WO2007145438A1 - Cosmetic or plastic composition comprising multipotent stem cells derived from human adipose tissue, fibroblast, and adipose or adiopocyte - Google Patents

Cosmetic or plastic composition comprising multipotent stem cells derived from human adipose tissue, fibroblast, and adipose or adiopocyte Download PDF

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WO2007145438A1
WO2007145438A1 PCT/KR2007/002641 KR2007002641W WO2007145438A1 WO 2007145438 A1 WO2007145438 A1 WO 2007145438A1 KR 2007002641 W KR2007002641 W KR 2007002641W WO 2007145438 A1 WO2007145438 A1 WO 2007145438A1
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adipose
stem cells
derived
fibroblasts
adult stem
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French (fr)
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Jeong Chan Ra
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Rnl Bio Co., Ltd
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    • 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
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0656—Adult fibroblasts
    • 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
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/60—Materials for use in artificial skin
    • 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
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0653—Adipocytes; Adipose tissue
    • 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
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/32—Amino acids
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/01—Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/33—Insulin
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/39—Steroid hormones

Definitions

  • Cosmetic or Plastic Composition Comprising Multipotent Stem Cells Derived from Human Adipose Tissue, Fibroblast, and Adipose or Adiopocyte
  • the present invention relates to a composition for cosmetic or plastic surgery, containing adult stem cells derived from human adipose tissue, fibroblasts and adipose or adiopocytes, and a method for preparing the same.
  • Stem cells refer to cells having both self-replication ability and the ability to differentiate into at least two cells, and can be classified into totipotent stem cells, pluripotent stem cells, and multipotent stem cells.
  • adipose tissue is a new source of multipotent stem cells (Cousin et al, BBRC, 301: 1016, 2003; Miranville et al, Circulation,
  • adipose tissue obtained by liposuction contains a group of undifferentiated cells which has the ability to differentiate into adipogenic, osteogenic, myogenic and chondrogenic cells (Zuk et al, Tissue Eng., 7:211, 2001; Rodriguez et al, BBRC, 315:255, 2004). Also, it was known through animal model experiments that adipose tissue-derived cells have the ability to regenerate muscles and stimulate the differentiation into neural blood vessels. Since this adipose tissue has an advantage in that it can be extracted on a large scale, it has been spotlighted as a new source of stem cells which overcomes the existing shortcomings.
  • the known adipose tissue-derived stem cells include human adipose- derived adult stem cells capable of epithelial differentiation (Brzoska et al., BBRC, 330:142, 2005), human adipose-derived adult stem cells capable of osteogenic and adipogenic differentiation (Cao et ah, BBRC, 332:370, 2005), human adipose-derived adult stem cells capable of neurogenic differentiation (Safford et al, BBRC, 294:371, 2002), rat adipose-derived stem cells capable of adipogenic differentiation (Ogawa et ah, BBRC, 319:511, 2004), rat adipose- derived stem cells capable of osteogenic and chondrogenic differentiation (Ogawa et ah, BBRC, 313:871, 2004), human adipose-derived stem cells capable of chondrogenic differentiation (Awad
  • the present inventors have made extensive efforts to use stem cells in the field of cosmetics and plastic surgery, including wrinkle improvement, protection of the flabby skin and the like, conducted an experiment by culturing adipose-containing suspension obtained by liposuction, collecting cell layers containing adult stem cells and fibroblasts, and mixing them with adipose, and found that the resulting composition is useful for cosmetic treatments and plastic surgery, thereby completing the present invention.
  • An object of the present invention is to provide a composition for cosmetic or plastic surgery, containing adult stem cells derived from human adipose tissue, fibroblasts and adipose or adiopocytes, and a method for preparing the same.
  • the present invention provides a method for preparing a composition for cosmetic or plastic surgery, containing (i) adult stem cells derived from human adipose tissue, (ii) fibroblasts and (iii) adipose or adiopocytes, the method comprising the steps of: (a) culturing an adipose- containing suspension obtained from human adipose tissue by liposuction in a culture flask to collect cell layers containing adipose-derived adult stem cells and fibroblasts, which are attached to the culture flask; and (b) mixing the collected cell layers containing adipose-derived adult stem cells and fibroblasts with adipose or adipocytes.
  • the present invention also provides a method for preparing a composition for cosmetic or plastic surgery, containing (i) adult stem cells derived from human adipose tissue, (ii) fibroblasts and (iii) adipose or adiopocytes, the method comprising the steps of: (a) preparing adipose-derived adult stem cells by culturing human adipose tissue-derived pellets in a medium containing N-acetyl- L-cysteine (NAC) to collect, adipose-derived stem cells and fibroblasts; and (b) mixing the selected adipose-derived adult stem cells and fibroblasts with adipose or adipocytes.
  • NAC N-acetyl- L-cysteine
  • the present invention provides a composition for cosmetic or plastic surgery, containing (i) adult stem cells derived from human adipose tissue, (ii) fibroblasts and (iii) adipose or adiopocytes.
  • FIG. 1 shows photographs of cells attached to a culture flask, which contains human adipose tissue-derived multipotent stem cells, fibroblasts and the like according to the present invention (original magnification, 10Ox).
