KR20170044999A

KR20170044999A - Composition for improving skin and preventing hairloss and method for preparing the same

Info

Publication number: KR20170044999A
Application number: KR1020150144819A
Authority: KR
Inventors: 김수; 이원종; 최은욱; 서민구; 우은영; 박은주
Original assignee: (주)프로스테믹스
Priority date: 2015-10-16
Filing date: 2015-10-16
Publication date: 2017-04-26

Abstract

The present invention relates to a composition comprising stem cells or exosomes derived from the culture thereof and having an effect of wound healing, skin improvement and hair loss prevention or treatment.
The stem cells or culture-derived exosomes provided by the present invention are excellent in skin permeability and excellent in improving the skin such as skin moisturization, skin whitening, wrinkle improvement and aging prevention during absorption, There is no safe. In addition, the stem cells provided from the present invention or exosomes derived from the culture thereof are excellent in hair growth and hair growth, and thus are excellent in preventing and treating hair loss, and are safe without ingestion or application to skin.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing or treating hair, a skin for improving and alleviating hair loss comprising stem cell-derived exosomes, and a method for preparing the same,

The present invention relates to stem cells or exosomes derived from the culture medium, and to a composition having the effect of wound healing, skin improvement and hair loss prevention or treatment, and a method for producing the same.

Stem cells are cells that have the ability to differentiate into all kinds of cells that make up the body, such as nerves, blood, There are two ways to obtain such stem cells. First, stem cells from embryos derived from embryos (embryonic stem cells), and stem cells from adult parts of the body (adult stem cells ). Although there are functional differences, embryonic stem cells and adult stem cells all have the ability to differentiate into different types of cells. Although embryonic stem cells have excellent differentiation potential and long telomeres, they have an ethical problem and it is difficult to acquire a large number of cells. However, adult stem cells can obtain a large number of cells, There is a drawback that the risk of infection or the ability to differentiate is relatively low.

Adult stem cells are very safe for medical applications. Specifically, cancer does not occur even when transplanted into the body for long-term regeneration. Since the immune rejection does not occur because it is in the adult body, autologous transplantation using its own cells is possible. In addition, there is a site-specific differentiation that differentiates itself according to the characteristics of the surrounding tissues. Since it does not cause cancer even if it is injected in undifferentiated state, besides producing cells necessary immediately after transplantation, It has the advantage of self-renewal ability to regenerate and store necessary undifferentiated stem cells. Therefore, the importance of adult stem cells has recently been highlighted, and various studies are under way to reveal its usefulness.

Recently, many hair growth inhibitors for baldness and hair loss prevention have been on the market worldwide, but there are still few drugs or technologies that can satisfy the therapeutic effect. The US Food and Drug Administration (FDA) has approved the FDA as the only drug approved as a hair growth product after minoxidil was originally marketed as a side effect, a side effect developed as a hypertension remedy and the first to be approved by the US federal government Baldness and alopecia. Another drug currently in use is Propecia, which is based on the hormone hypothesis of hair loss. The male hormone is converted to the metabolite dihydrotestosterone (DHT) by the action of 5-alpha reductase The material is known to atrophy and destroy hair follicles. The effect of blocking the 5-alpha reductase inhibits the production of DHT and inhibits the progression of male-type hair loss to the utmost. The effect of the drug is to prevent hair loss, not hair growth.

In addition, substances known to be effective in preventing hair growth and hair loss have been reported. For example, the compounds include cyclosporin derivatives (Korean Patent No. 10-0439467, Korean Patent No. 10-0695611), N-heterocyclic carboxylic acids and carbamates (Korean Patent Laid-Open No. 10-2001-0052502) (Korean Patent Laid-Open Publication No. 10-1999-0023754), crysine 7-O-crotonate (Korean Patent Laid-Open No. 10-2001-0009474), and 1,2-disubstituted benzenecarboxamide derivatives 1999-0037394) have been reported.

However, such a composition for treating hair loss or hair growth has a chemical composition that can cause fatal side effects to women and infants and adverse effects such as acceleration of hair loss due to skin inflammation are manifest in men. In many cases, Side effects that may occur during long-term use may also increase patient pain.

It is an object of the present invention to provide a composition for treating wounds that can treat or promote wounds stably using stem cells or culture-derived exosomes.

Another object of the present invention is to provide a composition for skin improvement which is safe and exerts excellent effects such as skin moisturizing, skin whitening, wrinkle reduction and aging prevention by using the stem cell or exosome derived from the culture medium.

Another object of the present invention is to provide a composition for prevention or treatment of hair loss which is safe and has a hair growth and hair growth effect using exosomes derived from the above-described stem cells or culture thereof.

