KR20120083166A

KR20120083166A - Enriched media of human adipose tissue-derived stem cells having skin elastic maintenance or antiaging effect and uses thereof

Info

Publication number: KR20120083166A
Application number: KR1020110004669A
Authority: KR
Inventors: 김동수; 박예형
Original assignee: (주)프로스테믹스
Priority date: 2011-01-17
Filing date: 2011-01-17
Publication date: 2012-07-25

Abstract

The present invention is a skin elastic maintenance or anti-aging composition containing the stem cell culture medium obtained by filtration after continuous culture in human fat-derived stem cells in serum medium and then cultured in serum-free medium, or a concentrate thereof as an active ingredient, the Skin elasticity maintenance or anti-aging method characterized in that the composition is applied to the skin topically or intradermal injection, skin elasticity maintenance or anti-aging treatment comprising the composition as an active ingredient and skin elasticity comprising the composition as an active ingredient It relates to a maintenance or anti-aging cosmetics.

Description

Enriched media of human adipose tissue-derived stem cells having skin elastic maintenance or antiaging effect and uses about

The present invention relates to a culture concentrate of human adipose derived stem cells having a skin elasticity or anti-aging effect and its use, and more particularly, to continuous culture in serum-free medium following passage of human adipose derived stem cells in serum medium. After that, the skin elasticity maintenance or anti-aging composition containing the stem cell culture solution or the concentrate obtained by filtration as an active ingredient, the skin elasticity maintenance or anti-aging, characterized in that the composition is applied to the skin locally or intradermal injection The present invention relates to a method for maintaining skin elasticity or anti-aging including the composition as an active ingredient, and to maintaining skin elasticity or anti-aging cosmetics comprising the composition as an active ingredient.

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 the 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, the importance of adult stem cells has recently been highlighted, and various studies are under way to reveal its usefulness.

Korean Patent Publication No. 2010-0114729 discloses a composition for skin regeneration using umbilical cord blood stem cell-derived angiogenic precursor cells, and Korean Patent Publication No. 2010-0032099 for skin wrinkle improvement using a mammalian stem cell culture solution or Disclosed is a composition for inhibiting skin aging.

The present invention is derived from the above requirements, the present inventors subcultured human adipose-derived stem cells in serum medium and cultured in serum-free medium, the stem cell culture obtained by filtration or the concentrate of the skin elasticity The present invention has been found to be effective in maintaining or preventing aging.

In order to solve the above problems, the present invention is subcultured in human adipose derived stem cells in serum medium and then cultured in serum-free medium, maintaining the skin elasticity containing the stem cell culture solution obtained by filtration or its concentrate as an active ingredient Or it provides an anti-aging composition.

In addition, the present invention provides a method for maintaining skin elasticity or anti-aging, characterized in that the composition is applied to the skin topically or intradermal injection.

The present invention also provides a therapeutic agent for maintaining skin elasticity or anti-aging comprising the composition as an active ingredient.

In addition, the present invention provides a skin elasticity maintenance or anti-aging cosmetics comprising the composition as an active ingredient.

Since the culture solution of human adipose derived stem cells of the present invention or the concentrate thereof significantly inhibits the activity of elastase, it is expected to be effectively used for maintaining skin elasticity or preventing aging.

Figure 1 shows the DNA expression of the elastin-related molecular group AAPE 44K-7.
Figure 2 shows the DNA expression of each group of elastin-related group AAPE 44K-7.
Figure 3 shows the inhibitory effect of AAPE on elastase activity (preliminary experiment).
Figure 4 shows the inhibitory effect of AAPE and ursolic acid on the elastase activity (this experiment).

In order to achieve the object of the present invention, the present invention is a skin containing a stem cell culture medium or its concentrate obtained by filtration after culturing human adipose derived stem cells in serum medium and then cultured in serum-free medium. It provides elasticity or anti-aging composition.

The stem cell culture or its concentrate is preferably

(a) passage of human adipose derived stem cells in serum medium followed by continuous culture in serum-free medium;

(b) applying any one or more physical stimuli selected from low oxygen culture, ultraviolet irradiation, low frequency treatment, nutrient deprivation, and mechanical friction to the cultured 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 production of stem cell culture concentrate, refer to Korean Patent Publication No. 2008-0109725, the entire contents of which are included in the present invention.

