor cells, and to provide exosomes isolated from dermal papilla progenitor cells and their various uses, which are excellent in preventing and improving treatment effects of hair loss, as well as improving skin and wound healing. can

1 shows the extraction value of the exosome cells according to the present invention.
Figure 2 shows the marker expression distribution of the exosomes according to the present invention for each derived cell.
3 shows the evaluation results of cell proliferation of the exosomes according to the present invention.
4a and 4b show the results of evaluation of the effect (hair follicle length growth) of the exosomes according to the present invention on the hair follicles.
5 shows the results of evaluating the effect of the exosomes according to the present invention on the hair growth cycle.
6 shows the evaluation results of cell proliferation in the hair follicles of the exosomes according to the present invention.
7 shows the results of evaluation of the expression of growth factors in the dermal papilla cells of the exosomes according to the present invention.
8a, 8b and 8c show the evaluation results of gene expression related to hair growth of exosomes according to the present invention.
Figures 9a and 9b shows the aging recovery evaluation results in the dermal papilla cells aging exosomes according to the present invention.
Figure 10 shows the results of evaluation of the cell mobility of the exosomes according to the present invention.

Hereinafter, the present invention will be described in detail by way of Examples and Experimental Examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Examples and Experimental Examples.

<Examples 1 and 2>

To isolate the exosomes of the present invention, dermal papilla progenitor cells were prepared with reference to the following literature [Gnedeva et al., PLoS One 10, e0116892, 2015]. Using the dermal papilla progenitor cells prepared according to the above method, Example 1 was cultured in a hypoxic condition of 1% oxygen, Example 2 was cultured in a normal culture condition of 5% carbon dioxide, 37°C in a cell incubator.

Incubate dermal papilla progenitor cells in an ultra-low cluster, 96-well culture dish at 10 ⁴ cells per well in 10% DMEM/F12 + glutamax (1:1) medium for 24 hours. After incubation, DMEM/F12 + glutamax (1:1) was replaced with serum-free medium and cultured for 48 hours. The medium was collected and centrifuged at 300 x g for 10 minutes to remove remaining cells and cell residues, and the supernatant. was taken, filtered using a 0.22 μm filter, and then centrifuged at 10,000 x g, 4°C for 70 minutes using a high speed centrifuge. The centrifuged supernatant was taken again and centrifuged for 90 minutes at 100,000 x g and 4°C using an ultracentrifuge to remove the supernatant, thereby separating the exosomes remaining in the lower layer to obtain exosomes.

<Comparative Examples 1 and 2>

The exosome separation method was the same as in Examples 1 and 2, but the exosomes were obtained by separating the dermal papilla cells of Comparative Example 1 or the dermal papilla cells of Comparative Example 2 instead of the dermal papilla progenitor cells.

<Experimental Example 1> Evaluation of characteristics of exosomes according to the present invention (exosome size)

In order to evaluate the characteristics of the exosomes of the present invention, the size of the exosomes isolated from dermal papilla progenitor cells was analyzed using Examples 1 and 2. As a result, the exosomes of the present invention were distributed between 50 and 150 nm, and the average size was found to be 63.6 nm.

<Experimental Example 2> Evaluation of characteristics of exosomes according to the present invention (number of exosomes extracted per cell)

In order to evaluate the characteristics of the exosomes of the present invention, the number of exosomes secreted per cell was comparatively evaluated using Examples and Comparative Example 2. As a result, the exosomes secreted from the dermal papilla progenitor cells according to the present invention were 5x10 ¹⁶ /cell, and the exosomes secreted from the dermal papilla cells of Comparative Example 2 were 2x10 ¹⁶ /cell, so the number of exosomes secreted from the dermal papilla progenitor cells was 2.5. It was found that it was more than twice as high (Fig. 1).

<Experimental Example 3> Evaluation of characteristics of exosomes according to the present invention (confirmation of expression markers)

In order to evaluate the characteristics of the exosomes of the present invention, the distribution of markers expressed in the exosomes was evaluated using Examples and Comparative Examples 1 and 2. As a result of the evaluation, exosome expression markers CD63, CD9, and CD81 were commonly expressed in each cell, but the expression ratio for each marker was different. In the exosomes of dermal papilla progenitor cells, which is an example of the present invention, the CD63 expression rate was about 90-95%. In the exosomes of the fibroblasts of Comparative Example 1, the expression rate of CD63 was 80%, CD81 was about 19%, and CD9 was about 1%. In the exosomes of the dermal papilla cells of Comparative Example 2, CD63 was found to be 98% or more, and the expression rates of CD9 and CD81 were found to be very low (FIG. 2).