  • FIG. 2 illustrates the results of the expression of Nestin, Oct4, SH2, SH3/4 in human adipose tissue-derived multipotent stem cells, which were sphere-cultured in a CORM-2-containing MEBM medium, using immunostaining (original magnification, 10Ox).
  • FIG. 3 displays photographs of adiopocytes differentiated from human adipose tissue-derived multipotent stem cells according to the present invention (A: phase contrast of a differentiated state; and B: adipocytes stained with oil red O staining, original magnification, 20Ox).
  • the present invention relates to a composition for cosmetic or plastic surgery, containing adult stem cells derived from human adipose tissue, fibroblasts and adipose or adiopocytes.
  • the composition according to the present invention can be preferably used for breast enlargement by the formation of new breast tissue or wrinkle removal, but is not limited to this purpose.
  • it can be used for the purpose of maintaining tightness of facial skin as well as removing glabellar frown lines and nasolabial folds through microlipoinjection and self-fat transplant.
  • human adipose tissue-derived adult stem cells, fibroblasts and adipose, which are contained in the composition according to the present invention, are self- derived cells.
  • composition for cosmetic or plastic surgery can be prepared by culturing an adipose-containing suspension obtained from human adipose tissue, resulting from liposuction and mixing the obtained adipose-derived adult stem cells and fibroblasts with other cells or tissues.
  • the composition for cosmetic or plastic surgery according to the present invention can be prepared by the following steps of: (a) culturing an adipose-containing suspension obtained from human adipose tissue by liposuction in a culture flask to collect cell layers containing adipose-derived adult stem cells and fibroblasts, which are attached to the culture flask; and (b) mixing the collected cell layers containing adipose-derived adult stem cells and fibroblasts with adipose or adipocytes.
  • the composition is prepared by culturing an adipose-containing suspension which is suspended in normal saline solution obtained by liposuction, and collecting after washing stem cells and fibroblasts, attached to a culture container such as a culture flask, with normal saline solution 1 time before collecting them so as to remove the remaining adipose and hemocytes of the supernatant and trypsinizing cell layers consisting of stem cells and fibroblasts which are attached to the bottom, directly collecting cells suspended in a small amount of normal saline by scrapping cell layers, or collecting the centrifuged cells by centrifugation, followed by mixing the collected cells with adipose.
  • the adult stem cells are preferably cells derived from mesoderm, and the mesoderm-derived cells are preferably adipogenic cells.
  • the adult stem cells are preferably cultured in an ce-MEM medium containing dexamethasone, indomethacin, insulin and IBMX to differentiate into adipogenic cells.
  • the composition for cosmetic or plastic surgery according to the present invention can be prepared by the following steps of: (a) culturing human adipose tissue-derived pellets in a medium containing N-acetyl- L-cysteine (NAC) to prepare adipose-derived adult stem cells and then collecting adipose-derived stem cells and fibroblasts; and (b) mixing the collected adipose- derived adult stem cells and fibroblasts with adipose or adipocytes.
  • NAC N-acetyl- L-cysteine
  • the liposuction which is a technique of cosmetic or plastic surgery for adipose removal, refers to extract fat by a vacuum pump or a vacuum syringe through an incision in the donor region.
  • adipose or adipocytes obtained incidentally from human adipose tissue by liposuction can be used, but is not limited thereto.
  • adipose includes adipose tissue.
  • adipose tissue contains l x l0 4 ⁇ l x l0 7 of adipose-derived stem cells and fibroblasts per ImI but when adipocytes are used, it is preferable to use adipocytes l ⁇ 50 fold greater than the total cell number of adipose-derived stem cells and fibroblasts.
  • fibroblasts are cells constituting an important component of fibrous connective tissue, which are also called fibrocytes. Tissue section of fibroblasts is flat and longish- shaped and often has irregular bulges. Fibroblast has a cytoplasm containing mitochondria, golgi complex, centrosome, a small basal body etc. but does not show other specific differentiation except them. A nucleus is weakly stained, elliptical and contains phosphorous. They are named a fibroblast because they are crowded around collagen fibers and considered to be related to the formation thereof. In present invention, the fibroblasts can be isolated from the process of separating stem cells from adipose tissue.
  • methods for obtaining multipotent stem cells expressing a target cell surface antigen from the obtained human adipose tissue-derived stem cell broth include a FACS method using a flow cytometer with a sorting function (Int. Immunol., 10(3):275, 1998), a method using magnetic beads, and a panning method using an antibody specifically recognizing multipotent stem cells (J. Immunol., 141(8):2797, 1998).
  • a method for obtaining multipotent stem cells from a large amount of culture broth includes a method in which antibodies specifically recognizing molecules expressed on the cell surface (hereinafter, referred to as "surface antigens”) are used alone or in combination as columns.
  • Flow cytometry sorting methods include a water drop charge method and a cell capture method and the like.
  • an antibody specifically recognizing an antigen on the cell surface is fluorescently labeled and the intensity of fluorescence from the labeled antigen-antibody complex is converted to an electric signal, thereby quantifying amounts of the antigen expressed. It is also possible to separate cells expressing a plurality of surface antigens by combining types of fluorescence used.
