WO2010008219A2 - Culture de cellules souches pluripotentes provenant de tissu adipeux et composition cosmétique contenant une protéine extraite de ces cellules - Google Patents

Culture de cellules souches pluripotentes provenant de tissu adipeux et composition cosmétique contenant une protéine extraite de ces cellules Download PDF

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WO2010008219A2
WO2010008219A2 PCT/KR2009/003923 KR2009003923W WO2010008219A2 WO 2010008219 A2 WO2010008219 A2 WO 2010008219A2 KR 2009003923 W KR2009003923 W KR 2009003923W WO 2010008219 A2 WO2010008219 A2 WO 2010008219A2
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adipose tissue
stem cells
cosmetic composition
derived
adult stem
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PCT/KR2009/003923
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English (en)
Korean (ko)
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WO2010008219A3 (fr
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라정찬
강성근
임자옥
김효은
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주식회사 알앤엘바이오
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • the present invention provides a protein extracted from (i) adipose tissue-derived adult stem cell culture and (ii) the adipose tissue-derived adult stem cell; Or it relates to a cosmetic composition containing a peptide or amino acid which is a hydrolyzate of the protein and a method for producing the same.
  • Stem cells are cells that have the ability to self-replicate and differentiate into two or more cells.
  • Totipotent stem cells pluripotent stem cells, and multipotent stem cells ( multipotent stem cells).
  • adipose tissue has been found to be a new source of multipotent stem cells (B. Cousin et al., BBRC , 301: 1016, 2003; A. Miranville et al ., Circulation , 110: 349, 2004; S. Gronthos et al., J. Cell Physiol., 189: 54, 2001; MJ Seo et al., BBRC , 328: 258, 2005).
  • adipose tissue obtained by human liposuction contains undifferentiated cell populations, which have differentiation ability to adipocytes, osteoblasts, myoblasts and chondrocytes in vitro (PA Zuk et al., Tissue Eng ., 7: 211, 2001; AM Rodriguez et al., BBRC , 315: 255, 2004).
  • liposuction contains undifferentiated cell populations, which have differentiation ability to adipocytes, osteoblasts, myoblasts and chondrocytes in vitro
  • PA Zuk et al., Tissue Eng ., 7: 211, 2001; AM Rodriguez et al., BBRC , 315: 255, 2004 PA Zuk et al., Tissue Eng ., 7: 211, 2001
  • AM Rodriguez et al., BBRC 315: 255, 2004.
  • adipose tissue-derived cells have the ability to promote muscle regeneration and neurovascular differentiation.
  • adipose derived stem cells include human adipose derived adult stem cells capable of differentiating into epithelial cells (M. Brzoska et al., BBRC , 330: 142, 2005), human adipose derived adult stem capable of bone formation and differentiation into adipose cells. Cells (Y. Cao et al., BBRC , 332: 370, 2005), human adipose derived stem cells capable of differentiation into neurons (KM Safford et al., BBRC, 294: 371, 2005), capable of differentiating into adipocytes Rat Adipose Stem Cells (R.
  • Rat Adipose Stem Cells capable of Differentiating to Bone Formation and Cartilage Cells R. Ogawa et al., BBRC , 313: 871, 2004
  • human adipose derived stem cells capable of differentiation into chondrocytes HA Awad et al., Biomaterials , 25: 3211, 2004
  • rat adipose derived stem cells capable of differentiating into neuronal cells J. Fujimura et al., BBRC , 333. : 116, 2005
  • adipose derived stem cells capable of differentiating into osteoblasts, chondrocytes, neurons or muscle cells US Pat. No. 6,777,231). Etc.
  • the present inventors have made efforts to use stem cells in the fields of cosmetics and cosmetics such as wrinkle improvement, prevention of sagging of skin, and cultured by obtaining adult stem cells from adipose tissue obtained from liposuction; After obtaining a protein or amino acid extracted from the adipose tissue-derived stem cells, it was confirmed that the composition containing it together is effective for cosmetic and molding, to complete the present invention.
  • the main object of the present invention is (i) culture of adult stem cells derived from mammalian adipose tissue, and (ii) proteins extracted from the adult stem cells derived from mammalian adipose tissue; Or to provide a cosmetic composition containing a peptide or amino acid that is a hydrolyzate of the protein as an active ingredient.
  • Another object of the present invention is to provide a method for producing a culture of adult stem cells derived from mammalian adipose tissue containing specific components.
  • the present invention is a mammalian adipose tissue-derived adult stem cell culture containing Fibronectin, VEGF and Procollagen, and a functional containing a protein or amino acid extracted from the adipose tissue-derived adult stem cells as an active ingredient It provides a cosmetic composition.
