WO2011136434A1 - Liquid culture concentrate of human adipose-derived stem cells having a skin-regenerating or wrinkle-improving effect and a use therefor - Google Patents

Abstract

The present invention relates to: a skin-regenerating or wrinkle-improving composition containing an active ingredient in the form of a stem-cell liquid culture concentrate obtained by culturing human adipose-derived stem cells in a serum medium and then passaging in a serum-free medium before then filtering; a skin-regenerating method in which the composition is topically coated or intradermally injected at a site of injury; a wrinkle-improving method in which the composition is topically coated or intradermally injected at a site where it is deemed that wrinkle lines need to be removed; a skin-regenerating or wrinkle-improving therapeutic agent comprising the composition as an active ingredient; and a skin-regenerating or wrinkle improving cosmetic comprising the composition as an active ingredient.

Description

Culture concentrate of human adipose derived stem cells having skin regeneration or wrinkle improvement effect and use thereof

The present invention relates to a culture concentrate of human adipose derived stem cells having a skin regeneration or wrinkle improvement effect, and more particularly, to culturing human adipose derived stem cells in a serum medium and then passaged in a serum-free medium. , A composition for skin regeneration or wrinkle improvement comprising the stem cell culture concentrate obtained by filtration as an active ingredient, the method for topical application or intradermal injection of the composition on the wound site, wrinkle removal of the composition Wrinkle improvement method, characterized in that the topical application or intradermal injection to the area that needs, a skin regeneration or wrinkle improvement treatment comprising the composition as an active ingredient, and skin regeneration or wrinkles comprising the composition as an active ingredient Relates to improving cosmetics.

Recently, with the improvement of medical technology and public health benefits, an aging society is rapidly arriving. As a result, the quality of life is being improved and the desire for the maintenance of youth is also increasing. Many studies have been conducted in various areas to delay and prevent aging, which is the first to appear on the skin.

The skin consists of the epidermis, the dermis and the subcutaneous tissue. The epidermis, the outer layer of skin, consists of the stratified squamous epithelium and acts as a protective barrier for the body to the outside 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, divided into papillary and reticular dermis, blood vessels, collagen, elastin fibers, pores, hair roots, sebaceous glands, Korean glands, various Sensory nerves, fibroblasts and macrophages are present and occupy the largest part of the skin.

The dermis consists primarily of collagen and elastin fibers, which support the skin. Therefore, when such a problem occurs in the dermis, wrinkles occur and skin elasticity is lost, thereby aging the skin. 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 that can contain a large amount of moisture, which serves to supply moisture to the dermis. When aging, the collagen loses its water-containing function and wrinkles increase. Collagen can also heal wounds by filling fibroblasts with sustained collagen production when the wound is injured.

Stem cells are cells that have been differentiated into specific cells without progress and, if necessary, have the ability to differentiate into all kinds of cells constituting the body such as nerves, blood, and cartilage. There are two ways to obtain such stem cells, firstly from embryos derived from fertilized eggs (embryonic stem cells) and secondly from stem cells (adult stem cells) stored in each part of our adult body. ) Is recovered. Although functionally different, both embryonic and adult stem cells have the ability to differentiate into different cell types. Embryonic stem cells have very good differentiation ability and long telomeres, but they have ethical problems and difficult to obtain a large amount of cells. On the other hand, adult stem cells can obtain a large number of cells, but can be transplanted to others. The risk of infection or differentiation 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, and since the immune rejection reaction does not occur because it is in an adult body, autologous transplantation using its own cells is possible. In addition, there is a site-specific differentiation ability to differentiate itself according to the characteristics of the surrounding tissue, and since injection does not cause cancer even when injected in undifferentiated state, in addition to producing the cells needed immediately after transplantation The advantage is the ability to self-renewal to regenerate and store the necessary undifferentiated stem cells. Therefore, adult stem cells have recently gained importance, and various studies are underway to reveal their usefulness.

Korean Patent No. 10-0883565 discloses an injection additive for tissue regeneration using adipose derived stem cells, and Korean Patent Publication No. 2008-0079256 discloses a composition for treating skin defects using mesenchymal stem cells.