  • the fluorescent substance which is usable in this case include FITC (fluorescein isothiocyanate), PE (phycoerythrin), APC (allo-phycocyanin), TR (Texas Red), Cy 3, CyChrome, Red 613, Red 670, TRI- Color, Quantum Red, etc.
  • FACS methods using a flow cytometer include; a method where the above stem cell broth is collected, from which cells are isolated by a method such as centrifugation, and directly staining antibodies; and a method where the cells are cultured and proliferated in a suitable medium and then staining antibodies.
  • the staining of cells is performed by mixing a primary antibody recognizing a surface antigen with a target cell sample and incubating the mixture on ice for 30 minutes to 1 hour. When the primary antibody is fluorescently labeled, the cells are isolated with a flow cytometer after washing.
  • the primary antibody When the primary antibody is not fluorescently labeled, cells reacted with the primary antibody and a fluorescent labeled secondary antibody having a binding activity to the primary antibody are mixed after washing, and incubated on ice water for 30 minutes to 1 hour. After washing, the cells having stained primary and secondary antibodies are isolated with a flow cytometer.
  • the inventive composition containing human adipose tissue-derived adult stem cells, fibroblasts and adipose is useful for cosmetic purposes due to the function of improving skin tightness, improving wrinkles such as nasolabial folds and glabellar frown lines as well as flabby skin, and restoring a depressed area through plastic surgery. Furthermore, since the human adipose tissue-derived adult stem cells can be differentiated into adipogenic cells, the composition according to the present invention is useful for the formation of new breast tissue.
  • Example 1 Isolation of multipotent adult stem cells from adipose tissue and and preparation of a composition for plastic surgery
  • Preparation example 1 An adipose-containing suspension suspended in normal saline solution obtained incidentally from human adipose tissue by liposuction was evenly re-suspended with a proper amount of normal saline, and the adequate amount of the re- suspended suspension was placed in a cell culture flask or a roller bottle for standing or shaking culture. At least, 6 ⁇ 12 hours were required for the standing culture. Next, cell layers (adipose-derived MSC, fibroblasts) attached to the flask surface were collected by treating with trypsin (FIG. 1).
  • the isolated cell layers contain adult stem cells and fibroblasts.
  • the composition for cosmetic or plastic surgery containing adult stem cells, fibroblasts, adipose and adipocytes, according to the present invention was prepared by mixing the isolated cell layers with adipose.
  • Multipotent stem cells were isolated and purified from human adipose tissue. That is, the isolated human adipose tissue was washed with PBS 3 times and then finely cut. The cut tissue was digested at 37 ° C for 1 hour in a DMEM-LG(low glucose) medium supplemented with collagenase type 1 (lmg/ml) at a ratio of 1:3 (adipose tissueiDMEM). After washing the digested tissue with PBS, centrifugation was performed at 1000 rpm for 5 minutes.
  • the supernatant was suctioned off, and the remaining pellets on the bottom were washed with a mixture solution of 10% FBS and DMEM-LG (low glucose) at a ratio of 1 :1, followed by another centrifugation at 1000 rpm for 5 minutes.
  • the resulting pellets were filtered through a 100 ⁇ m mesh to remove debris and washed with PBS.
  • the resulting cells were incubated in a DMEM medium (10% FBS, 2mM NAC, 0.2mM ascorbic acid).
  • K-NAC media Keratinocyte-SFM media + 2mM NAC + 0.2mM ascorbic acid + 0.09mM calcium + 5ng/ml rEGF + 50 jUg/ml BPE + 5 ⁇ g/ml insulin + 74ng/ml hydrocortisone
  • K-NAC media Keratinocyte-SFM media + 2mM NAC + 0.2mM ascorbic acid + 0.09mM calcium + 5ng/ml rEGF + 50 jUg/ml BPE + 5 ⁇ g/ml insulin + 74ng/ml hydrocortisone
  • Example 2 Immunological properties of adipose-derived multipotent stem cells
  • the adipose tissue-derived adult stem cells obtained in Example 1 were washed with PBS and treated with trypsin. The treated cells were collected and centrifuged at 1000 rpm at 4 ° C for 5 minutes. After the supernatant was discarded and the cells were washed with 2% FBS in PBS, centrifugation was carried out at ljOOOrpm, 4°C for 5 minutes. The supernatant was discarded, and the pellet was suspended with PBS to dispense I xIO 5 cells as many as sample numbers, followed by centrifugation at 1000 rpm at 4 ° C for 5 minutes.
  • the supernatant was discarded, and the cells were mixed well with 150 ⁇ Jl of blocking solution (5% FBS in PBS), and centrifuged at 1000 rpm at 4°C for 5 minutes. The supernatant was again discarded and the cells were well mixed with 100 ⁇ i of blocking solution (5% FBS in PBS) to incubate them at 4°C for 30 minutes. After that, the solution was centrifuged at 1000 rpm for 5 minutes at 4 ° C and the supernatant was removed, followed by adding 150 ⁇ Jl of blocking solution (5% FBS in PBS) to mix well.