  • the protein or amino acid is Tau, PEA (Phosphoethanolamine), Thr, Ser, Glu, ⁇ -AAA ( ⁇ -Aminoadipic acid), Gly, Ala, ⁇ -ABA ( ⁇ -Amino-n-butyric acid), Val Cys, Met, Cysthi (Cystathionine), Ile, Leu, Tyr, Phe, ⁇ -Ala, ⁇ -AiBA ( ⁇ -amino isobutyric acid), ⁇ -ABA (Gamma Amino-n-Butyric Acid), NH3 (Ammonia) , Hylys (hydroxylysine), Orn (Ornithine), Lys, His, Car, Arg, Asp and Pro may be one or more selected from the group consisting of, Thr, Ser, Glu, Gly, Ala, Val, Leu, Tyr, Phe, NH3 (Ammonia), Lys, characterized in that Asp and Pro.
  • the protein or amino acid extracted from the adipose tissue-derived adult stem cells can be suspended in a mammalian adipose tissue-derived adult stem cell culture.
  • cosmetic compositions include human adipose tissue-derived adult stem cell cultures containing TGF, bFGF, IGF, KGF, HGF, fibronectin, VEGF and Procollagen, and proteins derived from adult stem cells derived from human adipose tissue or Contains amino acids as active ingredients.
  • the present invention also provides a mammalian adipose tissue-derived adult stem cell culture containing TGF, bFGF, IGF, KGF, HGF, fibronectin, VEGF, and Procollagen, which are used as the main active ingredients of the cosmetic composition, and a method of preparing the same. .
  • 1 is a result of analyzing the secreted protein components of the adult adipose stem cells derived from human adipose tissue derived adult stem cells of the present invention.
  • Figure 2 is the result of analyzing the components of the secreted protein according to the Passage step of adult stem cells derived from human adipose tissue.
  • 3 is a graph of visual observation of changes in skin color according to the use of the cosmetic composition of the present invention.
  • Figure 4 is a graph of the L * value results measured by using a spectrophotomer skin color change according to the use of the cosmetic composition of the present invention.
  • Figure 5 is a photograph taken using the spectrophotomer skin color change according to the use of the cosmetic composition of the present invention.
  • Figure 6 is a graph showing the skin lifting effect using the cosmetic composition of the present invention.
  • Figure 7 is a facial contour picture showing the skin lifting effect according to the use of the cosmetic composition of the present invention.
  • 11 is a graph of visual observation of changes in skin wrinkles according to the use of the cosmetic composition of the present invention.
  • FIG. 12 is a graph of R-values measured by using a Visiometer skin wrinkle changes according to the use of the cosmetic composition of the present invention.
  • Figure 13 is a questionnaire evaluation results by the subject for the use of the cosmetic composition of the present invention.
  • the present invention in one aspect, mammalian adipose tissue-derived adult stem cell culture; It relates to a functional cosmetic composition containing a protein or amino acid extracted from the adult adipose tissue derived adult stem cells as an active ingredient.
  • Functional cosmetic composition of the present invention includes mammalian adipose tissue-derived adult stem cell culture.
  • Stem cells are cells that have the capacity of self-replicating and have the ability to differentiate into two or more cells.
  • Adult stem cells are stems that appear during the development or development of each organ of the embryo. It means a cell.
  • the term "mammal adipose tissue-derived adult stem cells” are undifferentiated adult stem cells isolated from adipose tissue of mammals, which are abbreviated herein as “fat stem cells”. This is done by culturing a suspension containing fat suspended in physiological saline obtained from liposuction, and then recovering the stem cell layer attached to the culture vessel such as a flask with trypsin and then recovering it, or scraping it with a scraper to recover the suspension in a small amount of physiological saline directly. It is obtained through the method or the like. In one embodiment of the present invention, preferably, adult stem cells derived from human adipose tissue can be used.
  • the term "mammal adipose tissue-derived adult stem cell culture” or “fat stem cell culture” refers to culturing the mammalian adipose tissue-derived adult stem cells in a medium containing a specific component, and then collecting the medium. After the removal of the cell debris (debris) refers to the remaining (broth). After all, the culture may be said to contain the secretion of the main components and fat stem cells contained in the medium. In one aspect of the present invention, preferably, adult stem cell culture derived from human adipose tissue can be used.
  • a medium used for obtaining the mammalian adipose stem cell culture a conventional medium known in the art to be suitable for culturing stem cells may be used.
  • DMEM Dense C Eagle medium
  • Keratinocyte-SFM Keratinocyte serum free
  • Adipose stem cell culture medium may be supplemented with additives that promote proliferation of the undifferentiated phenotype of adipose stem cells while inhibiting differentiation.
  • the medium generally contains neutral buffers (such as phosphates and / or high concentrations of bicarbonate) and protein nutrients (such as serum, such as FBS, serum substitutes, albumin, or essential and non-essential amino acids, such as glutamine) in isotonic solutions. can do.
