KR20070119497A - Cosmetic or plastic composition comprising multipotent stem cells derived from human adipose tissue, fibroblast and adipose or adiopocyte - Google Patents

Abstract

A composition comprising human adipose tissue-derived adult stem cells, fibroblasts and adipose or adipocytes is provided to be 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 due to the adult stem cells capable of being differentiated into fat cells. A composition for cosmetic or plastic surgery comprises human adipose tissue-derived adult stem cells, fibroblasts and adipose or adipocytes. A method for preparing the composition comprises 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 comprising both adipose-derived adult stem cells and fibroblasts, which are attached on the surface of the culture flask; and (b) mixing the collected cell layers with adipose or adipocytes, wherein the adult stem cells are mesoderm-derived cells and the adipose-derived adult stem cells are cultured in an alpha-MEM medium including dexamethasone, indomethacin, insulin and IBMX to differentiate into adipogenic cells.

Description

Method for producing skin cosmetic or cosmetic composition containing human adipose tissue-derived multipotent stem cells, fibroblasts and adipose or fat cells {Cosmetic or Plastic Composition Comprising Multipotent Stem Cells Derived from Human Adipose Tissue, Fibroblast and Adipose or Adiopocyte}

1 is a photograph taken at 100-fold magnification of cells attached to a flask including human adipose tissue-derived multipotent stem cells, fibroblasts, and the like according to the present invention.

2 is a 100-fold magnification of the expression of Nestin, Oct4, SH2, and SH3 / 4 using immunostaining after sphere culture of human adipose tissue-derived multipotent stem cells according to the present invention in CORM-2-containing MEBM medium. The picture shown.

Figure 3 shows the adipocytes differentiated from human adipose tissue-derived multipotent stem cells according to the present invention at a 200-fold magnification (A: phase contrast of the differentiated state, B: stained by Oil Red O staining method).

The present invention relates to a method for producing a composition for cosmetic or cosmetic skin containing adult stem cells, fibroblasts and fat or fat cells derived from human adipose tissue.

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).

Recently, adipose tissue has been found to be a new source of pluripotent 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). That is, it has been reported that undifferentiated cell populations are included in human adipose tissue obtained by liposuction (liposuction) and have differentiation ability into adipocytes, osteoblasts, myoblasts and chondrocytes in vitro. (PA Zuk et al ., Tissue Eng . , 7: 211, 2001; AM Rodriguez et al ., BBRC , 315: 255, 2004). In addition, it has been known through animal model experiments that adipose tissue-derived cells have the ability to promote muscle regeneration and neurovascular differentiation. These adipose tissues have the advantage that they can be extracted in large quantities, attracting attention as a source of new stem cells to supplement the existing disadvantages.

So far known 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 cells capable of bone formation and differentiation into adipocytes (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), mouse adipose derived stem cells capable of differentiating into adipocytes ( R. Ogawa et al ., BBRC , 319: 511, 2004), rat adipose derived stem cells capable of differentiating into bone formation and chondrogenic 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), mouse adipose derived stem cells capable of differentiating into neurons (J. Fujimura et. al ., BBRC , 333: 116, 2005) and adipose derived stem cells capable of differentiating into osteoblasts, chondrocytes, neurons or muscle cells (US Pat. No. 6,777,231).

Until now, efforts to use these fat-derived stem cells in the medical field including treatment of intractable diseases have been continued, but attempts to use stem cells for cosmetic or cosmetic purposes have been insufficient.

Accordingly, the present inventors have made efforts to use stem cells in cosmetic and cosmetic fields such as wrinkle improvement and prevention of sagging of skin. As a result, the adult stem cells and fibroblast-containing cell layers are recovered by culturing a fat-containing suspension obtained from liposuction. Then, as a result of mixing with fat, it was confirmed that it is effective for beauty and shaping, and completed the present invention.

An object of the present invention is to provide a composition for cosmetic or cosmetic containing adult stem cells, fibroblasts and fat or adipocytes derived from human adipose tissue and a method for producing the same.