  • blocking solution 5% FBS in PBS
  • Antibodies (R-phycoerythrin-conjugated mouse anti-human monoclonal antibodies) were placed into each well and incubated at 4°C for 30 minutes. After the incubation, they were centrifuged at 1000 rpm at 4 ° C for 5 minutes. The supernatant was removed, and the cells were washed with PBS and centrifuged at 1000 rpm for 5 minutes at 4°C . Once again, the supernatant was removed, and the cells were washed with PBS and centrifuged at 1000 rpm for 5 minutes at 4°C . After the supernatant was discarded, the cells were well mixed with 200 ⁇ of PBS and fixed with 1% paraformaldehyde, and finally analyzed using a flow cytometer.
  • Table 1 FACS analysis of surface antigens of adipose-derived stem cells
  • the adipose tissue-derived adult stem cells according to the present invention showed positive responses of 91% to CD73, 97% to CD90, 96% to CD29, 83% to CD44, and 80% to CD105. Also, as a result of FACS analysis to other antigens, the inventive stem cells showed negative immunological responses to all of CD33, CD34, CD45, CD4, CD31, CD62p, CD14 and HLA-DR.
  • Example 3 Immunostaining analysis of adipose tissue-derived stem cells
  • the adipose tissue-derived stem cell obtained in Example 1 were washed with PBS 3 times and fixed with 4% paraformaldehyde-containing PBS for 30 minutes.
  • the cells were washed with PBS 3 times and were permeabilized with PBS containing 0.1% Triton-XIOO for 10 minutes. After being washed with PBS 3 times, the cells were incubated with 10% NGS for 1 hour and then incubated with a primary antibody-containing PBS overnight.
  • the cells were also washed with PBS 3 times, and were allowed to react with a secondary antibody in a dark room for 1 hour. After being washed with PBS 3 times, the cells were mounted.
  • the multipotent stem cell spheres according to the present invention showed positive responses to all of Oct4 which can be regarded as a marker of undifferentiated cells, and SH2(CD105) and SH3/4(CD73) which are markers of mesenchymal stem cells.
  • Example 4 Differentiation of adipose-derived multipotent stem cells into adipocytes
  • Example 2 The adipose tissue-derived multipotent adult stem cells obtained in Example 1 were incubated in an ce-MEM medium containing 5% FBS, l ⁇ M dexamethasone,
  • Example 5 Animal analysis using adipose-derived multipotent stem cells
  • mice 12 mice for a control group and 12 mice for an experimental group were used.
  • Adipose (ImI) + adipose tissue-derived multipotent adult stem cells and fibroblasts (5> ⁇ 10 5 /200 ⁇ Ji saline) were injected into cephalic hypodermis, where subcutaneous adipose tissue is not present in a mouse, in the experimental group, whereas adipose (ImI) + vehicle (200 ⁇ JL saline) were injected in the control group.
  • adipose tissue was separated through a cephalic incision of mice, and the volume of adipose tissue was measured. As a result, while the volume of injected adipose was reduced to 31% in the control group, 45% of the injected adipose volume was preserved in the experiment group in which the composition prepared by Example 1 was injected. From this result, it was found that adipose tissue-derived stem cells and fibroblasts according the present invention have an effect of reducing adipose volume decrease resulting from liposuction.
  • the present invention has an effect to provide the composition for cosmetic or plastic surgery, containing human adipose tissue- derived adult stem cells, fibroblasts and adipose or adipocytes.
  • the composition for cosmetic or plastic surgery according to the present invention is useful not only for cosmetic purposes such as improving skin tightness, improving wrinkles and flabby skin, and restoring a depressed area through plastic surgery, but also for the formation of new breast tissue since the human adipose tissue-derived adult stem cells can be differentiated into adipogenic cells.

Abstract

The present invention relates to a composition for cosmetic or plastic surgery, containing adult stem cells derived from human adipose tissue, fibroblasts and adipose or adiopocytes, and a method for preparing the same. The composition for cosmetic or plastic surgery according to the present invention can be prepared by culturing an adipose-containing suspension obtained by liposuction and then collecting cell layers containing both adipose-derived adult stem cells and fibroblasts which are attached to the surface of the culture flask, followed by adding adipocytes to the collected cell layers. The composition according to the present invention is useful for cosmetic purposes such as skin tightness improvement, wrinkle and flabby skin improvement, and depressed area restoration through plastic surgery as well as useful for the formation of new breast tissue.

Description

Cosmetic or Plastic Composition Comprising Multipotent Stem Cells Derived from Human Adipose Tissue, Fibroblast, and Adipose or Adiopocyte
TECHNICAL FIELD
The present invention relates to a composition for cosmetic or plastic surgery, containing adult stem cells derived from human adipose tissue, fibroblasts and adipose or adiopocytes, and a method for preparing the same.
BACKGROUND ART
Stem cells refer to cells having both self-replication ability and the ability to differentiate into at least two cells, and can be classified into totipotent stem cells, pluripotent stem cells, and multipotent stem cells.