  • protein nutrients such as serum, such as FBS, serum substitutes, albumin, or essential and non-essential amino acids, such as glutamine
  • lipids fatty acids, cholesterol, HDL or LDL extracts of serum
  • other components found in most preservative media of this kind such as insulin or transferrin, nucleosides or nucleotides, pyruvate salts, any ionized form or salt
  • Sugar sources such as glucose, selenium, glucocorticoids such as hydrocortisone and / or reducing agents such as ⁇ -mercaptoethanol.
  • the medium also contains anti-clumping agents such as those sold by Invitrogen (Cat # 0010057AE) for the purpose of preventing cells from adhering to each other, adhering to the vessel wall, or forming too large a bundle. It can be beneficial to do so.
  • anti-clumping agents such as those sold by Invitrogen (Cat # 0010057AE) for the purpose of preventing cells from adhering to each other, adhering to the vessel wall, or forming too large a bundle. It can be beneficial to do so.
  • SCF Stem cell factor
  • Steel factor other ligands or antibodies that dimerize c-kit
  • other active agents of the same signal transduction pathway SCF, Steel factor
  • ⁇ Receptors of other tyrosine kinase related receptors such as platelet-Derived Growth Factor (PDGF), macrophage colony-stimulating factor, Flt-3 ligand and Vascular Endothelial Growth Factor (VEGF) Ligands for
  • ⁇ Factors that raise the cyclic AMP concentration such as forskolin
  • Hematopoietic stem growth factors such as thrombopoietin (TPO)
  • EGF epidermal growth factor
  • Neurotropins such as CNTF
  • N-acetyl-L-cysteine (NAC) N-acetyl-L-cysteine
  • the medium for culturing mammalian adipose stem cells of the present invention preferably contains NAC, ascorbic acid, calcium, insulin and hydrocortisone, more preferably FBS, NAC, ascorbic acid, calcium, rEGF, insulin and Hydrocortisone.
  • mammalian adipose stem cell culture of the present invention for example, human adipose stem cell culture, it can be obtained by the following method.
  • centrifugation and filtering of human adipose tissue-derived pellets from human adipose tissue obtained by liposuction or the like remove debris, and then in DMEM medium containing FBS, NAC and ascorbic acid. Incubate. Unattached cells were washed and incubated with Keratinocyte-SFM media containing FBS, NAC, ascorbic acid, calcium, rEGF, insulin and Hydrocortisone about every 2 days, Passage the isolated multipotent mesenchymal stem cells.
  • the adipose tissue-derived multipotent mesenchymal stem cells of Passage 3 were cultured at 90% confluency, and then cultured for about 120 hours in Keratinocyte-SFM media containing NAC, ascorbic acid, calcium, insulin, and hydrocortisone. After harvesting, centrifugation and filtering to remove cell debris yield pure human adipocyte stem cell culture.
  • human adipose tissue-derived adult stem cell cultures containing TGF, bFGF, IGF, KGF, HGF, fibronectin, VEGF and Procollagen comprising the following steps: Provide the manufacturing method:
  • Keratinocyte-SFM media containing FBS, NAC, ascorbic acid, calcium, rEGF, insulin and Hydrocortisone;
  • step (e) collecting the Keratinocyte-SFM media of step (d), followed by centrifugation and filtering to obtain a pure cell culture.
  • the fat stem cell culture of the present invention obtained by the method of the present invention which is different from the medium and components used for the isolation and cultivation of conventional fat stem cells, contains a fat stem contained in addition to the medium component. It contains the following protein components that cells secrete at high concentrations:
  • fibronectin (2402.3 pg / ml)
  • VEGF (6501.72 pg / ml)
  • TGF-b The transforming growth factor-beta (TGF-b) transforming growth factor affects tissue formation patterns, differentiation and proliferation, cell death, etc. in the embryo, and in adults, functions such as repairing wound tissue and regulating immune cell proliferation.
  • IGF-I Insulin-like Growth Factor-1
  • IGF-I Insulin-like growth factor
  • HGF hepatocyte growth factor
  • VEGF vascular endothelial growth factor
  • Vascular endothelial growth factor is a cytokine that increases the permeation of plasma proteins in capillaries and promotes cell division and migration and induces protease that causes cell reorganization.
  • the survival of newly formed blood vessels is maintained, and the antigen supply of neurons is suppressed to induce immune regulation to induce cell growth and division. It promotes the migration of vascular cells to promote the development and differentiation of new cells, thereby increasing the composition of the skin.
  • Procollagen is a protein component that makes up the dermal layer of the skin. It combines with moisture to maintain skin's moisture and elasticity.
  • such secreted proteins are proteins that are commonly secreted by stem cells derived from mammalian adipose tissue.
  • the US National Center for Biological Studies searched for similarities between human proteins and species.
  • chimpanzees were 99.2%, dogs 97.8%, cows 97.5%, mice 92.4%, and mice. Similarity was found in 92.8%, and in the case of Fibronectin 1, chimpanzees had 99.7%, dogs 95.2%, cows 94.0%, mice 92.2%, rats 92.3%, and VEGF type A 96.7%. , 94.7% of cows, 90.7% of mice, 90.7% of mice.7% of mice.