Wounds are sometimes referred to as wounds. Depending on the cause, the wounds can be divided into three types: incisal, jinjang, halcyon, acupuncture, fissure, (Bite wounds), and depending on the shape is divided into a line-shaped window, a plate-shaped window, and a missing window. Symptoms of a wound include pain, bleeding, and dysfunction. In the case of a wound, the body exhibits various physiological responses to healing it. In general, the malignant cells of the injured cells undergo death, migratory cells (migrating cells) from the surrounding tissues, and tissue fluid are leaked, and the deposition of fibrils and the granulation tissue (granulation tissue) are formed to heal wounds do.

In general, growth factors such as EGF (Epidermal Growth Factor), FGF (Fibroblast Growth Factor), TGF-α (Transforming Growth Factor-α), TGF-β and IGF- It is known to treat or promote wound healing.

However, even if a specific growth factor such as EGF is isolated and applied to clinical practice, it is difficult to expect satisfactory wound treatment or wound treatment promotion effect. In addition, when stem cells such as MSC are used directly, it is difficult to formulate cells while maintaining high cell survival rate, and it is still difficult to express the desired therapeutic effect in the same manner by showing various object deviations depending on the source of cells .

With the recent improvements in medical technology and public health benefits, an aging society is rapidly emerging. As a result, the quality of life is being improved and the desire for the maintenance of youth is increasing. Much research is underway in various areas to delay and prevent appearance, especially the aging that is first manifested in skin.

The skin consists of the epidermis, the dermis and the subcutaneous tissue. The outer skin of the skin consists of a stratified squamous epithelium, which acts as a protective barrier against the external environment. The epidermis is divided from the outside into stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum and stratum basale. The dermis is the layer between the epidermis and the subcutaneous tissue. It is divided into the papillary dermis and the reticular dermis. The dermis is composed of blood vessels, collagen, elastin fibers, pores, lipids, sebaceous glands, Sensory nerves, fibroblasts, and macrophages, and they occupy the largest portion of the skin.

The dermis consists mainly of collagen and elastin fibers, which play a role in supporting the skin. Therefore, when such a dermis is troubled, wrinkles occur and skin elasticity is lost and the skin ages. Collagen is known to play the most important role in skin regeneration, skin moisture content, wound healing and wrinkle improvement, and is produced from fibroblasts. Collagen has a function to contain a large amount of water, and serves to supply moisture to the dermis. When aging occurs, collagen's water-containing function is deteriorated and wrinkles are increased. Collagen also causes wound healing by filling the wound with continuous collagen production of fibroblasts when a wound is developed.

In addition, in the modern society, the living body is gradually damaged due to a lot of stress, and hair loss caused by this is a big social issue. Hair loss is caused by various factors such as poor growth of hair and lack of growth factors promoting growth of hair follicle cells.

Such hair loss reduces the quality of life due to mental stress and affects interpersonal relations and social life. In the social life, the image of the appearance is very impressive and the importance of the appearance is becoming an invisible power of self-esteem and social life, so that the cause of hair loss and treatment are getting more and more attention.

The inventors of the present invention have found that exosome derived from stem cells or culture thereof is excellent in skin permeability and excellent in effects such as wound healing, skin moisturization, skin whitening, wrinkle improvement and aging prevention upon absorption, And discovered the present invention, leading to the present invention.

According to one embodiment of the present invention, there is provided a pharmaceutical composition for treating a wound comprising an adult stem cell or an exosome derived from the culture medium as an active ingredient.

In the present invention, the exosome is preferably contained in an amount of 1 to 100 μg / ml, or 1 to 50 μg / ml. When the exosome is contained in an amount of less than 1 μg / ml, the wound treatment effect may be insignificant, and when it is contained in excess of 100 μg / ml, it may be economically problematic.

In addition, the exosome may be contained in a particle number of 1 × 10 ⁷ to 1 × 10 ¹² / ml. If the number of exosome particles is less than 1 X 10 ⁷ / ml, the effect of wound healing may be insignificant, and if the number of particles exceeds 1 X 10 ¹² / ml, economical problems may arise.

According to another embodiment of the present invention, there is provided a method for preparing a pharmaceutical composition for wound healing, comprising: primary centrifuging the adult stem cell culture solution at 100 to 500 g for 5 to 30 minutes; Recovering the supernatant after the primary centrifugation and performing secondary centrifugation at 1,000 to 3,000 g for 10 to 30 minutes; Recovering the supernatant after the secondary centrifugation and subjecting it to tertiary centrifugation at 5,000 to 20,000 g for 20 to 60 minutes; Recovering the supernatant after the tertiary centrifugation, and performing quadratic centrifugation at 100,000 to 120,000 g for 60 to 120 minutes; And recovering the pellet after the quaternary centrifugation, followed by fifth centrifugation at 50,000 to 150,000 g for 60 to 120 minutes to recover the pellet.

The present invention can obtain the exosome having excellent wound healing effect with high purity and content through the 5 step centrifugation process.