In order to achieve the above object, the present invention (a) culturing the fat-containing suspension obtained by liposuction of human adipose tissue in a culture vessel, and then adult stem cells and fibers derived from fat attached to the culture vessel Recovering the blast-containing cell layer; And (b) mixing the recovered adipose derived adult stem cells and fibroblast-containing cell layers with fat or adipocytes, (i) human adipose tissue derived adult stem cells, (ii) fibroblasts and (iii) It provides a method for producing a skin cosmetic or cosmetic composition containing fat or fat cells.

The present invention also comprises the steps of (a) culturing human adipose tissue-derived pellets in NAC (N-acetyl-L-cysteine) -containing medium to produce adult stem cells, and then recovering adipose derived stem cells and fibroblasts. ; And (b) mixing the recovered adipose derived adult stem cells and fibroblasts with fat or adipocytes, (i) human adipose tissue derived adult stem cells, (ii) fibroblasts and (iii) fat or It provides a method for producing a skin cosmetic or molding composition containing fat cells.

The present invention also provides a composition for skin beauty or shaping, comprising (i) adult stem cells derived from human adipose tissue, (ii) fibroblasts and (iii) fat or fat cells.

The present invention, in one aspect, relates to a composition for cosmetic or cosmetic containing adult stem cells, fibroblasts and fat or fat cells derived from human adipose tissue.

The composition according to the present invention may be characterized in that for breast enlargement or wrinkle removal through breast tissue formation, but is not limited thereto. For example, it may be used for the purpose of removing nasolabial folds and forehead wrinkles and maintaining elasticity of the face of the face such as microfat graft and autologous graft. The human adipose tissue-derived adult stem cells, fibroblasts and fat contained in the composition according to the present invention are preferably autologous cells.

The cosmetic or cosmetic composition according to the present invention can be prepared by mixing fat-derived adult stem cells and fibroblasts obtained by culturing a fat-containing suspension obtained by liposuction of human adipose tissue with other cells or tissues. .

In one embodiment, the skin cosmetic or cosmetic composition may be prepared by the following steps: (a) culturing the fat-containing suspension obtained by liposuction of human adipose tissue in a culture vessel, and then culturing the culture vessel Recovering the adipose derived adult stem cells and fibroblast-containing cell layers attached thereto; And (b) mixing the recovered adipose derived adult stem cells and fibroblast-containing cell layers with fat or adipocytes.

More specifically, after culturing a suspension containing fat suspended in physiological saline obtained from liposuction, the stem cells and fibroblasts attached to the culture vessel such as a flask are washed once with physiological saline immediately before recovery, and the remaining fat of the supernatant is recovered. And removing the blood cells and the like, and recovering the stem cells and fibroblast layers attached to the bottom with trypsin, or recovering them by scraping them with a scraper, or directly recovering the suspended solids in a small amount of physiological saline, or centrifuging the cell layer. Is collected and mixed with fat to prepare.

In the present invention, the adult stem cells may be characterized in that the mesoderm derived cells, the mesoderm derived cells may be characterized in that the adipocytes.

In the present invention, the adult stem cells can be characterized by differentiation into adipocytes by culturing in α-MEM medium containing dexamethasone (indamethasone), indomethacin (indomethacin), insulin and IBMX.

In another embodiment, the skin cosmetic or cosmetic composition may be prepared by the following steps: (a) Adipose-derived adult stem cells by culturing pellets derived from human adipose tissue in a medium containing N-acetyl-L-cysteine (NAC) After preparing, recovering the adipose derived stem cells and fibroblasts; And (b) mixing the recovered adipose derived adult stem cells and fibroblasts with fat or adipocytes.

The liposuction is a technique of cosmetic or plastic surgery of fat removal, which means cutting the fat portion and extracting the fat by a vacuum pump or by the pressure of a syringe.

In the present invention, fat or adipocytes may be used that is obtained incidentally in liposuction of human adipose tissue, but is not limited thereto. Fat in the present invention includes adipose tissue.

When the skin cosmetic or cosmetic composition of the present invention uses fat, the fat tissue preferably contains 1 × 10 ⁴ to 1 × 10 ⁷ fat-derived stem cells and fibroblasts per 1 ml. It is preferable to use 1 to 50 times the fat cells with respect to the total cell number of the adipose derived stem cells and fibroblasts.