  • the effect of the present invention contains Fibronectin, VEGF and Procollagen It can be seen to include all the cases of mammalian adipose stem cell culture.
  • the functional cosmetic composition of the present invention in addition to the mammalian adipose tissue-derived adult stem cell culture containing Fibronectin, VEGF and Procollagen, a protein extracted from mammalian adipose tissue-derived adult stem cells; Or a peptide or amino acid that is a hydrolyzate of the protein. It can also be called a purified product of mammalian adipose stem cells.
  • the amino acids derived from human adipose stem cells analyzed in one embodiment of the present invention are also mammalian adipose stem cells.
  • the range can be extended to amino acids derived from. That is, the present invention includes not only human adipose stem cells but also amino acids extracted from other mammalian adipose stem cells.
  • proteins or amino acids extracted from adult stem cells derived from human adipose tissue can be obtained by the following method.
  • This method relates to a method of producing a state of maintaining the activity by salt precipitation, dialysis of the proteins constituting the fat stem cells.
  • the adipose stem cells After washing the adipose stem cells so that residual components of the adipose stem cell culture solution do not remain, the adipose stem cells are crushed and Ammonium Sulfate is added to the supernatant little by little to precipitate the protein to recover the protein pellets.
  • dialysis is performed by means of removing impurities. After the dialysis is completed, the protein is recovered by centrifugation, the supernatant is recovered, and lyophilized to extract protein components of fat stem cells.
  • low-molecular amino acids and peptides can be prepared by hydrolyzing the proteins constituting the adipose stem cells.
  • Fatty stem cells obtained by leaving no residual components of the adipose stem cell culture solution were treated with a protease, stirred and subjected to enzymatic digestion. After this process, the proteins that make up the adipose stem cells are converted into peptide and amino acid states.
  • the amino acids extracted from the adipose tissue-derived adult stem cells of the present invention obtained by the above method include the following amino acids. That is, the protein or amino acid is Tau, PEA (Phosphoethanolamine), Thr, Ser, Glu, ⁇ -AAA ( ⁇ -Aminoadipic acid), Gly, Ala, ⁇ -ABA ( ⁇ -Amino-n-butyric acid), Val, Cys, Met, Cysthi (Cystathionine), Ile, Leu, Tyr, Phe, ⁇ -Ala, ⁇ -AiBA ( ⁇ -amino isobutyric acid), ⁇ -ABA (Gamma Amino-n- Butyric Acid), NH3 (Ammonia), Hylys (hydroxylysine), Orn (Ornithine), Lys, His, Car, Arg, Asp and Pro may be one or more selected from the group, Preferably, Thr, Ser, Glu, Gly, Ala, Val, Leu, Tyr , Phe,
  • the freeze-dried fat stem cell extract protein, low molecular peptide or amino acid obtained by the above method can be suspended in the adipose stem cell culture described above to make a cosmetic composition of the present invention.
  • a functional cosmetic can be prepared and used by using the culture mixture of the present invention as an active ingredient.
  • the mammalian adipose tissue-derived adult stem cell culture mixture in which the adipose stem cell extract protein, the low molecular peptide or the amino acid is suspended, may be contained in the range of 1 to 100% by weight based on the total weight of the cosmetic composition. It is more preferably contained in the range of 1 to 10% by weight, more preferably 1 to 5% by weight.
  • cosmetics include lotion, milky lotion, cream, gel, essence (essence), pack cosmetics, and the like.
  • Cosmetics containing an effective amount of the composition of the present invention is promoted the regeneration of the skin tissue of the applied site, in particular, it is possible to maintain a young and healthy skin by the wrinkle improvement and / or anti-aging effect.
  • skin 'wrinkle improvement and / or anti-aging' collectively refers to all skin moisturizing, skin elasticity enhancement, skin thickness increase, skin wrinkle improvement, skin irritation and skin damage recovery action.
  • Cosmetic water is generally a clear liquid cosmetic applied to the skin surface in order to keep the skin clean and healthy.
  • the basic function of the lotion is to supply moisture or moisturizing ingredients to the stratum corneum of the skin, and the lotion also has the function of softening the skin. Dissolves and stabilizes substances that are difficult to dissolve in water to make the appearance transparent. Transparent or translucent using microemulsion or lipid nanosphere, O / W type (oil in water type), The opaque lotion emulsified by W / O type or W / S type
  • known “cosmetics” can be appropriately used depending on the purpose thereof.
  • lotion contains the following components, for example. Namely, purified water for dissolving water-soluble components such as ion-exchanged water and supplying water to the stratum corneum; alcohol for dissolving and sterilizing oil-soluble components such as ethanol and propanol to give a refreshing feeling; glycerin, PEG, hyal Moisturizers for moisturizing the stratum corneum, such as lonic acid; emollients (oil components that prevent moisture from evaporating); moisturizing agents such as ester oils and vegetable oils; and raw material components such as polyoxyethylene oleyl alcohol ethers.