<Experimental Example 4> Cell proliferation evaluation

In order to evaluate the hair growth and skin improvement effect of the exosome according to the present invention, dermal papilla cells (DPC), outer root-sheath cells (ORSC), which are known to be important for hair follicle formation and hair growth, Cell proliferation was evaluated for keratinocytes (KC), which are skin cells.

The control group was not treated with any treatment, the positive control group was minoxidil used for hair loss treatment, and the comparative example was treated with fibroblast-derived exosomes, and the evaluation results are shown in FIG. 3 . Experimental method is to incubate 5 x 10 ³ dermal papilla cells per well in a 96-well culture plate in 10% DMEM medium for 24 hours. After 24 hours, it was replaced with a serum-free DMEM medium and treated with the exosomes of Example 1 (1.0x10 ⁹ particles) and minoxidil (1 uM) as a positive control. After 48 hours of incubation, 75 μl of serum-free medium and 25 μl of MTT solution were added, incubated for 3 hours, and then absorbance was measured at 570 nm to confirm cell proliferation. The cell proliferation rate is a graph showing the increase rate of cell proliferation compared to the negative control for the absorbance measurement result.

The outer hair root sheath cells and keratinocytes of the hair follicles are cultured in Epilife medium at 1 x 10 ⁴ cells per well in a 96 well plate for 24 hours. After culturing for 24 hours, the medium was replaced with a medium without supplements, and the exosomes of Example 1 (1.0x10 ⁹ particles) and minoxidil (1 μM) as a positive control were treated, followed by the same conditions as above.

As a result of the evaluation, in Example 1 according to the present invention, proliferation was active in all dermal papilla cells, lateral hair root sheath cells, and keratinocytes, and in particular, it can be seen that Example 1 has an excellent cell proliferation rate compared to minoxidil used as a hair loss treatment ( 3).

<Experimental Example 5> Evaluation of hair follicle growth effect through human hair follicle organ culture test

In order to evaluate the effect of the exosomes of the present invention on human hair follicle growth, a human hair follicle organ culture test (ex vivo hair follicle organ culture) was performed. More specifically, the scalp tissue was separated into hair follicles, and the remaining hair follicles were used by cutting the part below the sebaceous gland of the separated hair follicles. The prepared hair follicles were treated with Examples 1 and 2 and minoxidil as a positive control, and the length of the hair growing on the 3rd and 6th days was measured, and then compared to the negative control group to evaluate the length growth and growth cycle of human hair follicles.

5-1. Hair follicle length growth evaluation 1

To evaluate the effect of exosomes derived from dermal papilla progenitor cells isolated by different cells and culture conditions on the length growth of hair follicles, human hair follicle tissue was treated with 2 mM L-glutamine, 100 U/ml streptomycin, 10 ng/ml hydrocortisone, Cultured in Williams E culture medium containing 10 μg/ml insulin, exosomes of Example 1 (1.0x10 ⁹ particles), exosomes of Example 2 (1.0x10 ⁹ particles), and minoxidil as a positive control (1 μM ) and the exosomes of Comparative Example 2 (1.0x10 ⁹ particles) were treated and cultured in a carbon dioxide incubator at 37°C. The length of the hair follicles grown on the 3rd and 6th days of culture was measured using a microscope ruler. The evaluation result is shown in FIG. 4A.

As a result of the evaluation, in the control group and Comparative Example 1 without any treatment, hair grew by 1 mm for 6 days, and the positive control group grew by about 1.5 mm. On the other hand, the hypoxic cultured Example 1 grew about 2.2 mm for 6 days, and the general cultured Example 2 grew 1.8 mm. can

5-2. Hair follicle length growth evaluation 2

To evaluate the effect of exosomes derived from dermal papilla progenitor cells isolated by different cell and culture methods on the length growth of hair follicles, human hair follicle tissue was treated with 2 mM L-glutamin, 100 U/ml streptomycin, 10 ng/ml hydrocortisone, Exosomes of Example 2 (1.0x10 ⁹ particles) cultured in Williams E culture medium containing 10 μg/ml insulin, and three-dimensionally cultured exosomes of Comparative Example 2 (1.0x10 ⁹ particles) And the exosomes of Example 2 (1.0x10 ⁹ particles) mixed with three-dimensional culture and keratinocytes were treated and cultured in a carbon dioxide incubator at 37°C. The length of the hair follicles grown on the 3rd and 6th days of culture was measured using a microscope ruler. The evaluation results are shown in FIG. 4B .