The present invention is derived from the above requirements, the present inventors cultured human adipose derived stem cells in serum medium and then subcultured in serum-free medium, the stem cell culture concentrate obtained by filtration is skin regeneration or wrinkles The present invention has been found to be effective for improvement.

In order to solve the above problems, the present invention cultivated human adipose derived stem cells in serum medium and then passaged in serum-free medium, skin regeneration or wrinkle improvement containing the stem cell culture concentrate obtained by filtration as an active ingredient It provides a composition for.

In addition, the present invention provides a method for regenerating the skin, characterized in that the composition is applied topically or intradermal injection to the wound site.

In addition, the present invention provides a method for improving wrinkles, characterized in that the composition is applied topically or intradermal injection to the site that needs to remove wrinkles.

The present invention also provides a therapeutic agent for skin regeneration or wrinkle improvement comprising the composition as an active ingredient.

In addition, the present invention provides a skin regeneration or wrinkle improvement cosmetics comprising the composition as an active ingredient.

Since the culture concentrate of human adipose derived stem cells of the present invention has an activity of inducing wound healing and production of type 1 procollagen, it is expected to be effectively used for skin regeneration or wrinkle improvement.

1 shows the wound healing effect of AAPE in artificially wounded hairless mice.

Figure 2 shows the results of analyzing the wound healing effect of AAPE by measuring the area of the wound site remaining in the hairless mouse artificially induced the wound.

Figure 3 shows the wound healing effect of AAPE in the hairless mouse artificially induced wounds through histological analysis.

Figure 4 shows the effect of AAPE and L- ascorbic acid on the production rate of type 1 procollagen in human dermal fibroblasts. **: Significance test of the test group against the negative control (Dunnett's t-test, p <0.01).

In order to achieve the object of the present invention, the present invention is a skin regeneration containing the stem cell culture concentrate obtained by filtration after culturing human adipose derived stem cells in serum medium and then subcultured in serum-free medium or It provides a composition for improving wrinkles.

The stem cell culture concentrate is preferably

(a) culturing human adipose derived stem cells in serum medium and then subcultured in serum-free medium;

(b) applying any one or more physical stimuli selected from low oxygen culture, ultraviolet irradiation, low frequency treatment, nutrient deprivation, mechanical friction, etc. to the passaged stem cells;

(c) culturing by adding at least one vitamin selected from vitamin A, vitamin B, vitamin C and vitamin D to the culture medium; and

(d) may be prepared by filtering the culture solution.

Steps (b) and (c) may be performed in combination under conditions in which maximum production of human growth factors occurs. For the preparation of stem cell culture concentrate, refer to Korean Patent Publication No. 2008-0109725, which is incorporated herein by reference in its entirety.

In the composition of the present invention, the serum-free medium may preferably be a mixed medium (see Korean Patent Publication No. 2008-0109725) of Dulbecco's Modified Eagle's Medium (DMEM) and Ham's F-12 nutrient mixture, The mixing ratio may preferably be 1: 1 with DMEM and Ham's F-12 nutrient mixture.

The composition of the present invention may exhibit skin regeneration or wrinkle improvement by increasing the production of type 1 procollagen in dermal fibroblasts.

The stem cells refer to undifferentiated cells having the ability to differentiate into various types of tissue cells, and are separated from embryonic stem cells and adult tissues separated from the inner cell mass of the blastocyst. It can be broadly classified into adult stem cells. Adult stem cells refer to undifferentiated cells having mutipotency derived from adult tissues of mammals including humans, preferably humans. For example, various stem cells such as bone marrow, blood, brain, skin, fat, umbilical cord blood, etc. Can be derived from adult cells.

The human adipose derived stem cells are a kind of adult stem cells isolated from human adipose tissue. Acquisition of adipose tissue can be obtained incidentally in the process of liposuction, which is conventionally performed, and thus has an advantage of easily obtaining and culturing a sufficient amount of stem cells, and is also superior in safety to bone marrow harvesting.