  • purified water for dissolving water-soluble components such as ion-exchanged water and supplying water to the stratum corneum
  • alcohol for dissolving and sterilizing oil-soluble components such as ethanol and propanol to give a refreshing feeling
  • glycerin, PEG, hyal Moisturizers for moisturizing the stratum corneum, such
  • Solubilizer for solubilizing; buffering agent for adjusting pH of products such as citric acid, lactic acid, amino acids, etc .; for adding flavors such as vanillin, orange flavor, lemon flavor, milk flavor geraniol, linarol Flavoring agent; Antiseptic for inhibiting microorganisms such as methylparaben and phenoxy ethanol to prevent decay; Colorant for coloring; Metal ion sequestrant, Ultraviolet ray Hand et al., Fading agent to prevent fading or discoloration; a drug, such as astringents and, fungicides, energetic agents, anti-inflammatory agents, or whitening agent, may contain a mixture of two or more.
  • the composition of the present invention may contain 0.00 to 20% by weight, preferably 0.01 to 10% by weight, more preferably about 0.1 to 3% by weight.
  • Emmulsion is an intermediate between a lotion and a cream and is generally a fluid emulsion.
  • Latex is a cosmetic mainly used to supply moisture, moisturizer, oil, etc. to the skin in order to maintain the skin's moisture balance, moisturizing and flexibility.
  • the components contained in the milky milk are similar to the ingredients contained in the creams described later. However, since the milky milk is fluid, the amount of solid oil and lead is less than that of the creams.
  • a well-known "oil emulsion" can be used suitably according to the use etc.
  • the emulsion may contain, for example, 1 to 20% by weight, preferably 1 to 10% by weight, more preferably 1 to 5% by weight of the composition of the present invention.
  • “Cream” is a type of emulsion in which one side of a liquid that does not mix with each other, such as water and oil, is dispersed in a stable state in the other dispersion medium.
  • cream can be used suitably according to the use etc. These creams include emollient creams for moisturizing and softening of the skin, massage creams for promoting blood circulation, cleansing creams for cleansing the skin, hair removers for hair loss, deodorant creams for odor removal, and keratin softening. Keratin softening cream and the like.
  • the cream contains, for example, the following ingredients: That is, the aqueous phase components included in the cream include purified water such as ion-exchanged water; alcohols for dissolving and sterilizing oil-soluble components such as ethanol and propanol to give a refreshing feeling; Humectants; and mucus such as queen's seed, pectin, cellulose derivatives, and the like.
  • Oil components included in the cream include hydrocarbons such as squalene, liquid paraffin, petrolatum and solid paraffin; oils such as olive oil, almond oil, cacao fat and castor oil; lead such as beeswax, lanolin and jojoba oil; stearic acid, oleic acid and palmitic acid Higher alcohols such as fatty acids such as cetanol and stearyl alcohol, esters such as IPM, glycerin triester, pentaerythritol tetraester, silicone oils such as polysiloxanes, and the like.
  • hydrocarbons such as squalene, liquid paraffin, petrolatum and solid paraffin
  • oils such as olive oil, almond oil, cacao fat and castor oil
  • lead such as beeswax, lanolin and jojoba oil
  • stearic acid oleic acid and palmitic acid
  • Higher alcohols such as fatty acids such as cetanol and stearyl alcohol, esters
  • the cream may comprise 1 to 100%, preferably 1 to 20%, 1 to 10%, more preferably 5 to 10% by weight of the composition of the present invention.
  • Gels are cosmetics with a uniform appearance, such as gels or sols, and are transparent to translucent cosmetics.
  • a known "gel” can be appropriately used depending on the purpose thereof.
  • an aqueous gel for replenishing and moisturizing the skin an oily gel for maintaining the moisture of the skin, and replenishing oil, a water-soluble gel for promoting blood circulation, and a gel for washing.
  • the gel may be prepared by using a gelling agent containing a water-soluble polymer such as carboxyvinyl polymer and methyl cellulose.
  • Emulsion is basically a component such as the lotion, emulsion and cream described above, and includes an emulsion, a solubilizer, a moisturizer, water, and other drugs.
  • the well-known cosmetic liquid in the treatment method of this invention can be used.
  • Emulsions included in the cosmetic liquid include natural vegetable oils such as olive oil, camellia oil, sesame oil, sunflower oil, sweet almond oil and jojoba oil; diglycerin esters or triglycerin esters of fatty acids such as capric acid, myristic acid, oleic acid and isostearic acid. Etc. can be mentioned.
  • Such emulsions mainly function as emollients.
  • Solubilizers included in the cosmetic liquid include propylene glycol fatty acid esters such as glycerin fatty acid ester, polyglycerol fatty acid ester, sorbitan fatty acid ester, and propylene glycol monostearate, glycerin, diglycerol, ethylene glycol, propylene glycol, 1,3-butyl Polyhydric alcohols, such as len glycol, etc. are mentioned.