In the present invention, the adult stem cell culture solution may be prepared by culturing adult stem cells in a medium containing 800 to 1,200 mg / L of D glucose, 500 to 600 mg / L of L-glutamine, 100 to 200 mg / L of sodium pyruvate, 5 to 15% Of fetal bovine serum (FBS) and 0.5-1.5 volume% penicillin-streptomycin, and then cultured in DMEM medium supplemented with 5% -10% CO 2 at 80-95% humidity and 30-40 ° C temperature ₂ subculture; And subcultured cultures were diluted 1: 0.5 with DMEM and Ham's F-12 nutrient mixture supplemented with 350-400 mg / L L-glutamine, 10-20 mM HEPES and 50-60 mg / L sodium pyruvate To 2% by weight of a mixed medium, followed by culturing for 60 to 120 hours under hypoxia conditions containing 1 to 5% by volume of oxygen.

According to another embodiment of the present invention, there is provided a cosmetic composition for skin improvement comprising an adult stem cell or an exosome derived from the culture medium as an active ingredient.

According to another embodiment of the present invention, there is provided a method for producing a cosmetic composition for skin improvement, comprising the steps of: primary centrifuging the adult stem cell culture solution at 100 to 500 g for 5 to 30 minutes; Recovering the supernatant after the primary centrifugation and performing secondary centrifugation at 1,000 to 3,000 g for 10 to 30 minutes; Recovering the supernatant after the secondary centrifugation and subjecting it to tertiary centrifugation at 5,000 to 20,000 g for 20 to 60 minutes; Recovering the supernatant after the tertiary centrifugation, and performing quadratic centrifugation at 100,000 to 120,000 g for 60 to 120 minutes; And recovering the pellet after the quaternary centrifugation, followed by fifth centrifugation at 50,000 to 150,000 g for 60 to 120 minutes to recover the pellet.

The present invention can obtain exosome having excellent skin improving effect with high purity and content through the 5 step centrifugation process.

According to another embodiment of the present invention, there is provided a pharmaceutical composition for preventing or treating hair loss comprising adult stem cells or an exosome derived from the culture medium as an active ingredient.

According to another embodiment of the present invention, there is provided a cosmetic composition for preventing or improving hair loss comprising adult stem cells or an exosome derived from the culture medium as an active ingredient.

According to another embodiment of the present invention, there is provided a composition for preventing or improving hair loss comprising adult stem cells or an exosome derived from the culture medium as an active ingredient.

According to another embodiment of the present invention, there is provided a method for producing a pharmaceutical composition for preventing or treating hair loss, comprising the steps of: primary centrifuging the adult stem cell culture solution at 100 to 500 g for 5 to 30 minutes; Recovering the supernatant after the primary centrifugation and performing secondary centrifugation at 1,000 to 3,000 g for 10 to 30 minutes; Recovering the supernatant after the secondary centrifugation and subjecting it to tertiary centrifugation at 5,000 to 20,000 g for 20 to 60 minutes; Recovering the supernatant after the tertiary centrifugation, and performing quadratic centrifugation at 100,000 to 120,000 g for 60 to 120 minutes; And recovering the pellet after the quaternary centrifugation, followed by fifth centrifugation at 50,000 to 150,000 g for 60 to 120 minutes to recover the pellet.

The present invention can obtain exosome having excellent hair growth and hair growth effect with high purity and content through a 5 step centrifugation process.

In the present invention, the adult stem cells are undifferentiated cells having multipotency derived from mammals including humans, preferably human adult cells. Examples of the stem cells include bone marrow, blood, brain, skin, fat , Adipose tissue or fat cells), umbilical cord blood, umbilical cord blood, Wharton's jelly, and the like. In the present specification, the "adult stem cells" include mesenchymal stem cells derived from adult cells.

As the adult stem cells, fat-derived stem cells which can be obtained by using fat tissues which are commonly used in the liposuction process and which do not require invasive procedures can be preferably used. The adipose-derived stem cells are obtained from mammals including humans, preferably human adipose tissue or adipocytes, according to known methods (for example, International Patent Publication Nos. WO 2000/53795 and WO 2005/042730) Liposuction and sedimentation, treatment of enzymes such as collagenase, removal of floating cells such as red blood cells by centrifugation, and the like. The adipose tissue includes brown or white tissue derived from subcutaneous, mesenteric, visceral, breast gonad or other adipose tissue areas and can be easily obtained from conventional liposuction.

In the present invention, an exosome refers to a small follicle having a membrane structure that is secreted from various cells. The diameter of the exosome is approximately 30 to 1,000 nm, which means the follicle that is fused to the plasma membrane and released to the extracellular environment. Representative expression markers of exosomes correspond to HSP70, CD63 and CD9.