Recently, it was found that adipose tissue is a new source of multipotent stem cells (Cousin et al, BBRC, 301: 1016, 2003; Miranville et al, Circulation,
110:349, 2004; Gronthos et al, J. Cell Physiol., 189:54, 2001; Seo et al, BBRC,
328:258, 2005). Namely, it was reported that human adipose tissue obtained by liposuction contains a group of undifferentiated cells which has the ability to differentiate into adipogenic, osteogenic, myogenic and chondrogenic cells (Zuk et al, Tissue Eng., 7:211, 2001; Rodriguez et al, BBRC, 315:255, 2004). Also, it was known through animal model experiments that adipose tissue-derived cells have the ability to regenerate muscles and stimulate the differentiation into neural blood vessels. Since this adipose tissue has an advantage in that it can be extracted on a large scale, it has been spotlighted as a new source of stem cells which overcomes the existing shortcomings.
i Until now, the known adipose tissue-derived stem cells include human adipose- derived adult stem cells capable of epithelial differentiation (Brzoska et al., BBRC, 330:142, 2005), human adipose-derived adult stem cells capable of osteogenic and adipogenic differentiation (Cao et ah, BBRC, 332:370, 2005), human adipose-derived adult stem cells capable of neurogenic differentiation (Safford et al, BBRC, 294:371, 2002), rat adipose-derived stem cells capable of adipogenic differentiation (Ogawa et ah, BBRC, 319:511, 2004), rat adipose- derived stem cells capable of osteogenic and chondrogenic differentiation (Ogawa et ah, BBRC, 313:871, 2004), human adipose-derived stem cells capable of chondrogenic differentiation (Awad et ah, Biomaterials, 25:3211, 2004), rat adipose-derived stem cells capable of neural differentiation (Fujimura et ah, BBRC, 333:116, 2005), and adipose-derived stem cells capable of osteogenic, chondrogenic, neural or muscular differentiation (US Patent 6,777,231).
Although many efforts have been made to use adipose-derived stem cells in the medical field including treatment of incurable diseases up until now, there have not been many efforts to try to use stem cells for use in cosmetics and plastic surgery.
Accordingly, the present inventors have made extensive efforts to use stem cells in the field of cosmetics and plastic surgery, including wrinkle improvement, protection of the flabby skin and the like, conducted an experiment by culturing adipose-containing suspension obtained by liposuction, collecting cell layers containing adult stem cells and fibroblasts, and mixing them with adipose, and found that the resulting composition is useful for cosmetic treatments and plastic surgery, thereby completing the present invention. SUMMARY OF THE INVENTION
An object of the present invention is to provide a composition for cosmetic or plastic surgery, containing adult stem cells derived from human adipose tissue, fibroblasts and adipose or adiopocytes, and a method for preparing the same.
In order to achieve the above object, the present invention provides a method for preparing a composition for cosmetic or plastic surgery, containing (i) adult stem cells derived from human adipose tissue, (ii) fibroblasts and (iii) adipose or adiopocytes, the method comprising the steps of: (a) culturing an adipose- containing suspension obtained from human adipose tissue by liposuction in a culture flask to collect cell layers containing adipose-derived adult stem cells and fibroblasts, which are attached to the culture flask; and (b) mixing the collected cell layers containing adipose-derived adult stem cells and fibroblasts with adipose or adipocytes.
The present invention also provides a method for preparing a composition for cosmetic or plastic surgery, containing (i) adult stem cells derived from human adipose tissue, (ii) fibroblasts and (iii) adipose or adiopocytes, the method comprising the steps of: (a) preparing adipose-derived adult stem cells by culturing human adipose tissue-derived pellets in a medium containing N-acetyl- L-cysteine (NAC) to collect, adipose-derived stem cells and fibroblasts; and (b) mixing the selected adipose-derived adult stem cells and fibroblasts with adipose or adipocytes.
Furthermore, the present invention provides a composition for cosmetic or plastic surgery, containing (i) adult stem cells derived from human adipose tissue, (ii) fibroblasts and (iii) adipose or adiopocytes.
Another features and embodiments of the present invention will be more clarified from the following detailed description and the appended claims.
BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 shows photographs of cells attached to a culture flask, which contains human adipose tissue-derived multipotent stem cells, fibroblasts and the like according to the present invention (original magnification, 10Ox).
FIG. 2 illustrates the results of the expression of Nestin, Oct4, SH2, SH3/4 in human adipose tissue-derived multipotent stem cells, which were sphere-cultured in a CORM-2-containing MEBM medium, using immunostaining (original magnification, 10Ox).
FIG. 3 displays photographs of adiopocytes differentiated from human adipose tissue-derived multipotent stem cells according to the present invention (A: phase contrast of a differentiated state; and B: adipocytes stained with oil red O staining, original magnification, 20Ox).
DETAILED DESCRIPTION OF THE INVENTION, AND PREFERRED EMBODIMENTS
In one aspect, the present invention relates to a composition for cosmetic or plastic surgery, containing adult stem cells derived from human adipose tissue, fibroblasts and adipose or adiopocytes.
The composition according to the present invention can be preferably used for breast enlargement by the formation of new breast tissue or wrinkle removal, but is not limited to this purpose. For example, it can be used for the purpose of maintaining tightness of facial skin as well as removing glabellar frown lines and nasolabial folds through microlipoinjection and self-fat transplant. It is desirable that human adipose tissue-derived adult stem cells, fibroblasts and adipose, which are contained in the composition according to the present invention, are self- derived cells.