As a result of the evaluation, the exosomes (1.0x10 ⁹ particles) isolated in Example 2 cultured by a general culture method grew 0.6 mm in hair for 6 days, and the exosomes isolated in Comparative Example 2 cultured in three dimensions (1.0x10 9 particles) x10 ⁹ particles) grew about 1.1 mm. On the other hand, the exosomes isolated in 3D cultured Example 2 (1.0x10 ⁹ particles) grew 1.2 mm, and the exosomes isolated in Example 2 were mixed with 3D culture and keratinocytes (1.0x10 ⁹ particles). ) grew about 1.4 mm, and unlike the general culture method, the hair follicle length growth rate of the exosomes isolated after culturing by the tertiary culture method is superior, and it can be seen that there is a synergistic effect when treated with keratinocytes. have.

5-3. Hair growth cycle evaluation

In order to evaluate the effect of the present invention on the hair growth cycle, only the anagen hair follicles were isolated from the hair follicle tissue and exosomes isolated in Examples 1 and 2 (1.0x10 ⁹ particles), the control group, the positive control group (1 μM) and the comparative example In order to evaluate the growth cycle of hair follicles after treatment with the exosomes isolated in 1 (1.0x10 ⁹ particles), an experiment was conducted with reference to the following literature [Langan et al., Experimental dermatology, 2015]. Growth cycle classification criteria were classified by comparing the distance between the hair shaft and dermal papilla cells after observing human hair follicles under a microscope. The growth phase and the degenerative phase 1 to 3 were divided by scores, and the results are shown in FIG. 5 .

As a result of the evaluation, most of the hair follicles treated with the control group were converted to a catagen phase without a growth phase, and the hair follicles treated with the exosomes isolated in Comparative Example 1 had a shorter catagen phase than the control group, but no growth phase. The hair follicles treated with minoxidil also showed similar results to the hair follicles treated with the exosomes isolated in Comparative Example 1. In contrast, in the hair follicles treated with the exosomes isolated in Example 2 (normal culture conditions) of the present invention, the catagen stage was very short at 20%, and the initial catagen stage was maintained at 40% or more. The hair follicles treated with the exosomes isolated in Example 1 (hypoxic conditions) did not appear at all in the degenerative phase, and 60% in the initial anagen phase and 20% in the anagen phase, indicating that the hair continues to grow.

5-4. Evaluation of hair follicle stromal cell proliferation in hair follicles

In order to evaluate the effect of the exosomes of the present invention on the proliferation of hair matrix cells involved in the direct length growth of hair, only anagen hair follicles were isolated from the hair follicle tissue and used in this experiment. Human hair follicle tissue was cultured in Williams E culture medium containing 2 mM L-glutamin, 100 U/ml streptomycin, 10 ng/ml hydrocortisone, and 10 μg/ml insulin as a control group, a positive control group (1 μM) and three-dimensional culture. The exosomes (1.0x10 ⁹ particles) isolated in Examples 1 and 2 were cultured in a carbon dioxide incubator at 37° C. for 2 days. After culturing, human hair follicles were frozen and cut, followed by immunofluorescence staining for Ki-67. As the number of Ki-67 positive cells in the hair follicle stromal cells increased, it was evaluated that hair follicle growth was increased, and the results are shown in FIG. 6 .

As a result of the evaluation, more Ki-67-positive cells (red) were expressed in the hair follicle tissue treated with the exosomes isolated in Examples 1 and 2 of the present invention compared to minoxidil, which is a control group and a positive control group. It can be seen that active

<Experimental Example 6> Hair growth-related growth factor expression evaluation

In order to evaluate the effect of the exosomes of the present invention on hair growth, the expression of genes important for hair growth in dermal papilla cells was confirmed. In a 6-well culture plate (well plate), 5.0x10 ⁴ dermal papilla cells per well were cultured in 10% DMEM culture medium for 24 hours. After washing twice using serum-free DMEM medium, cultured in serum-free DMEM medium for 24 hours. Exosomes isolated in Examples 1 and 2 (1.0x10 ⁹ particles), a control group and a positive control group (1 μM) were treated for 2 hours, then RNA was extracted and cDNA was synthesized. Expression of FGF7, FGF10, IGF1 and PDGF genes, which are growth factors involved in hair growth, was evaluated using real-time PCR, and the results are shown in FIG. 7 .