The human adipose derived stem cells may be cultured in a conventional manner using a stem cell culture medium, for example, a serum medium. Preferably, by incubating human adipose derived stem cells in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum, and finally in a 1: 1 mixed medium of serum-free DMEM and Ham's F-12 nutrient mixtures. In addition, the protein content in the culture broth obtained can be increased. The serum-free medium step minimizes the differentiation by exposing the stem cells isolated from adipose tissue to a specific extreme environment and allows the recovery of as much protein as possible from the culture medium.

In addition, the present invention provides a method for regenerating the skin, characterized in that the composition is applied topically or intradermal injection to the wound site. The dosage form may preferably be topical application or intradermal injection, but is not limited thereto.

In addition, the present invention provides a method for improving wrinkles, characterized in that the composition is applied topically or intradermal injection to the site that needs to remove wrinkles. The dosage form may preferably be topical application or intradermal injection, but is not limited thereto.

The present invention also provides a therapeutic agent for skin regeneration or wrinkle improvement comprising the composition as an active ingredient.

The therapeutic agent for skin regeneration or wrinkle improvement of the present invention can be applied not only to the skin for the treatment of wounds such as wounds and burns, but also to areas requiring wrinkle removal.

Pharmaceutically acceptable carriers included in the therapeutic agents for skin regeneration or anti-wrinkle of the present invention are commonly used in the preparation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate Gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil It is not limited to this.

The therapeutic agent for skin regeneration or wrinkle improvement of the present invention is formulated using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by those skilled in the art to which the present invention pertains. It may be prepared in the form or incorporated into a multi-dose container, and may further include a dispersant or stabilizer.

In addition, the present invention provides a skin regeneration or wrinkle improvement cosmetics comprising the composition as an active ingredient.

Ingredients included in the skin regeneration or wrinkle improvement cosmetics of the present invention may include components commonly used in cosmetic compositions in addition to stem cell culture concentrate as an active ingredient, for example, antioxidants, stabilizers, solubilizers, vitamins, Conventional adjuvants and carriers such as pigments and perfumes.

The skin rejuvenation or antiwrinkle cosmetics of the present invention may be prepared in any formulations commonly prepared in the art, for example solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, cleansing and It may be formulated in a spray or the like, but is not limited thereto.

When the formulation of the present invention is a paste, cream or gel, animal oils, vegetable oils, waxes, paraffins, starches, trachants, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicas, talc or zinc oxide may be used as carrier components. Can be.

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used, in particular in the case of a spray, additionally chlorofluorohydrocarbon, propane Propellant such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as the carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 Fatty acid esters of, 3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan.

When the formulation of the present invention is a suspension, liquid carrier diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol esters and polyoxyethylene sorbitan esters, and microcrystals are used as carrier components. Soluble cellulose, aluminum metahydroxy, bentonite, agar or tracant and the like can be used.

When the formulation of the present invention is a cleansing agent, the carrier component is aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide ether sulfate, alkyl Amidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.

In addition, the skin regeneration or wrinkle improvement cosmetics of the present invention may further contain other components for the conventional skin regeneration or wrinkle improvement in addition to the stem cell culture concentrate, the type and content of these existing components can be easily selected by those skilled in the art Can be used.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1: Obtaining Adipose Stem Cells

The patient received the subcutaneous fat removal procedure, and the adipose tissue was removed from the patient by liposuction method. The extracellular matrix of adipose tissue was treated with 0.075% collagenase for 45 minutes at 37 ° C. in a 5% CO ₂ incubator, and then the resulting adipose tissue was centrifuged at about 1200 × g for 5 minutes to stroma. Sex vessel fractions were obtained. The obtained fractions were washed with PBS (phosphate buffered saline), other tissues were removed through a 70 μm nylon cell strainer, and histopaque-1077 (Sigma, St. Louis, MO, USA) was used to separate cell debris and mononuclear cells including red blood cells. .

Isolated mononuclear cells were cultured with DMEM (Dulbecco's Modified Eagle's Medium; Lonza, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA), 1% penicillin-streptomycin (Gibco, USA). After incubation for 24 hours at 37 ℃, 5% CO ₂ incubator, the non-adhesive cells were removed to isolate the adipocyte stem cells.