In addition, fibroblasts (fibroblast) is an important component of the fibrous connective tissue, also called fibroblasts. Observed by tissue section, it has a flat and elongated appearance and often shows irregular projections. The cytoplasm contains mitochondria, Golgi apparatus, centrosomes, small fat bodies and the like, and does not exhibit any special differentiation. The nucleus is weakly stainable, elliptical, and contains phosphorus. In many cases, it is closely related to collagen fibers, and because it is thought to be related to its formation, the same name is given. In the present invention, the fibroblasts can be separated together in the process of separating stem cells from adipose tissue.

On the other hand, the FACS method using a flow cytometer with a sorting function ( Int . Immunol . , 10 (3) was used as a method for obtaining multipotent stem cells expressing the surface antigens of interest from the stem cell fluid derived from human adipose tissue obtained above . : 275, 1998), methods using magnetic beads, and panning methods using antibodies that specifically recognize multipotent stem cells ( J. Immunol . , 141 (8): 2797, 1998). In addition, as a method for obtaining multipotent stem cells from a large amount of culture solution or the like, antibodies which are expressed on the surface of cells and specifically recognize molecules (hereinafter referred to as surface antigens) are used alone or in combination and used as columns. There is a way.

Examples of the flow cytometer sorting method include a water charge method, a cell capture method, and the like. Any method can quantitate the amount of antigen expressed in a cell by labeling an antibody that specifically recognizes the surface antigen of the cell with fluorescence, and converting the fluorescence intensity into an electrical signal by measuring the fluorescence of a conjugate of the labeled antibody and antigen. have. In addition, by combining the kinds of fluorescent materials to be used, it is possible to separate cells expressing a plurality of surface antigens. Fluorescent materials usable herein include FITC (fluorescein isothiocyanate), PE (phycoerythrin), APC (allo-phycocyanin), TR (TexasRed), Cy3, CyChrome, Red613, Red670, TRI-Color, QuantumRed and the like.

As a FACS method using a flow cytometer, the stem cell solution obtained above is collected, the cells are separated by centrifugation or the like, and then stained with an antibody directly, and cultured and propagated in a suitable medium once, followed by antibody. The method of dyeing can be used. To stain the cells, firstly, the primary antibody recognizing the surface antigen and the desired cell sample are mixed and incubated for 30 minutes to 1 hour on ice. If the primary antibody is labeled with fluorescence, it is separated by a flow cytometer after washing. In the case where the primary antibody is not fluorescently labeled, after washing, the fluorescently labeled secondary antibody having a binding activity to the primary antibody and the cells reacted with the primary antibody are mixed and incubated in ice water for 30 minutes to 1 hour. After washing, the cells stained with the primary antibody and the secondary antibody are separated by flow cytometry.

The composition containing the adult adipose stem cells, fibroblasts and fat derived from human adipose tissue according to the present invention improves skin elasticity, improves wrinkles, especially the nasolabial folds and brow wrinkles and skin sagging, and restores the dents through molding. It is effective for beauty. In addition, since the adult adipose stem cells derived from human adipose tissue can be differentiated into adipocytes, the composition according to the present invention is also useful for forming breast tissue.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example  1: in adipose tissue Multiplicity  Isolation and Molding of Adult Stem Cells

Production Example  One

The fat-containing suspension suspended in physiological saline obtained by liposuction of human adipose tissue is resuspended evenly with an appropriate amount of physiological saline and placed in an appropriate amount in a cell culture flask or roller bottle, followed by stationary culture or rotary culture. Was done. For stationary cultivation, it was left for at least 6 to 12 hours. Next, the cell layer (fat-derived MSC, fibroblast) attached to the flask surface was recovered by treatment with trypsin (FIG. 1). At this time, to directly recover the suspended in a small amount of physiological saline, or to reduce the volume of the cell layer, only the pellet layer was used by centrifuging the cell layer recovered with physiological saline for 10 minutes at 1000 rpm. The separated cell layer contains adult stem cells and fibroblasts. The separated cell layer was mixed with fat to prepare a composition for skin beauty or molding containing adult stem cells, fibroblasts and fat or fat cells according to the present invention.