The composition for cosmetic or plastic surgery according to the present invention can be prepared by culturing an adipose-containing suspension obtained from human adipose tissue, resulting from liposuction and mixing the obtained adipose-derived adult stem cells and fibroblasts with other cells or tissues.
In one embodiment, the composition for cosmetic or plastic surgery according to the present invention can be prepared by the following steps of: (a) culturing an adipose-containing suspension obtained from human adipose tissue by liposuction in a culture flask to collect cell layers containing adipose-derived adult stem cells and fibroblasts, which are attached to the culture flask; and (b) mixing the collected cell layers containing adipose-derived adult stem cells and fibroblasts with adipose or adipocytes.
More particularly, the composition is prepared by culturing an adipose-containing suspension which is suspended in normal saline solution obtained by liposuction, and collecting after washing stem cells and fibroblasts, attached to a culture container such as a culture flask, with normal saline solution 1 time before collecting them so as to remove the remaining adipose and hemocytes of the supernatant and trypsinizing cell layers consisting of stem cells and fibroblasts which are attached to the bottom, directly collecting cells suspended in a small amount of normal saline by scrapping cell layers, or collecting the centrifuged cells by centrifugation, followed by mixing the collected cells with adipose.
In the present invention, the adult stem cells are preferably cells derived from mesoderm, and the mesoderm-derived cells are preferably adipogenic cells. In the present invention, the adult stem cells are preferably cultured in an ce-MEM medium containing dexamethasone, indomethacin, insulin and IBMX to differentiate into adipogenic cells.
In another embodiment, the composition for cosmetic or plastic surgery according to the present invention can be prepared by the following steps of: (a) culturing human adipose tissue-derived pellets in a medium containing N-acetyl- L-cysteine (NAC) to prepare adipose-derived adult stem cells and then collecting adipose-derived stem cells and fibroblasts; and (b) mixing the collected adipose- derived adult stem cells and fibroblasts with adipose or adipocytes.
The liposuction, which is a technique of cosmetic or plastic surgery for adipose removal, refers to extract fat by a vacuum pump or a vacuum syringe through an incision in the donor region.
In the present invention, as adipose or adipocytes, adipose or adipocytes obtained incidentally from human adipose tissue by liposuction can be used, but is not limited thereto. In the present invention, adipose includes adipose tissue.
When adipose is used as the composition for cosmetic or plastic surgery according to the present invention, it is preferable that adipose tissue contains l x l04~l x l07 of adipose-derived stem cells and fibroblasts per ImI but when adipocytes are used, it is preferable to use adipocytes l~50 fold greater than the total cell number of adipose-derived stem cells and fibroblasts.
Moreover, fibroblasts are cells constituting an important component of fibrous connective tissue, which are also called fibrocytes. Tissue section of fibroblasts is flat and longish- shaped and often has irregular bulges. Fibroblast has a cytoplasm containing mitochondria, golgi complex, centrosome, a small basal body etc. but does not show other specific differentiation except them. A nucleus is weakly stained, elliptical and contains phosphorous. They are named a fibroblast because they are crowded around collagen fibers and considered to be related to the formation thereof. In present invention, the fibroblasts can be isolated from the process of separating stem cells from adipose tissue.
Meanwhile, methods for obtaining multipotent stem cells expressing a target cell surface antigen from the obtained human adipose tissue-derived stem cell broth include a FACS method using a flow cytometer with a sorting function (Int. Immunol., 10(3):275, 1998), a method using magnetic beads, and a panning method using an antibody specifically recognizing multipotent stem cells (J. Immunol., 141(8):2797, 1998). Also, a method for obtaining multipotent stem cells from a large amount of culture broth includes a method in which antibodies specifically recognizing molecules expressed on the cell surface (hereinafter, referred to as "surface antigens") are used alone or in combination as columns.
Flow cytometry sorting methods include a water drop charge method and a cell capture method and the like. In any of these methods, an antibody specifically recognizing an antigen on the cell surface is fluorescently labeled and the intensity of fluorescence from the labeled antigen-antibody complex is converted to an electric signal, thereby quantifying amounts of the antigen expressed. It is also possible to separate cells expressing a plurality of surface antigens by combining types of fluorescence used. The fluorescent substance which is usable in this case include FITC (fluorescein isothiocyanate), PE (phycoerythrin), APC (allo-phycocyanin), TR (Texas Red), Cy 3, CyChrome, Red 613, Red 670, TRI- Color, Quantum Red, etc.
FACS methods using a flow cytometer include; a method where the above stem cell broth is collected, from which cells are isolated by a method such as centrifugation, and directly staining antibodies; and a method where the cells are cultured and proliferated in a suitable medium and then staining antibodies. The staining of cells is performed by mixing a primary antibody recognizing a surface antigen with a target cell sample and incubating the mixture on ice for 30 minutes to 1 hour. When the primary antibody is fluorescently labeled, the cells are isolated with a flow cytometer after washing. When the primary antibody is not fluorescently labeled, cells reacted with the primary antibody and a fluorescent labeled secondary antibody having a binding activity to the primary antibody are mixed after washing, and incubated on ice water for 30 minutes to 1 hour. After washing, the cells having stained primary and secondary antibodies are isolated with a flow cytometer.