  • the solubilizer is preferable to use glycerin or diglycerin and to increase the thermal effect in the massage. Distilled water and deionized water are mentioned as water contained in a cosmetic liquid.
  • Other pharmaceutical agents include fungicides, preservatives, vitamins, natural extracts from plants, colorants, fragrances and the like.
  • adopt a well-known component amount when the total amount of the cosmetic liquid is 100% by weight, the composition of the present invention may contain 1 to 20% by weight, preferably 1 to 10% by weight, more preferably about 1 to 5% by weight. In addition, 65 to 90% by weight of the emulsion, 1 to 10% by weight of the solubilizer, 1 to 30% by weight of the moisturizer, about 0 to 10% by weight of other drugs, and the rest may include water. What is necessary is just to manufacture a cosmetic liquid according to a well-known manufacturing method.
  • Pack cosmetic is to apply to the target site for the purpose of moisturizing the skin, promoting blood circulation, and cleansing.
  • Pack cosmetics generally include a jelly, paste, powder, creamy liquid, and the like, and may be applied to the face to form a film and then peeled off, wiped off after application, or washed off after application. .
  • Powdered materials are used by dissolving or suspending uniformly in water or the like during use.
  • the pack cosmetics which impregnated cosmetics to the nonwoven fabric and collagen sheets of shapes, such as an eye mask, the pack cosmetics etc. which are immersed in cosmetics at the time of use may be sufficient.
  • Pack cosmetics may contain 1 to 20% by weight, preferably 1 to 10% by weight, more preferably 1 to 5% by weight, most preferably about 2 to 3% by weight of the composition of the present invention.
  • the other composition may be a known composition used for a pack cosmetic, and may be prepared according to a known production method.
  • the above cosmetics may be used according to the method in which each is commonly used.
  • the present invention is carried out in the case of human adipose derived stem cells, it will be apparent to those skilled in the art that other mammalian adipose derived stem cells such as sheep can also be used.
  • Human adipose tissue obtained from abdominal fat by liposuction was isolated and washed with PBS.
  • the tissue was chopped and digested at 37 ° C. for 2 hours using DMEM media to which collagenase type 1 (1 mg / ml) was added. After washing with PBS and centrifuged for 5 minutes at 1000rpm. The supernatant was sucked and the remaining pellets were washed with PBS and centrifuged at 1000 rpm for 5 minutes. After filtering to 100 ⁇ m mesh to remove the debris and washed with PBS, and incubated in DMEM (10% FBS, 2mM NAC, 0.2mM ascorbic acid) medium.
  • Adipose tissue-derived multipotent mesenchymal stem cells were isolated by subculture with changing media every 2 days.
  • the above-obtained adipose tissue-derived multipotent mesenchymal stem cells of Passage 3, 5, 8, and 10 were cultured at 90% confluency in T75-flask, respectively, and then 2 mM NAC, 0.2 mM ascorbic acid, 0.09 mM calcium, The cells were incubated for 120 hours by exchange with Keratinocyte-SFM media containing 5 ⁇ g / ml insulin and 74 ng / ml Hydrocortisone. After incubation, the medium was collected, centrifuged (1500 rpm, 5 minutes), filtered (0.2um), and cell debris were removed to obtain pure cell culture solution 1.
  • the multipotent mesenchymal stem cells derived from adipose tissue of Passage 3, 5, 8, and 10 obtained above were incubated at 90% confluency in T75-flask, respectively, and then 1 to 100ng / ml EGF, 0.5 to 20 per passage. 120 hours were incubated with Keratinocyte-SFM media containing% human UCB Plasma-derived Serum, 2mM NAC, 0.2mM ascorbic acid, 0.09mM calcium, 5 ⁇ g / ml insulin and 74ng / ml Hydrocortisone. After incubation, the medium was collected, centrifuged (1500 rpm, 5 minutes), filtered (0.2 ⁇ m), and cell debris were removed to obtain pure cell culture solution 2.
  • Example 1 The components in the adipocyte stem cells 1 and 2 obtained in Example 1 were analyzed.
  • each of the adipose stem cell cultures 1 and 2 were sampled and centrifuged to remove cell debris. Immediately tested or aliquoted, the samples were stored at -20 ° C, but the samples were frozen. In later experiments, the process of freezing and melting was avoided.
  • Standard and samples for drawing the reagents and standard reagent control lines used for the test were prepared.
  • a stock solution of the protein to be measured was serially diluted in a polypropylene tube, and a total of seven concentrations were set between the highest concentration and the lowest concentration.
  • Antimicrobial test microplate strips were prepared, and serial dilution standard reagent control lines and aliphatic stem cell culture supernatant were aliquoted onto the microplate for reaction. After the reaction, the standard reagent and the sample were collected, and the microplate was washed three times with washing buffer. The conjugate for the antibody of the protein to be measured was added to each plate and reacted. After the reaction, the conjugate was recovered and washed three times with washing buffer. Substrate solution was added to each plate and allowed to react.