As a result of the evaluation, the exosomes isolated in Examples 1 and 2 of the present invention had a higher expression rate of all growth factors compared to minoxidil, a positive control, and in particular, the expression of the growth factors treated with the exosomes isolated in Example 1 was the highest. it can be seen that

<Experimental Example 7> Evaluation of gene expression related to hair follicle formation ability

In order to evaluate how much the exosome of the present invention maintains hair follicle formation performance, the expression of proteoglycan, a Wnt target gene, and a dermal papilla cell marker gene, which are genes involved in the hair follicle formation performance of dermal papilla cells, was checked.

In a 6-well culture dish (well plate), 5.0x10 ⁴ dermal papilla cells per well were cultured in 10% DMEM culture medium for 24 hours. After washing twice using serum-free DMEM medium, cultured in serum-free DMEM medium for 24 hours. Exosomes isolated in Examples 1 and 2 (1.0x10 ⁹ particles), a control group and a positive control group (1 μM) were treated for 4 hours, then RNA was extracted and cDNA was synthesized. Expression of each gene was evaluated using real-time PCR, and the results are shown in FIGS. 8A to 8C.

7-1. Evaluation of proteoglycan-related gene expression

Proteoglycans are polymorphic macromolecules present in skin and hair, and are known to interact with growth factors and collagen. In addition, it has been found that in the hair growth cycle, it decreases in the resting hair follicles and increases in the anagen hair follicles (Couchman, 2017, Journal of Dermatological Science). The expression of Versican, Biglycan, and Syndecan1 genes involved in proteoglycan expression was evaluated in the dermal papilla cells treated with the exosomes isolated in Examples 1 and 2, the control group and the positive control group using real-time PCR, and the results are shown in Fig. 8a shown in

As a result of the evaluation, the exosomes isolated in Example 2 of the present invention expressed genes at or above the level of the positive control group. It can be seen that in the exosomes isolated in Example 1 of the present expression, the expression of all genes was higher than that of the positive control group.

7-2. Wnt target gene expression evaluation

The Wnt signaling system is a key material in the process of hair follicle formation and differentiation, and it is well known that the activation of related genes is important for entering the growth phase from the resting phase in the hair follicle growth cycle (Lim & Nusse, 2013). In order to confirm whether the exosome of the present invention regulates the expression of major target genes of the Wnt signaling system, the exosomes isolated in Examples 1 and 2, the control group and the dermal papilla cells treated with the positive control group were subjected to real-time PCR Thus, the gene expression levels of LEF1, Axin2, beta-catenin, and Wnt5a, which are involved in the expression of Wnt target genes, were evaluated. The results are shown in Figure 8b.

As a result of the evaluation, the exosomes isolated in Examples 1 and 2 of the present invention showed higher expression rates of LEF1, Axin2, beta-catenin, and Wnt5a compared to the control group and the positive control group. It was more than 2 times higher, and it can be seen that beta-catenin and Wnt5a were higher than 30-40%.

7-3. Evaluation of dermal papilla cell representative gene expression

It is known that as the expression rate of BMP4, SOX2, and Corin, which are well known markers of dermal papilla progenitor cells, increases, the ability to form hair follicles increases. The dermal papilla cells treated with the exosomes isolated in Examples 1 and 2, the control group and the positive control group were evaluated for expression of BMP4, SOX2, and Corin genes, which are representative dermal papilla cells using real-time PCR, and the results are shown in FIG. 8c indicated.

As a result of the evaluation, it can be seen that the expression rates of the exosomes BMP4, SOX2, and Corin genes isolated in Examples 1 and 2 of the present invention were at least 3 times higher, and at most 10 times higher than that of the control group and the positive control group.

<Experimental Example 8> Aging recovery evaluation in aged dermal papilla cells

As the dermal papilla cells are subcultured, they become senescent (cellular senescence) and lose their ability to form hair follicles. It was confirmed whether the exosomes of the present invention are effective in the hair follicle-forming ability and cellular aging recovery of aged dermal papilla cells that have lost the hair follicle-forming ability.

8-1. Evaluation of rejuvenation of hair follicles

Alkaline phosphatase (AP) is a representative marker of hair follicle-forming ability, and as the dermal papilla cells age, the activity of alkaline phosphatase decreases. In a 6-well culture dish (well plate), 3.0x10 ⁴ dermal papilla cells were cultured with 10% DMEM for 24 hours. After washing twice with serum-free DMEM medium, the exosomes isolated in Examples 1 and 2 and Comparative Example 1 (1.0x10 ⁹ particles) and the control group were treated. After 48 hours, washed twice with TBS (Tris-buffered saline), fixed with cold acetone for 10 minutes, and NBT/BCIP in TN (0.01M Tris-HCl and 0.1M NaCl, pH8.0) 1: Put the mixed solution in a ratio of 50 and react for 10 minutes. After washing twice again with TBS (Tris-buffered saline), it is observed under a microscope, and the activity of AP can be confirmed to the extent that it is dyed purple. The results are shown in Figure 9a.