The inventive composition containing human adipose tissue-derived adult stem cells, fibroblasts and adipose is useful for cosmetic purposes due to the function of improving skin tightness, improving wrinkles such as nasolabial folds and glabellar frown lines as well as flabby skin, and restoring a depressed area through plastic surgery. Furthermore, since the human adipose tissue-derived adult stem cells can be differentiated into adipogenic cells, the composition according to the present invention is useful for the formation of new breast tissue.
Examples
Hereinafter, the present invention will be described in more detail by examples. It will be obvious to a person skilled in the art, however, that these examples are for illustrative purpose only and are not construed to limit the scope of the present invention.
Example 1: Isolation of multipotent adult stem cells from adipose tissue and and preparation of a composition for plastic surgery
Preparation example 1 An adipose-containing suspension suspended in normal saline solution obtained incidentally from human adipose tissue by liposuction was evenly re-suspended with a proper amount of normal saline, and the adequate amount of the re- suspended suspension was placed in a cell culture flask or a roller bottle for standing or shaking culture. At least, 6~12 hours were required for the standing culture. Next, cell layers (adipose-derived MSC, fibroblasts) attached to the flask surface were collected by treating with trypsin (FIG. 1). At this time, cells suspended in a small amount of normal saline solution were directly collected to use, or the cell layers collected from normal the saline solution were centrifuged at lOOOrpm for 10 minutes to use only precipitated pellets in the case where the volume of cell layers should be reduced. The isolated cell layers contain adult stem cells and fibroblasts. The composition for cosmetic or plastic surgery containing adult stem cells, fibroblasts, adipose and adipocytes, according to the present invention was prepared by mixing the isolated cell layers with adipose.
Preparation example 2
Multipotent stem cells were isolated and purified from human adipose tissue. That is, the isolated human adipose tissue was washed with PBS 3 times and then finely cut. The cut tissue was digested at 37 °C for 1 hour in a DMEM-LG(low glucose) medium supplemented with collagenase type 1 (lmg/ml) at a ratio of 1:3 (adipose tissueiDMEM). After washing the digested tissue with PBS, centrifugation was performed at 1000 rpm for 5 minutes. The supernatant was suctioned off, and the remaining pellets on the bottom were washed with a mixture solution of 10% FBS and DMEM-LG (low glucose) at a ratio of 1 :1, followed by another centrifugation at 1000 rpm for 5 minutes. The resulting pellets were filtered through a 100 μm mesh to remove debris and washed with PBS. The resulting cells were incubated in a DMEM medium (10% FBS, 2mM NAC, 0.2mM ascorbic acid). One night later, unattached cells were washed with PBS, and attached cells were cultured in K-NAC media (Keratinocyte-SFM media + 2mM NAC + 0.2mM ascorbic acid + 0.09mM calcium + 5ng/ml rEGF + 50 jUg/ml BPE + 5 μg/ml insulin + 74ng/ml hydrocortisone) while the media are replaced every two days, thereby obtaining multipotent stem cells derived from human adipose tissue and fibroblasts. By mixing the obtained cells with adipose, the composition for cosmetic or plastic surgery, containing adult stem cells, fibroblasts, and adipose or adipocytes, according to the present invention was prepared.
Example 2: Immunological properties of adipose-derived multipotent stem cells
The adipose tissue-derived adult stem cells obtained in Example 1 were washed with PBS and treated with trypsin. The treated cells were collected and centrifuged at 1000 rpm at 4°C for 5 minutes. After the supernatant was discarded and the cells were washed with 2% FBS in PBS, centrifugation was carried out at ljOOOrpm, 4°C for 5 minutes. The supernatant was discarded, and the pellet was suspended with PBS to dispense I xIO5 cells as many as sample numbers, followed by centrifugation at 1000 rpm at 4°C for 5 minutes. The supernatant was discarded, and the cells were mixed well with 150 μJl of blocking solution (5% FBS in PBS), and centrifuged at 1000 rpm at 4°C for 5 minutes. The supernatant was again discarded and the cells were well mixed with 100 μi of blocking solution (5% FBS in PBS) to incubate them at 4°C for 30 minutes. After that, the solution was centrifuged at 1000 rpm for 5 minutes at 4°C and the supernatant was removed, followed by adding 150 μJl of blocking solution (5% FBS in PBS) to mix well. Antibodies (R-phycoerythrin-conjugated mouse anti-human monoclonal antibodies) were placed into each well and incubated at 4°C for 30 minutes. After the incubation, they were centrifuged at 1000 rpm at 4°C for 5 minutes. The supernatant was removed, and the cells were washed with PBS and centrifuged at 1000 rpm for 5 minutes at 4°C . Once again, the supernatant was removed, and the cells were washed with PBS and centrifuged at 1000 rpm for 5 minutes at 4°C . After the supernatant was discarded, the cells were well mixed with 200 μϋ of PBS and fixed with 1% paraformaldehyde, and finally analyzed using a flow cytometer.