In addition, the pharmaceutical composition of the present invention may further comprise an appropriate carrier, excipient or diluent according to a conventional method. Examples of carriers, excipients and diluents that can be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate But are not limited to, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In addition, the pharmaceutical composition according to the present invention may be formulated in the form of oral, granule, tablet, capsule, suspension, emulsion, syrup, aerosol or the like, oral preparation, suppository or sterilized injection solution, Can be used. In detail, when formulating the composition, it can be prepared using a diluent or an excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant and the like which are usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like. Such solid preparations can be prepared by mixing the pharmaceutical composition of the present invention with at least one excipient such as starch, calcium carbonate, Sucrose, lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, syrups and the like, and various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. in addition to commonly used diluents such as water and liquid paraffin . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The route of administration of the pharmaceutical compositions according to the present invention may be, but is not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, Sublingual or rectal. Oral or parenteral administration is preferred. The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. The pharmaceutical compositions of the present invention may also be administered in the form of suppositories for rectal administration.

The pharmaceutical composition of the present invention varies depending on various factors including the activity of the specific compound used, age, weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease to be prevented or treated. And the dose of the pharmaceutical composition may be appropriately selected by a person skilled in the art depending on the condition of the patient, the body weight, the degree of disease, the type of drug, the route of administration and the period of time, and may be 0.0001 to 50 mg / kg or 0.001 to 50 mg / kg. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way. The pharmaceutical compositions according to the present invention can be formulated into pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions.

The food composition of the present invention may be prepared in the form of various foods such as beverages, gums, tea, vitamin complexes, powders, granules, tablets, capsules, confectionery, rice cakes and breads. Since the food composition of the present invention is composed of a plant extract having little toxicity and side effects, it can be safely used for prolonged use even for prophylactic purposes.

When the exosome contained as an active ingredient in the present invention is contained in the food composition, the amount thereof may be added in a proportion of 0.1 to 50% of the total weight.

Here, when the food composition is prepared in a beverage form, there are no particular limitations other than those containing the food composition at the indicated ratios and may contain various flavors or natural carbohydrates such as ordinary beverages as an additional ingredient. That is, natural carbohydrates include monosaccharides such as glucose, disaccharides such as fructose, sucrose and the like and sugar sugars such as polysaccharide, dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol can do. Examples of the above-mentioned flavors include natural flavors (such as tau martin and stevia extract (for example, rebaudioside A and glycyrrhizin) and synthetic flavors (for example, saccharine and aspartame).

In addition, the food composition of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants, pectic acid and its salts, alginic acid and its salts, , a pH adjusting agent, a stabilizer, a preservative, a glycerin, an alcohol, a carbonating agent used in a carbonated drink, and the like.

These components may be used independently or in combination. The proportion of such additives is not so critical, but is generally selected in the range of 0.1 to about 50 parts by weight per 100 parts by weight of the food composition of the present invention.

In addition, the cosmetic composition of the present invention can be used in cosmetics, nutritional lotions, nutritional essences, massage cream, cosmetic bath additives, body lotion, body milk, bath oil, baby oil, baby powder, shower gel, Creams, suntan creams, skin lotions, skin creams, sunscreen cosmetics, cleansing milks, depilatory creams, facial and body lotions, face and body creams, skin whitening creams, hand lotions, hair lotions, cosmetic creams, jasmine Oil, bath soap, water soap, soap, shampoo, hand cleanser, medicinal soap {non-medical use}, cream soap, facial wash, whole body cleanser, scalp cleaner, hair rinse, makeup soap, tooth whitening gel, toothpaste And the like. To this end, the composition of the present invention may further comprise a solvent commonly used in the production of a cosmetic composition, or a suitable carrier, excipient or diluent.

For example, water, saline solution, DMSO, or a combination thereof may be used. Examples of the carrier, excipient or diluent include purified water, oil, wax But are not limited to, fatty acids, fatty acid alcohols, fatty acid esters, surfactants, humectants, thickeners, antioxidants, viscosity stabilizers, chelating agents, buffers, lower alcohols and the like. Further, if necessary, it may contain a whitening agent, a moisturizing agent, a vitamin, an ultraviolet screening agent, a perfume, a dye, an antibiotic, an antibacterial agent, and an antifungal agent.

As the oil, hydrogenated vegetable oil, castor oil, cottonseed oil, olive oil, palm oil, jojoba oil and avocado oil may be used. Examples of the wax include wax, wax, carnauba, candelilla, montan, ceresin, liquid paraffin, Can be used.

As the fatty acid, stearic acid, linoleic acid, linolenic acid and oleic acid may be used. As the fatty acid alcohol, cetyl alcohol, octyldodecanol, oleyl alcohol, panthenol, lanolin alcohol, stearyl alcohol and hexadecanol may be used As fatty acid esters, isopropyl myristate, isopropyl palmitate, and butyl stearate may be used. As the surfactant, a cationic surfactant, an anionic surfactant and a nonionic surfactant known in the art can be used, and a surfactant derived from a natural material is preferably used.