In the composition of the present invention, the serum-free medium may preferably be a mixed medium of DMEM (Dulbecco's Modified Eagle's Medium) and Ham's F-12 nutrient mixture (see Korean Patent Publication No. 2008-0109725), 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 elasticity or anti-aging effects by inhibiting the activity of elastase.

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, the human adipose derived stem cells are cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum, and finally cultured 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 maintaining skin elasticity or anti-aging, characterized in that the composition is applied to the skin topically or intradermal injection. The dosage form may preferably be topical application or intradermal injection, but is not limited thereto.

Pharmaceutically acceptable carriers used in the maintenance of skin elasticity or anti-aging therapeutic agents of the present invention are commonly used in the preparation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, know Nate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oils It may be, but is not limited thereto.

Skin elasticity maintenance or anti-aging therapeutic agent of the present invention is formulated using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily carried out by those skilled in the art to which the present invention belongs It may be prepared in a dosage form or incorporated into a multi-dose container and may further include a dispersant or stabilizer.

Ingredients included in the skin elasticity maintenance or anti-aging cosmetics of the present invention as an active ingredient may include components commonly used in cosmetic compositions in addition to the stem cell culture concentrate, for example, antioxidants, stabilizers, solubilizers, vitamins And conventional adjuvants and carriers such as pigments and fragrances.

The skin firming or anti-aging cosmetics of the present invention may be prepared in any formulation 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, an animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier component .

In the case where 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 as a carrier component. Especially, in the case of a spray, a mixture of chlorofluorohydrocarbons, propane / Propane 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, as a carrier component, an aliphatic alcohol sulfate, an aliphatic alcohol ether sulfate, a sulfosuccinic acid monoester, isethionate, an imidazolinium derivative, a methyltaurate, a sarcosinate, a fatty acid amide ether sulfate, an alkyl Amidobetaines, fatty 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 elasticity maintenance or anti-aging cosmetics of the present invention may further contain other conventional skin elasticity or anti-aging components in addition to the stem cell culture concentrate, the type and content of these conventional components can be easily You can choose to use it.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example 1: Obtaining Adipose Stem Cells

Consent was obtained from a patient who had undergone a subcutaneous fat removal procedure, and the adipose tissue was removed from the patient by liposuction. The extracellular matrix of adipose tissue was treated with 0.075% collagenase for 45 minutes at 37 ° C., 5% CO ₂ incubator, and then the resulting adipose tissue was centrifuged at about 1200 × g for 5 minutes. Stromal vascular fractions were obtained. The obtained fractions were washed with PBS (phosphate buffered saline), other tissues were removed through a 70 μm nylon cell filter, 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 in a 37 ℃, 5% CO ₂ incubator, the non-adhesive cells were removed to isolate the fat stem cells.

Example 2: Culture of Stem Cells

Adipose stem cells 1.25 × 10 ⁵ cells obtained in Example 1 were obtained 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. Inoculated at a concentration of 1.2 × 10 ⁶ cells / dish or 1.2 × 10 ⁸ cells / cell factory in a 1: 1 mixed medium (DMEM / F-12) of DMEM and Ham's F-12 nutrient mixtures, under hypoxia conditions. Incubated for a time ~ 1 week.

Example 3: Culture and Concentration of Stem Cells

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 sunk 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 (hereinafter referred to as AAPE) was divided into small portions, dispensed, lyophilized, or concentrated in half to give a concentrated solution of 5 mL. The resulting concentrate was lyophilized and stored at -70 ° C.

Example 4: AAPE Composition Profiling Results (Elastin Related Molecular Groups AAPE 44K-7)

Table 1 shows the expression of AAPE DNA by human whole genome DNA chip (Agilent 44K microarray), where elastin-related molecules were present in the expression profile, and 7 of the molecules were selected to select the main molecular group (AAPE 44K). -7). Lysyl oxidase is a protein necessary to form cross-linked tertiary stereostructures by oxidizing lysyl present in collagen and elastin, and TIMP (tissue inhibitor of metalloproteinase) is an extracellular matrix. matrix) is thought to play a role in protecting the structure by inhibiting enzymes involved in the degradation of the collagen and elastin structures.