  • the stop solution was added, and the value was measured using a spectrometer having an absorbance of 540-570 nm within 30 minutes. Further details follow the ELISA Kit information used for protein determination.
  • VEGF R & D system Cat.No.DVE00
  • TGF-b1 (R & D system Cat.No.DB100B)
  • IGF-1 (R & D system Cat.No.DG100)
  • Adipose stem cell secretion protein components were analyzed, and as shown in FIG. 1 in both cultures 1 and 2, TGF (166.7 pg / ml), bFGF (916.15 pg / ml), IGF (2490 pg / ml), and KGF (1054 pg). / ml), HGF (20778pg / ml), fibronectin (2402.3pg / ml), VEGF (6501.72pg / ml), Procollagen (842.23ng / ml), Fibronectin (not shown) was found to be secreted in high concentration .
  • Fibronectin 1 showed similarity to chimpanzees 99.7%, dogs 95.2%, cows 94.0%, mice 92.2%, and mice 92.3%.
  • chimpanzees had a similarity of 96.7%, bovine 94.7%, mouse 90.7%, and rat 90.7%.
  • the effect of the present invention is a mammalian adipose tissue-derived adult stem cells Cells are included in all cases, and adult stem cells derived from human adipose tissue are representative examples.
  • the concentration of VEGF was increased as the passage was 434.62 GF / ml increased to 940.77 pg / ml, but HGF (mean 391.0 pg / ml), TGF (mean 552.7 pg / ml), IGF (mean 207.5 pg / ml), Procollagen (mean 556.6 ng / ml) Did not change much by Passage.
  • Protein was extracted from the adipose stem cells obtained in Example 1. At this time, the proteins constituting the adipose stem cells were prepared in a state of maintaining activity by salt precipitation and dialysis.
  • dialysis was performed by adding 1 ml of Tri-HCl buffer (pH 7.2) to remove impurities. 2% (M / V) Na-bicarbonate and 1mM EDTA (pH8.0) were added, and the dialysis bag was pretreated at 100 ° C for 10 minutes, washed with distilled water, and treated with 1mM EDTA (pH 8.0) at 100 ° C for 10 minutes. Washed once again. Adipose stem cell precipitate was added to the dialysis bag, and then completely sealed. Tris Buffer was added and stirred for 24 hours.
  • Tri-HCl buffer pH 7.2
  • the protein was recovered and centrifuged at 370 x g for 10 minutes to recover the supernatant and lyophilized.
  • Proteins extracted from adipose stem cells obtained in Example 3 were hydrolyzed to prepare peptides and amino acids of low molecular weight.
  • Example 3 The freeze-dried adipose stem cell extract protein obtained in Example 3 was suspended in the adipose stem cell culture solution prepared in Example 1, and then the protein was encapsulated using lecitin to increase skin absorption.
  • Dr.JUCRE Million Stem Cell Magic Concentrate in cream form prepared in Example 5 was evaluated for skin color, elasticity improvement and pore contraction effect evaluation.
  • Derma Pro / Dermatology Research Institute 12 healthy female subjects aged 18 to 60 years (mean age 37.2 ⁇ 5.6 years), was allowed to use the test product once a day for about 8 weeks before and after use. At each time point (after 4 and 8 weeks) the skin improvement was evaluated.
  • the evaluation items were visual evaluation, skin color (L * Value), pore contraction, and facial contour photographs .
  • a questionnaire evaluation was conducted by subjects at 4 and 8 weeks after use.
  • the improvement rate was calculated as (Initial treatment (0W) -After treatment (4W, 8W)) / Initial treatment x 100.
  • a decrease in mean value means an improvement in skin color. That is, as shown in Fig. 3, as a result of visual evaluation, a significant skin color improvement effect was observed at each evaluation point when compared to before using the product.
  • FIGS. 4 and 5 The results are shown in FIGS. 4 and 5. Increasing the mean value means improving skin color. That is, as shown in Figure 4, also in the L * value measurement results, significant skin tone improvement effect was observed at each evaluation point when compared to before using the product. As shown in FIG. 5, as a result of confirming with a spectrophotomer (only one subject is shown as an example), the skin color gradually became clear.
  • Skin lifting is a term that collectively refers to the effect of increasing the elasticity of the skin (tightening), or face lifting.
  • the skin lifting effect was examined by observing the elasticity of the skin weekly before and after use (after 4 and 8 weeks).
  • the improvement rate was calculated as (Initial treatment (0W) -After treatment (4W, 8W)) / Initial treatment x 100.
  • FIGS. 6 and 7. Increasing the mean value means increasing the skin lifting effect. That is, as shown in Figure 6, significant skin lifting effect was observed at each time of evaluation compared with before using the product. And from the facial contour observation results shown in FIG. 7 (only one subject is shown as an example), it was confirmed that the facial contours were raised after using the test product (lifting effect).