In addition, it may contain a hygroscopic agent, a thickening agent, an antioxidant and the like widely known in the field of cosmetics, and the kind and amount thereof are well known in the art.

The stem cells or culture-derived exosomes provided by the present invention have excellent wound healing or wound healing-promoting activity by promoting cell migration to wound sites.

It has excellent skin permeability and is excellent in skin improving effect such as skin moisturization, skin whitening, wrinkle improvement and aging prevention during absorption, and is free from irritation or side effects to the skin and is safe.

In addition, the stem cells provided from the present invention or exosomes derived from the culture thereof are excellent in hair growth and hair growth, and thus are excellent in preventing and treating hair loss, and are safe without ingestion or application to skin.

FIG. 1 (a) is a TEM photograph of Exosome in Experimental Example 1, and FIG. 1 (b) is a graph showing an analysis of size of particles present in exosome.
FIG. 2 (a) is a photograph showing the expression of HSP70, CD63 and CD9 markers of exosome using Western blot in Experimental Example 1. FIG. 2 (b) is a photograph showing the expression of exonsome CD24 and AQP2 markers (C) shows the expression of CD63, CD9 and CD81 markers of exosomes using FACS.
FIG. 3 is a photograph showing migration of human skin fibroblasts to the wound area after treatment of the exosome obtained in Example 3 in Experimental Example 2 and the adipose-derived stem cell culture solution in which exosome obtained in Comparative Example 1 is removed.
FIG. 4 is a graph showing the ratio of exosome obtained in Example 3 in Experimental Example 2 and human skin fibroblasts migrated to the injured region after treatment of the adipose-derived stem cell culture liquid obtained in Comparative Example 1 .
FIG. 5 is a graph showing the degree of expression of procollagen, KGF and CD34 after treatment with exoose obtained in Example 3 in Experimental Example 3 and with adipose-derived stem cell culture obtained in Comparative Example 1. FIG.
6 (a) and 6 (b) are graphs showing the degree of expression of procollagen and elastin after concentration of the exosome obtained in Example 3 in Experimental Example 4. FIG.
FIGS. 7 (a) to 7 (c) are graphs showing the expression levels of KGF, CD34 and VEGF by concentration after treatment with exosomes obtained in Example 3 in Experimental Example 5. FIG.
8 is a graph showing the degree of expression of LEF-1, PTC-1 and KGF after treatment of the exosome obtained in Example 3 in Example 6 and the adipose-derived adipose-derived stem cell culture obtained in Comparative Example 1 will be.

Hereinafter, preferred embodiments of the present invention will be described. However, the embodiments of the present invention can be modified into various other forms, and the scope of the present invention is not limited to the embodiments described below. Further, the embodiments of the present invention are provided to more fully explain the present invention to those skilled in the art.

[ Example 1] Obtaining of fat-derived stem cells

We obtained consent from the patient who underwent subcutaneous fat removal and removed the abandoned fat tissue from the patient through liposuction method. The extracellular matrix of the adipose tissue was treated with 0.075% collagenase for 45 minutes at about 37 ° C in a 5% CO ₂ incubator, and the obtained adipose tissue was centrifuged at about 1200 g for 5 minutes to obtain a stromal A blood vessel fraction was obtained. The obtained fractions were washed with PBS and other tissues were removed through a 70-μm nylon cell filter, and then cell debris including mononuclear cells was separated with Histopaque-1077 (Sigma Co.).

Separated mononuclear cells were cultured in DMEM (Dulbecco's Modified Eagle's Medium) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin at about 37 ° C in a 5% CO ₂ incubator For 24 hours, and then non-adherent cells were removed to isolate adipose-derived stem cells.

[ Example 2] Culture of adipose-derived stem cells

1.25 × 10 ⁵ cells of the adipose-derived stem cells obtained in Example 1 were cultured in a culture medium containing 1000 mg / L of D glucose, 584 mg / L of L-glutamine, 110 mg / L of sodium pyruvate, 10% -Streptomycin supplemented DMEM medium and subcultured in a 5 volume% CO ₂ incubator for 30 days at a temperature of about < RTI ID = 0.0 > 90% < / RTI >

The culture was removed from the subcultured cultures using a pipette, washed three times with phosphorylation buffer, and supplemented with 365 mg / L L-glutamine, 15 mM HEPES, 55 mg / L sodium pyruvate The cells were inoculated at a concentration of 1.2 × 10 ⁶ cells / dish in a 1: 1 (by weight) mixed medium of DMEM and Ham's F-12 nutrient mixture and cultured for 72 hours under hypoxia conditions.