Figure 1 shows the results of RNA extracted from AASC-producing ADSC (human adipose derived stem cells), and then trace back the protein profile expressed by DNA microarray. Figure 1 shows data on AAPE producing intracellular expression levels (expression levels in hypoxic environment versus oxygen environment culture) of the AAPE 44K-7 molecule group believed to be related to elastin (control: normal oxygen partial pressure ( normoxia)), LOXL1-3 required for collagen-elastin cross-linking show very strong expression.

FIG. 2 is a graph showing the expression pattern of three AAPE 44K-7 molecular groups, and the introduced value is the expression level measured on the DNA chip. The results indicate that overexpression of certain related molecular groups is induced by the hypoxic environment introduced during AAPE production, and therefore AAPE can be used for inhibiting degeneration of skin tissue.

Example 5: AAPE of Elastase ( elastase Activity inhibition efficacy test

The test was conducted to evaluate the inhibitory effect of elastase activity by AAPE using elastase isolated from porcine pancreas. The treatment concentration of the test substance for evaluating the inhibitory effect of elastase activity was set with reference to the result of calculating the approximate IC50 obtained by performing the preliminary test. In the preliminary test, the treatment concentration of the test substance was set to 4 µg / mL as the highest concentration, and then diluted in azeotropic 4 to set 5 concentrations (0.0157, 0.0626, 0.25, 1, and 4 µg / mL).

In the preliminary test, the test substances at 0.0157, 0.0626, 0.25, 1 and 4 μg / mL concentrations showed that the activity of elastase was 92.8, 93.3, 79.2, 48.1 compared to the negative control group (100%), respectively. And 33.6%, with an approximate IC50 of 1 μg / mL (FIG. 3 and Table 2). Accordingly, the treatment concentrations of the test substance in this test were set to 0.5, 1 and 2 ㎍ / mL, and the application concentration of the positive control ursolic acid was set to 100 ㎍ / mL.

As a result of treatment of 0.5, 1 and 2 μg / mL concentrations in this study, the activity of elastase was statistically significantly reduced concentration-dependently to 86.0, 51.6 and 42.7%, respectively, compared to the negative control group. It became. The elastase activity in the positive control ursolic acid treated group was significantly reduced as 57.2% compared to the negative control treated group (Fig. 4 and Table 3).

In conclusion, in the activity inhibitory efficacy test of elastase isolated from porcine pancreas, the test substance AAPE was found to have a significant inhibitory effect on elastase activity at a concentration of 0.5 μg / mL or more.

Claims

Subcutaneous culture of human fat-derived stem cells in serum medium and then cultured in serum-free medium, the skin elasticity maintenance or anti-aging composition containing the stem cell culture obtained by filtration or its concentrate as an active ingredient.

The method of claim 1, wherein the stem cell culture or its concentrate
Subcultured human adipose derived stem cells in serum medium followed by continuous culture in serum-free medium;
Applying any one or more physical stimuli selected from low oxygen culture, ultraviolet irradiation, low frequency treatment, nutrient deprivation, and mechanical friction to the cultured 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
Skin elasticity maintenance or anti-aging composition, characterized in that prepared by filtering the culture solution.

The composition of claim 1, wherein the serum-free medium is a mixed medium of DMEM and Ham's F-12 nutrient mixture.

The composition for maintaining skin elasticity or anti-aging according to claim 1, wherein the composition exhibits skin elasticity maintaining or anti-aging effect by inhibiting the activity of elastase.

A method for maintaining skin elasticity or anti-aging, wherein the composition of claim 1 is applied to the skin topically or intradermally.

A therapeutic agent for maintaining skin elasticity or anti-aging comprising the composition of claim 1 as an active ingredient.

Skin elasticity maintenance or anti-aging cosmetics comprising the composition of claim 1 as an active ingredient.