Example 2: Culture of Stem Cells

1.25 × 10 ⁵ cells of the adipose stem cells obtained in Example 1 were prepared with 1000 mg / L of D glucose, 584 mg / L of L-glutamine, 110 mg / L of sodium pyruvate, 10% fetal bovine serum, It was added to DMEM medium containing 1% penicillin-streptomycin and passaged for 30 days in a 5% CO ₂ incubator under about 90% humidity and about 37 ° C. temperature conditions. The culture medium was removed from the passaged culture using a pipette and washed three times with PBS, containing 365 mg / L of L-glutamine, 15 mM HEPES, and 55 mg / L of sodium pyruvate. DMEM and Ham's F-12 nutrient mixtures were inoculated at a concentration of 1.2 × 10 ⁶ cells / dish in a 1: 1 mixed medium (DMEM / F-12) and incubated for 72 hours under hypoxia conditions.

Example 3: Preparation of a culture concentrate of stem cells and fractions thereof

500 mL of the culture solution finally obtained in Example 2 was centrifuged at 300 x g for 5 minutes to remove stem cells that had settled into pellets. The resulting supernatant was filtered with a 0.22 μm syringe filter to remove residual stem cells and unknown macromolecules. The obtained solution was divided in half and concentrated under reduced pressure to obtain 5 mL of concentrate (hereinafter referred to as AAPE). The resulting concentrate was lyophilized and stored at -70 ° C.

The remaining solution from which the remaining stem cells and the unknown macromolecules are removed is passed through a membrane having a pore size of 30 kDa, and the filtrate that has not passed through the membrane is washed three times with PBS to obtain a macromolecule larger than 30 kDa. After separation into molecular fractions and fractions up to 30 kDa, solutions containing up to 30 kDa and washed PBS were again passed through a membrane having a pore size of 10 kDa to a concentrated fraction of 10 to 30 kDa and up to 10 kDa Separated into fractions. Separated 10-30 kDa fractions and at least 30 kDa fractions were stored at -70 ° C.

Example 4 Wound Healing and Skin Regeneration Effects of AAPE in Wound-induced Hairless Mice

Female hairless mice were used to test the wound healing effect of AAPE and were performed according to the Animal Laboratory Guidelines of the National Institutes of Health (NIH). Two circular wounds with a diameter of 8 mm were artificially induced on the dorsal skin of the mouse, and then only the medium (DMEM / F-12, control) was applied to the left side, and the AAPE stock solution collected in Example 3 was applied to the right side. At the same time, the fractions obtained in Example 3 (30 kDa or more and 10 to 30 kDa) were respectively applied to the wounds of the mice. The wound healing effect was analyzed by measuring the area of the remaining wound area. In observing the external repair pattern of the dermatitis-induced wounds, the control group coated with the medium only showed a significantly lower recovery at the early stage of the observation period compared to the experimental group treated with AAPE stock solution or fractions (FIGS. 1 and 2). . In the histological examination, inflammatory cells were infiltrated in the control group and flattening of the basal layer of the epidermis was observed. Also no ridge formation was observed and the treatment was not complete (FIG. 3). In the case of AAPE stock treatment, the skin regeneration was relatively superior to the control group during the observation period. In particular, the AAPE stock treatment group showed a better effect than the other experimental group as the skin regeneration after the first three days (Figs. 1 and 2). In addition, the histological findings showed that the number of inflammatory cells was significantly reduced compared to the control group, and the epidermal layer was relatively thick and the formation of rete ridges was observed, indicating that the skin regeneration effect was good (FIG. 3).