[ Example 3] Origin of adipose-derived stem cell culture fluid Exosome detach

50 ml of the adipose stem cell culture obtained in Example 2 was transferred to a centrifuge tube and centrifuged at 4 ° C and 300 g for 10 minutes. The supernatant was collected and transferred to a new centrifuge tube, which was then centrifuged at 2,000 g for 20 minutes at 4 ° C. The resulting supernatant was transferred to a tube capable of a new high-speed centrifuge and subjected to high-speed centrifugation at 10,000 g for 30 minutes . The supernatant was transferred to a new ultra-high speed centrifuge polycarbonate bottle and ultracentrifuged centrifugation was performed at 110,000 g at 4 ° C for 70 minutes using an OPTIMA XE-90 ultracentrifuge from Beckman Coulter to remove the supernatant . The settled pellet was resuspended in 1000 μl PBS in a tube using a micropipette. The resuspended tubes were centrifuged at 100,000 g for 70 min at 4 ° C using an OPTIMA MAX-XP ultracentrifuge from Beckman Coulter, all possible supernatants were removed and the pellets were collected. A small amount of PBS was added to the recovered pellet, and after resuspending, 100 μl of the solution was separated and stored at -80 ° C.

[ Comparative Example 1] From the adipose-derived stem cell culture fluid Exosomatic remove

50 ml of the adipose stem cell culture obtained in Example 2 was transferred to a centrifuge tube and centrifuged at 4 ° C and 300 g for 10 minutes. The supernatant was collected and transferred to a new centrifuge tube and centrifuged at 4 ° C and 2,000 g for 20 minutes. The supernatant, which had been centrifuged at 2,000 g for 20 minutes at 4 ° C, was transferred to a tube capable of a new high-speed centrifuge and subjected to high-speed centrifugation at 10,000 g for 30 minutes at 4 ° C. The supernatant was transferred to a polycarbonate bottle for a new ultrahigh-speed centrifuge and subjected to ultracentrifugation at 110,000 g at 4 ° C for 70 minutes using an OPTIMA XE-90 ultracentrifuge of Beckman Coulter, followed by removal of the pellet and removal of the supernatant. The removed fat-derived stem cell culture solution was prepared.

[ Experimental Example 1] Derived from adipose-derived stem cell culture fluid Exosomatic Character analysis

The exosome obtained in Example 3 was observed through a transmission electron microscope (TEM), and the photograph was shown in Fig. 1 (a). The exosome was observed through nanoparticle tracking analysis (NTA) And the results are shown in the graph of FIG. 1 (b).

Expression of representative exosomatic markers HSP70, CD63 and CD9 was confirmed for the exosomes obtained in Example 3 and the fat-derived stem cell lysates as a control group using Western blot. The results are shown in FIG. 2 (a). In addition, the expression levels of the exosomes obtained in Example 3 and the exo-somatic cells of fat-derived stem cells as a control group were confirmed using real-time PCR, and the results are shown in FIG. 2 (b) . Lastly, the exosome surface markers CD63, CD9 and CD81 were expressed in the exosome obtained in Example 3 using a flow cytometer (FACS), and the results are shown in FIG. 2 (c).

As shown in FIG. 1 (a), it can be confirmed that the exosome isolated from adipose-derived stem cell culture liquid has a fine spherical structure in the nanometer scale. As shown in FIG. 1 (b) Is mainly distributed in the range of 30 to 150 nm.

Also, as shown in Figs. 2 (a) to 2 (c), it can be confirmed that the exosomes isolated from the adipose-derived stem cell culture solution have all the typical markers of exosomes.

Thus, it can be seen that the exosome isolated from the adipose stem cell culture fluid has typical exosome characteristics.

[ Experimental Example 2] Migration effect of human skin fibroblast from wound area

Human skin fibroblasts were attached to a culture dish at a concentration of 5 X 10 ⁴ cells / well and scratches were made at regular intervals. Then, the exosome obtained in Example 3 and the exosome obtained in Comparative Example 1 The oocyte-free adipose-derived adipose-derived stem cell culture solution was treated for 48 hours, and the degree of cell migration was confirmed. The results are shown in FIG. 3 and FIG.

As shown in FIG. 3, it can be visually confirmed that many cells migrate to the injured area when the exoose-treated adipose-treated adipose-derived stem cell culture liquid is treated.

Also in FIG. 4, it can be confirmed that when the exosome is treated, the cell migration effect is more than twice as much as that in the case of treating the adoseose-eliminated adipose-derived stem cell culture solution.

[ Experimental Example 3] Prevention of skin aging and increase of gene expression related to regeneration

Human skin fibroblasts were adhered to a culture dish at a concentration of 5 X 10 ⁴ cells / well, and the exosomes obtained in Example 3 and the adipose-derived adipose-derived stem cell cultures obtained in Comparative Example 1 were treated. After incubation for 48 hours, RNA was extracted from cells of each group, and then complementary DNA (cDNA) was synthesized using intron premix and real-time PCR was performed using the synthesized cDNA. Expression of procollagen, KGF, and CD34 was confirmed as genes associated with skin aging prevention and regeneration in the cells of each treatment group, and the results are shown graphically in Fig.