As a result of the evaluation, the ratio of cells stained with purple was higher in the exosomes isolated in Examples 1 and 2 of the present invention compared to the exosomes isolated in the control and Comparative Example 1. Through this, it can be seen that the exosomes of the present invention increase the activity of AP and also increase the ability to form hair follicles.

8-2. Anti-aging evaluation in dermal papilla cells

In order to confirm whether the exosome of the present invention is effective in cellular senescence recovery (rejuvenation) of aged dermal papilla cells, senescence-associated β-galactosidase (SA-β-gal) staining was performed, a cellular senescence marker. In a 6-well culture dish (well plate), 3.0x10 ⁴ dermal papilla cells were cultured with 10% DMEM for 24 hours. After washing twice with serum-free DMEM medium, the exosomes isolated in Example 1 (1.0x10 ⁹ particles), a control group and a positive control group minoxidil (1 uM) were treated for 48 hours. Stained according to the protocol using Senescence β-galactosidase Staining Kit. It can be confirmed that the aging of the cells increased as the amount of SA-β-gal staining (blue) increased, and the results are shown in FIG. 9B .

As a result of the evaluation, it was confirmed that the ratio of cells stained with SA-β-gal was low in the exosomes isolated in Example 1 of the present invention compared to the control group and the positive control group. Through this, it was found that the exosomes isolated in Example 1 had excellent anti-aging effects through the inhibition of cell aging.

<Experimental Example 9> Cell motility evaluation

In order to evaluate the effect of exosomes according to the present invention on wound healing and skin improvement, the cell migration ability of keratinocytes was confirmed. After putting 1.0x10 ⁵ keratinocytes per well in a 6-well culture dish (well plate), they were cultured for 24 hours. The cells in culture in the culture dish were scraped using a sterile 10 μl tip to make a wound, and then washed with PBS (phosphate buffer saline). Epilife medium was treated with the exosomes isolated in Example 1 (1.0x10 ⁹ particles), EGF (epidermal growth factor), which is an epidermal growth factor as a control group and a positive control group, and the degree of cell migration was observed using a microscope after 12 hours. Cell migration ability was evaluated by observing the degree of filling the wounded area under a microscope, and the results are shown in FIG. 10 .

As a result of the evaluation, compared to the control group, it can be seen that the cell migration ability increased by the exosomes isolated in Example 1, and thus the wound area decreased, and the cell migration ability was almost similar to that of the positive control, EGF.

Claims (10)

A cosmetic composition for skin improvement comprising, as an active ingredient, exosomes isolated from dermal papilla progenitor cells or dermal papilla progenitor cell cultures,
The skin improvement is a cosmetic composition for improving skin at least one selected from wrinkle improvement, elasticity enhancement, or skin regeneration. The cosmetic composition for skin improvement according to claim 1, wherein the dermal papilla progenitor cells are derived from embryonic stem cells, adult stem cells, induced pluripotent stem cells, hematopoietic stem cells, neural stem cells, or mesenchymal stem cells. The cosmetic composition for skin improvement according to claim 1, wherein the dermal papilla progenitor cells are differentiated or produced from embryonic stem cells, adult stem cells, induced pluripotent stem cells, hematopoietic stem cells, neural stem cells, or mesenchymal stem cells. The cosmetic composition for skin improvement according to claim 1, wherein the exosomes have a size of 50 to 150 nm. The cosmetic composition for skin improvement according to claim 1, wherein the exosome expresses at least one marker among CD63, CD9, and CD81. The cosmetic composition for skin improvement according to claim 5, wherein the expression rate of CD63 in the exosome is 80% or more. The cosmetic composition for skin improvement according to claim 1, wherein the exosomes contain 1x10 ¹⁶ /cell to 1x10 ¹⁷ /cell. The cosmetic composition for skin improvement according to claim 1, wherein the dermal papilla progenitor cells are cultured under 1 to 2% oxygen conditions. According to claim 1, wherein the dermal papilla progenitor cells are three-dimensional cultured or keratinocytes, outer hair root sheath cells, melanin-forming cells, the cosmetic composition for skin improvement, characterized in that the mixed culture with any one or more cells selected from the group consisting of fibroblasts. . The cosmetic composition for skin improvement according to claim 1, wherein the exosomes are contained in an amount of 1.0x10 ⁶ particles to 1.0x10 ¹² particles.

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