In the case of fraction treatment of 30 kDa or more showed a relatively good skin regeneration effect compared to the control during the observation period (Fig. 1 and 2). In addition, the histological findings showed almost no inflammation at the epidermal / dermal border, and the cell density of the epidermal layer was high and the formation of reticulum into the dermal layer was completely normalized (FIG. 3). In the case of 10-30 kDa fraction treatment, it showed the best skin regeneration effect in the early 3 days after application (FIGS. 1 and 2). In addition, the histological findings showed a similar healing effect as the group treated with 30 kDa or more fractions, but the inflammatory infiltration was somewhat higher than the group treated with 30 kDa or more fractions (FIG. 3). The results indicate that AAPE, a culture of adipose stem cells, exhibits a different regeneration effect according to the molecular composition of each fraction, and has excellent therapeutic activity of skin regeneration. Furthermore, the entire AAPE fraction constituting all of them is differentiated through the first half and the second half during skin regeneration. It suggests that it has a good therapeutic activity by operating. In addition, AAPE stock solution showed sufficient regeneration effect compared to the concentrated component fraction. Therefore, it is expected that the whole fraction of adipose stem cell cultured AAPE can be usefully used for symptom improvement and treatment of damaged skin.

Example 5 Evaluation of Type 1 Collagen Production Promoting Effect Using Normal Human Dermal Fibroblasts

DMEM mixed with 1% of an excipient penicillin-streptomycin was added to 20 μg of lyophilized AAPE, and then dissolved by vortexing. To a total of 6 mL by adding an excipient to 0.192 mL of the prepared AAPE solution (10 μg / mL), a stock solution having a final concentration of 0.32 μg / mL was prepared. The stock solution was diluted by azeotropy 4 to prepare a total of three concentrations (0.32, 0.08 and 0.02 μg / mL). In addition, ascorbic acid (L-ascorbic acid; Sigma, USA) as a positive control was dissolved in the excipient was prepared at a concentration of 35.22 μg / mL (200 μM), the negative control was administered to the excipient.

The cell lines used in the present invention are normal human dermal fibroblasts (7F3802, Clonetics, USA), and the cell line is a complete medium (DMEM / containing 10% fetal bovine serum and 1% penicillin-streptomycin). After subculture in F-12), it was used in the experiment at 5 passages. Human dermal fibroblasts cultured in the complete medium were divided into 400 μl (1 × 10 ⁵ cells / 400 μl / well) in 48 well plates, respectively, for 24 hours under 37 ° C. and 5% CO ₂ conditions. Incubated. After completion of the culture, the culture solution of each well was removed, and 500 μl of D-PBS (Dulbecco's phosphate buffered saline) was added to each well and washed. 800 μl of each preparation of the AAPE and the positive control was added to each well, and 800 μl of the excipient was added to the well of the negative control, followed by incubation for 48 hours at 37 ° C. and 5% CO _2. It was. After completion of the culture, the culture solution of each well was recovered and centrifuged at 25 ° C. and 3000 rpm for 10 minutes, and then the supernatant was taken and used for quantification of procollagen type I (procollagen type I). 1 mL of D-PBS was added to each well of the plate from which the culture solution was removed, and then, 300 μl of cell lysis buffer was added to each well. Cells were hemolyzed by storing the plates in a deep freezer (deep freezer, -70 ° C) for 2 hours and freezing, then thawing again at room temperature. Cell lysates of each well were collected and centrifuged at 13,000 rpm for 30 minutes at 4 ° C., and then the supernatant was taken and used for quantification of total protein.

For quantification of total protein, 40 μl of the supernatant obtained was placed in each well of a 96 well plate. In addition, the standard solution provided in the BCA protein quantitative kit (Pierce Biotechnology Inc., USA) was diluted to prepare a 250, 125, 50, 25, 5 and 0 ㎍ / mL, and then 40 ㎕ each to another well. Subsequently, 160 μl of the reagent mixture provided in the protein quantification kit was added to each well containing 40 μl of the supernatant and standard solution, and then reacted in a 60 ° C. incubator for 30 minutes. After completion of the reaction, the absorbance of each well was measured at 562 nm using an ELISA reader (PowerWave XS, BioTek, Instruments, Ins., USA). The absorbance of each well was substituted into the standard curve formula to calculate the total protein amount of the wells to which AAPE, positive control and negative control were added.