However, the above-mentioned procollagen maintains skin elasticity and corresponds to a gene involved in collagen synthesis necessary for the main protein formation of the skin. KGF (keratinocyte growth factor) is a major growth factor that stimulates differentiation, growth and physiological activity of skin cells. It is a gene that prevents skin aging, synthesizes collagen, regulates skin regeneration, and is involved in anti-aging. Finally, CD34 is a factor involved in vascular regeneration and corresponds to a gene involved in skin regeneration.

As shown in FIG. 5, the expression of procollagen, KGF, and CD34 was significantly increased in the case of treatment with exoose-treated adipose-derived adipose-derived stem cell culture solution.

[ Experimental Example 4] Increased expression of skin elasticity-related genes

Human skin fibroblasts were adhered to a culture dish at a concentration of 5 X 10 ⁴ cells / well, and the exosomes obtained in Example 3 were treated at a concentration of 5 and 10 μg / ml, respectively. After incubation for 48 hours, RNA was extracted from the cells of each group and cDNA was synthesized using intron pre-mix, and real-time PCR was performed using the synthesized cDNA. The degree of expression of procollagen and elastin was confirmed as a gene involved in the increase in the anti-aging skin elasticity in the cells of each treatment group, and the results are shown graphically in Fig. However, exosomes were not treated as a control.

As shown in FIGS. 6 (a) and 6 (b), when the exosomes were treated, the level of mRNA expression of procollagen and elastin was significantly higher than that of the control, and the level of expression increased with increasing treatment concentration Can be confirmed.

Thus, it can be seen that the exosome obtained from the fat-derived stem cell culture solution according to the present invention plays an important role in maintaining skin elasticity and preventing aging.

[ Experimental Example 5] Increased expression of genes related to skin regeneration

Human skin fibroblasts were attached to a culture dish at a concentration of 5 X 10 ⁴ cells / well, and the exosomes obtained in Example 3 were treated at a concentration of 5 and 10 μg / ml, respectively. After incubation for 48 hours, RNA was extracted from the cells of each group and cDNA was synthesized using intron pre-mix, and real-time PCR was performed using the synthesized cDNA. The expression levels of KGF, CD34 and VEGF were examined as genes involved in skin regeneration in the cells of each treatment group, and the results are shown graphically in FIG. However, exosomes were not treated as a control.

As shown in FIGS. 7 (a) to 7 (c), when the exosomes were treated, the expression level of KGF, CD34 and VEGF mRNA was significantly higher than that of the control group. .

Thus, it can be seen that the exosome obtained from the adipose stem cell culture according to the present invention plays an important role in skin regeneration.

[ Experimental Example 9] Dairy cattle Increased expression of genes related to hair follicular growth and development of cells

5 × 10 ⁵ human dermal papilla cells were attached to a 60 mm (SPL, Korea) culture dish, and the exosome obtained in Example 3 and the adipose-derived adipose-derived stem cell culture obtained in Comparative Example 1 were treated . After 48 hours of culture, the cells were recovered, RNA was isolated using an RNA isolation kit (Quiagen, USA), and cDNA was synthesized against 1 ug of the separated RNA using a cDNA synthesis kit (Intronbio, Korea). Each primer was synthesized in Cosmos Tech, the primer was used at a concentration of 10 pmol / ul, and real time PCR was detected by adding 2X Cyber Green Master Mix (Takara, Japan). The expression level of LEF-1, PTC-1, and KGF was examined as a gene involved in hair follicle growth and development in the cells of each treatment group, and the results are shown graphically in FIG.

However, the lymphoid enhancer-binding factor-1 (LEF-1) promotes morphogenesis and differentiation of hair follicles. PTC-1 (patched protein, Sonic Hedgehog (Shh) receptor) It is a gene that regulates growth. KGF (keratinocyte growth factor) is a gene that plays an important role in the growth, development and differentiation of hair follicles.

As shown in FIG. 8, the expression of LEF-1, PTC-1 and KGF was significantly increased in the case of treatment with exoose-treated adipose-derived stem cell culture medium.

Thus, it can be seen that the exosome obtained from the adipose stem cell culture according to the present invention plays an important role in the growth, development and differentiation of hair follicles.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is clearly understood that the same is by way of illustration and example only and is not to be taken by way of limitation, It is to be understood that the invention may be practiced within the scope of the appended claims.

Claims

A pharmaceutical composition for treating a wound comprising an adult stem cell or an exosome derived from the culture as an active ingredient.