  • test product was used once a day for 8 weeks, and then the volume of pores was observed weekly at each time point before and after use (after 4 and 8 weeks). Improvement rate was calculated as (Initial treatment (0W) -After treatment (4W, 8W)) / Initial treatment x 100.
  • FIGS. 8 and 9 Reducing the mean value means the effect of reducing the skin pore volume. That is, as shown in FIGS. 8 and 9 (only one subject is shown as an example), as a result of measuring the pore size using PRIMOS, a significant reduction in pore volume was observed at 8 weeks compared to before use of the product. .
  • a skin wrinkle evaluation test was performed on the cream-like Dr.JUCRE Million Stem Cell Magic Concentrate prepared in Example 5.
  • the Derma Pro / Dermatology Research Institute 21 female subjects with eye wrinkles over 30 years of age, randomly assigned test and control products to the left and right eyes for 8 weeks.
  • the effects of skin wrinkle improvement were evaluated using visual evaluation, skin wrinkle measurement (R-value) using Visiometer, and questionnaire evaluation by subjects at 4 and 8 weeks before and after using the product.
  • test and control products were randomly assigned to the left and right eyes, and then the change of skin wrinkles was visually observed before and after 4 and 8 weeks of use.
  • Saline solution was used as a control.
  • a decrease in mean value means an improvement in wrinkles. That is, as shown in Figure 11, the results of the visual evaluation, the test group showed a tendency to reduce the skin wrinkles compared to the control group at 4 and 8 weeks after the product use. In addition, the test group was significantly improved after 8 weeks of product use compared to before using the product (p ⁇ 0.05).
  • test and control products were randomly assigned to the left and right eyes, and the wrinkle wrinkle R-values were measured using a Visiometer before and after 4 weeks and 8 weeks.
  • a decrease in mean value means an improvement in wrinkles. That is, as shown in FIG. 12, as a result of skin wrinkle (R-value) analysis, the product R1 and R2 parameters were measured at 4 weeks and 8 weeks after product use, and the R5 parameter at 8 weeks after product use. Compared to that, skin wrinkles tended to decrease significantly (p ⁇ 0.05). In addition, the test group significantly improved the R1 and R2 parameters at 4 and 8 weeks after using the product, and R3, R4 and R5 at 8 weeks after using the product (p ⁇ 0.05).
  • test and control products were randomly assigned to the left and right eyes, and then questionnaires were evaluated by the subjects at 4 and 8 weeks of use. The results are shown graphically in FIG. 13.
  • the cosmetic composition of the present invention has an excellent effect on skin wrinkle improvement.
  • composition for cosmetics of this invention promotes the regeneration of the skin tissue of the apply

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Abstract

La présente invention concerne une culture de cellules souches adultes provenant de tissu adipeux et une composition cosmétique contenant comme principe actif, soit une protéine extraite desdites cellules souches adultes provenant de tissu adipeux, soit des hydrolysats de peptides ou d'acides aminés de ces protéines. Étant donné les effets synergiques et mutuels de l'amélioration des rides et de la prévention du vieillissement desdites cultures de cellules souches adultes provenant du tissu adipeux et des protéines ou acides aminés extraits de ces cultures, la composition cosmétique de la présente invention convient à la fabrication de produits cosmétiques fonctionnels.
PCT/KR2009/003923 2008-07-16 2009-07-16 Culture de cellules souches pluripotentes provenant de tissu adipeux et composition cosmétique contenant une protéine extraite de ces cellules WO2010008219A2 (fr)

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JP2016517696A (ja) * 2013-05-09 2016-06-20 アール バイオ カンパニー リミテッドＲ Ｂｉｏ Ｃｏ．， Ｌｔｄ． 幹細胞の再生能向上のための培地組成物及びこれを利用した幹細胞の培養方法

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KR101582469B1 (ko) * 2013-09-11 2016-01-06 (주)아프로존 피부세포 재생용 화장품의 제조 방법 및 장치
WO2016072660A1 (fr) * 2014-11-07 2016-05-12 주식회사 티아라줄기세포연구소 Procédé de préparation d'extrait de constituant de cellules souches et procédé de préparation d'agent de traitement de la perte des cheveux à l'aide de celui-ci
KR101709702B1 (ko) * 2015-04-24 2017-02-23 한국생산기술연구원 화장품 소재 및 이의 제조 방법
KR20210045103A (ko) * 2019-10-16 2021-04-26 시니어 과학생명 주식회사 머츄어링을 통한 고농도 사이토카인 함유 항노화 에센스 생산방법 및 그에 따른 사이토카인 생산 시스템 모듈
KR20230139010A (ko) 2022-03-25 2023-10-05 주식회사 래디안 섬유아유사세포 또는 그 배양액을 유효성분으로 포함하는 항노화 및 주름 억제용 조성물

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