For quantification of type 1 procollagen, the obtained supernatant was placed in 100 μl into each well of a 96 well plate provided in type 1 procollagen C-peptide EIA kit (Takara Bio Inc., Japan). In addition, the standard solution provided in the kit was diluted in steps to prepare 640, 320, 160, 80, 40, 20, 10 and 0 ng / mL, and then 100 μl of each was added to another well. Thereafter, the reaction was carried out in a 37 ° C. incubator for 2 hours. After completion of the reaction, the reaction solution of each well was removed, and 400 μl of PBS was added to each well, followed by washing (4 times). 100 μl of TMBZ (tetramethylbenzidine) substrate solution provided in the kit was added to each well, followed by reaction at 30 ° C. for 15 minutes. After completion of the reaction by adding 100 μl of a stop solution (1N H ₂ SO ₄ ) to each well, the absorbance of each well was measured at 450 nm using an ELISA reader. Absorbance was substituted into the standard curve equation to calculate the amount of type 1 procollagen in wells with AAPE, positive control and negative control. Synthesis rate of procollagen type I was calculated by substituting the corrected amount of type 1 procollagen into the following formula.

% Production of type 1 procollagen = A / B × 100

A: Procollagen type 1 of the test substance or positive control.

B: Procollagen type 1 of negative control.

The production rate of type 1 procollagen obtained in the present invention was assayed using a statistical program SAS (version 9.1.3, SAS Institute Inc., Cary, NC, USA). One-way analysis of variance was performed (significance level: 5%), and a multiple test of Dunnett's t-test was performed to confirm the significance of the test group to the control group (significance level; unilateral 5% and 1%). .

As a result, there was no statistically significant difference (0.02 μg / mL; 108.0 ± 0.6%) in the type 1 procollagen production rate of the AAPE-treated group at 0.02 μg / mL compared to the negative control (100.0 ± 0.0%). The AAPE administration groups at 0.08 and 0.32 μg / mL concentrations were significantly increased (0.08 μg / mL; 114.8 ± 1.2%, 0.32 μg / mL; 142.7 ± 5.5%). The type 1 procollagen production rate of the positive control at the concentration of 35.2 μg / mL (200 μM) was statistically significantly increased (35.2 μg / mL; 161.0 ± 9.8%) compared to the negative control (FIG. 4).

The present invention was conducted to evaluate the type 1 collagen production promoting effect of the test substance AAPE using normal human dermal fibroblasts. Treatment of human dermal fibroblasts with AAPE at 0.02, 0.08 and 0.32 μg / mL concentrations increased the production rate of type 1 procollagen in a dose-dependent manner, and the AAPE group at 0.08 and 0.32 μg / mL concentrations was negative. Statistically significant increase compared to the control. Therefore, the test material AAPE is considered to have the effect of promoting the production of collagen type 1.

Claims (8)

1. A composition for skin regeneration or wrinkle improvement comprising the stem cell culture concentrate obtained by filtration after culturing human adipose derived stem cells in a serum medium and then subcultured in a serum-free medium.
2. According to claim 1, wherein the stem cell culture concentrate is

   Culturing human adipose derived stem cells in serum medium and then subcultured in serum-free medium;

   Applying one or more physical stimuli selected from hypoxic culture, ultraviolet irradiation, low frequency treatment, nutrient deprivation, and mechanical friction to the passaged stem cells;

   Culturing by adding one or more vitamins selected from vitamin A, vitamin B, vitamin C and vitamin D to the culture medium, and

   The composition for skin regeneration or wrinkle improvement, which is prepared by filtering the culture solution.
3. The composition of claim 1, wherein the serum-free medium is a mixed medium of DMEM and Ham's F-12 nutrient mixture.
4. The composition for skin regeneration or wrinkle improvement according to claim 1, wherein the composition exhibits skin regeneration or wrinkle improvement by increasing the production of type 1 procollagen in dermal fibroblasts.
5. A method for regenerating the skin, wherein the composition of claim 1 is applied topically or intradermally to the wound.
6. The method for improving wrinkles according to claim 1, wherein the composition of claim 1 is applied topically or intradermally to a site requiring wrinkle removal.
7. A therapeutic agent for skin regeneration or wrinkle improvement comprising the composition of claim 1 as an active ingredient.
8. Skin regeneration or wrinkle improvement cosmetics comprising the composition of claim 1 as an active ingredient.

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