The method according to claim 1,
Wherein the adult stem cells are derived from at least one of bone marrow, blood, brain, skin, fat, umbilical cord blood, and umbilical cord blood wharton's jelly.

The method according to claim 1,
Wherein the exosome is contained in an amount of 1 to 100 占 퐂 / ml.

The method according to claim 1,
Wherein the exosome is contained in a particle number of 1 × 10 ⁷ to 1 × 10 ¹² / ml.

A cosmetic composition for improving skin comprising adult stem cells or an exosome derived from the culture medium as an active ingredient.

6. The method of claim 5,
Wherein the adult stem cells are derived from at least one of bone marrow, blood, brain, skin, fat, umbilical cord blood, and umbilical cord blood wharton's jelly.

6. The method of claim 5,
Wherein the exosome is contained in an amount of 1 to 100 占 퐂 / ml.

6. The method of claim 5,
Wherein the exosome is contained in a particle number of 1 × 10 ⁷ to 1 × 10 ¹² / ml.

A pharmaceutical composition for preventing or treating hair loss comprising adult stem cells or an exosome derived from the culture medium as an active ingredient.

10. The method of claim 9,
Wherein said adult stem cells are derived from at least one of bone marrow, blood, brain, skin, fat, umbilical cord blood, and umbilical cord blood wharton's jelly.

10. The method of claim 9,
Wherein the exosome is contained in an amount of 1 to 100 占 퐂 / ml.

10. The method of claim 9,
Wherein the exosome is contained in a particle number of 1 × 10 ⁷ to 1 × 10 ¹² / ml.

A composition for preventing or improving hair loss comprising adult stem cells or an exosome derived from the culture medium as an active ingredient.

A cosmetic composition for preventing or improving hair loss comprising adult stem cells or an exosome derived from the culture medium as an active ingredient.

Culturing the adult stem cell culture solution at 100 to 500 g for 5 to 30 minutes;
Recovering the supernatant after the primary centrifugation and performing secondary centrifugation at 1,000 to 3,000 g for 10 to 30 minutes;
Recovering the supernatant after the secondary centrifugation and subjecting it to tertiary centrifugation at 5,000 to 20,000 g for 20 to 60 minutes;
Recovering the supernatant after the tertiary centrifugation, and performing quadratic centrifugation at 100,000 to 120,000 g for 60 to 120 minutes; And
Recovering the pellet after the quaternary centrifugation, and recovering the pellet by 5 < th > centrifugation at 50,000 to 150,000 g for 60 to 120 minutes.

16. The method of claim 15,
The adult stem cell culture solution is prepared by culturing adult stem cells in a medium containing 800 to 1,200 mg / L of D glucose, 500 to 600 mg / L of L-glutamine, 100 to 200 mg / L of sodium pyruvate, 5 to 15% After inoculation in DMEM medium supplemented with serum (Fetal Bovine Serum, FBS) and penicillin-streptomycin of 0.5-1.5 volume%, the cells were cultured in a 5-10% CO ₂ incubator under a humidity of 80-95% Subculturing; And subcultured cultures were diluted 1: 0.5 with DMEM and Ham's F-12 nutrient mixture supplemented with 350-400 mg / L L-glutamine, 10-20 mM HEPES and 50-60 mg / L sodium pyruvate To 2% by weight of a mixed medium, and culturing the cells for 60 to 120 hours under hypoxia conditions containing 1 to 5% by volume of oxygen.

Culturing the adult stem cell culture solution at 100 to 500 g for 5 to 30 minutes;
Recovering the supernatant after the primary centrifugation and performing secondary centrifugation at 1,000 to 3,000 g for 10 to 30 minutes;
Recovering the supernatant after the secondary centrifugation and subjecting it to tertiary centrifugation at 5,000 to 20,000 g for 20 to 60 minutes;
Recovering the supernatant after the tertiary centrifugation, and performing quadratic centrifugation at 100,000 to 120,000 g for 60 to 120 minutes; And
Recovering the pellet after the quaternary centrifugation, and then centrifuging the pellet at 50,000 to 150,000 g for 60 to 120 minutes to recover the pellet.

Culturing the adult stem cell culture solution at 100 to 500 g for 5 to 30 minutes;
Recovering the supernatant after the primary centrifugation and performing secondary centrifugation at 1,000 to 3,000 g for 10 to 30 minutes;
Recovering the supernatant after the secondary centrifugation and subjecting it to tertiary centrifugation at 5,000 to 20,000 g for 20 to 60 minutes;
Recovering the supernatant after the tertiary centrifugation, and performing quadratic centrifugation at 100,000 to 120,000 g for 60 to 120 minutes; And
Recovering the pellet after the quaternary centrifugation, and collecting the pellet by centrifugation at 50,000 to 150,000 g for 60 to 120 minutes for 5 to 100 minutes.