Table 1 : FACS analysis of surface antigens of adipose-derived stem cells
Figure imgf000012_0001
As a result, as shown in Table 1, the adipose tissue-derived adult stem cells according to the present invention showed positive responses of 91% to CD73, 97% to CD90, 96% to CD29, 83% to CD44, and 80% to CD105. Also, as a result of FACS analysis to other antigens, the inventive stem cells showed negative immunological responses to all of CD33, CD34, CD45, CD4, CD31, CD62p, CD14 and HLA-DR.
Example 3: Immunostaining analysis of adipose tissue-derived stem cells
The adipose tissue-derived stem cell obtained in Example 1 were washed with PBS 3 times and fixed with 4% paraformaldehyde-containing PBS for 30 minutes. The cells were washed with PBS 3 times and were permeabilized with PBS containing 0.1% Triton-XIOO for 10 minutes. After being washed with PBS 3 times, the cells were incubated with 10% NGS for 1 hour and then incubated with a primary antibody-containing PBS overnight. The cells were also washed with PBS 3 times, and were allowed to react with a secondary antibody in a dark room for 1 hour. After being washed with PBS 3 times, the cells were mounted.
As a result, as shown in FIG. 2, the multipotent stem cell spheres according to the present invention showed positive responses to all of Oct4 which can be regarded as a marker of undifferentiated cells, and SH2(CD105) and SH3/4(CD73) which are markers of mesenchymal stem cells.
Example 4; Differentiation of adipose-derived multipotent stem cells into adipocytes
The adipose tissue-derived multipotent adult stem cells obtained in Example 1 were incubated in an ce-MEM medium containing 5% FBS, lμM dexamethasone,
200μM indomethacin, 10 //g/ml insulin and 0.5mM IBMX (3-isobutyl-l- methylxanthine) for two weeks to induce adipogenic differentiation and then analyzed using an oil red O staining method. As a result, as shown in FIG. 3, it was found that the human adipose tissue-derived multipotent stem cells were differentiated into adipogenic cells.
Example 5; Animal analysis using adipose-derived multipotent stem cells
The ability to preserve a volume of adipose tissue by adipose-derived stem cells was examined using mice. 12 mice for a control group and 12 mice for an experimental group were used. Adipose (ImI) + adipose tissue-derived multipotent adult stem cells and fibroblasts (5>< 105/200 μJi saline) were injected into cephalic hypodermis, where subcutaneous adipose tissue is not present in a mouse, in the experimental group, whereas adipose (ImI) + vehicle (200 μJL saline) were injected in the control group. 15 days after the injection, adipose tissue was separated through a cephalic incision of mice, and the volume of adipose tissue was measured. As a result, while the volume of injected adipose was reduced to 31% in the control group, 45% of the injected adipose volume was preserved in the experiment group in which the composition prepared by Example 1 was injected. From this result, it was found that adipose tissue-derived stem cells and fibroblasts according the present invention have an effect of reducing adipose volume decrease resulting from liposuction.
INDUSTRIAL APPLICABILITY
As described in detail above, the present invention has an effect to provide the composition for cosmetic or plastic surgery, containing human adipose tissue- derived adult stem cells, fibroblasts and adipose or adipocytes. The composition for cosmetic or plastic surgery according to the present invention is useful not only for cosmetic purposes such as improving skin tightness, improving wrinkles and flabby skin, and restoring a depressed area through plastic surgery, but also for the formation of new breast tissue since the human adipose tissue-derived adult stem cells can be differentiated into adipogenic cells.
Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only for a preferred embodiment and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims

THE CLAIMS
What is claimed is:
L A composition for cosmetic or plastic surgery, containing (i) human adipose tissue-derived adult stem cells, (ii) fibroblasts and (iii) adipose or adipocytes.
2. The composition according to claim 1, wherein the composition is for the formation of new breast tissue or wrinkle removal.
3. A method for preparing the composition for cosmetic or plastic surgery, containing (i) human adipose tissue-derived adult stem cells, (ii) fibroblasts and (iii) adipose or adipocytes, the method comprising the steps of:
(a) culturing an adipose-containing suspension obtained from human adipose tissue by liposuction in a culture flask and then collecting cell layers containing both adipose-derived adult stem cells and fibroblasts, which are attached to the surface of the culture flask; and
(b) mixing the collected cell layers containing adipose-derived adult stem cells and fibroblasts with adipose or adipocytes.
4. The method according to claim 3, wherein the adult stem cells are mesoderm- derived cells.
5. The method according to claim 3, wherein the adipose-derived adult stem cells are cultured in an α-MEM medium containing dexamethasone, indomethacin, insulin and IBMX to differentiate into adipogenic cells.
6. A method for preparing the composition for cosmetic or plastic surgery, containing (i) human adipose tissue-derived adult stem cells, (ii) fibroblasts and (iii) adipose or adipocytes, the method comprising the steps of: (a) culturing human adipose tissue-derived pellets in a medium containing N-acetyl-L-cysteine (NAC) to prepare adipose-derived adult stem cells and then collecting adipose-derived stem cells and fibroblasts; and
(b) mixing the collected adipose-derived adult stem cells and fibroblasts with adipose or adipocytes.
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