Production Example  2

Multipotent stem cells were isolated and purified from human adipose tissue. In other words, separated human adipose tissue was washed three times with PBS, and then the tissue was chopped, and then the fat and DMEM were 1: 3 using DMEM-LG (low glucose) medium to which collagenase type1 (1 mg / ml) was added. The mixture was mixed at a ratio and digested at 37 ° C. for 1 hour. After washing with PBS, centrifugation was performed for 5 minutes at 1000 rpm. The supernatant was suctioned and the pellet remaining on the bottom was washed with 10% FBS and DMEM-LG (low glucose) mixed 1: 1 and centrifuged at 1000 rpm for 5 minutes. After filtering to 100㎛ mesh to remove the debris and washed with PBS. Incubated in DMEM (10% FBS, 2 mM NAC, 0.2 mM ascorbic acid) medium. After overnight, non-stick cells were washed with PBS and K-NAC media (Keratinocyte-SFM media + 2 mM NAC + 0.2 mM ascorbic acid + 0.09 mM calcium + 5 ng / ml rEGF + 50 µg / ml BPE + 5 µg / ml insulin) + 74ng / ml hydrocortisone) was replaced every 2 days to obtain multipotent stem cells and fibroblasts derived from human adipose tissue. Fat was mixed with the obtained cells to prepare a composition for cosmetic or molding skin containing adult stem cells, fibroblasts and fat or adipocytes according to the present invention.

Example  2: derived from fat Multiplicity  Immunological Characteristics of Stem Cells

Adipose tissue-derived adult stem cells obtained in Example 1 were washed with PBS, trypsinized, and the cells were harvested and centrifuged at 4 ° C. at 1000 rpm for 5 minutes. The supernatant was discarded and washed with a mixture of 2% FBS and PBS, followed by centrifugation at 4 ° C. for 5 minutes at 1000 rpm. After discarding the supernatant, the cells were suspended in PBS, and 1 × 10 ⁵ cells were dispensed as many as the number of samples. After centrifugation at 4 ° C. for 5 minutes at 1000 rpm, the supernatant was removed and 150 μl of blocking solution (5% FBS). in PBS) and mixed well and centrifuged at 4 ° C. for 5 minutes at 1000 rpm. The supernatant was removed again, 100 μl of blocking solution (5% FBS in PBS) was added and mixed well, followed by reaction at 4 ° C. for 30 minutes. After centrifugation at 1000 ° C. for 5 minutes at 4 ° C., the supernatant was removed, and 150 μl of blocking solution (5% FBS in PBS) was added, mixed well, and antibody (R-phycoerythrin-conjugated mouse) was added to each well. anti-human monoclonal antibody) was added and reacted at 4 ° C. for 30 minutes. After the reaction, the mixture was centrifuged at 4 ° C. for 5 minutes at 1000 rpm. The supernatant was removed, washed with PBS, and centrifuged at 4 ° C. for 5 minutes at 1000 rpm. Once again, the supernatant was removed, washed with PBS and centrifuged at 4 ° C. for 5 minutes at 1000 rpm. After removing the supernatant and mixed well in 200μl of PBS, 1% paraformaldehyde was added to single and analyzed using a flow cytometer.

Surface antigen analysis of adipose derived stem cells (FACS analysis) Antigen AD-MSCs CD73 + CD90 + CD29 + CD44 + CD105 + CD33 - CD34 - CD45 - CD4 - CD31 - CD62p - CD14 - HLA-DR -

As a result, as shown in Table 1, the adipose tissue-derived adult stem cells of the present invention were 91% for CD73, 97% for CD90, 96% for CD29, 83% for CD44, 80% for CD105. It was positive. In addition, as a result of confirming the immunophenotype for other antigens, all showed negative immunological characteristics for CD33, CD34, CD45, CD4, CD31, CD62p, CD14 and HLA-DR.

Example  3: Immunostaining Analysis of Stem Cells Derived from Adipose Tissue

The adipose tissue-derived stem cells obtained in Example 1 were washed three times with PBS and fixed for 30 minutes with PBS containing 4% paraformaldehyde. After washing three times with PBS, it is permeabilized with PBS containing 0.1% Triton-X100 for 10 minutes. After washing three times with PBS, it was reacted with 10% NGS for 1 hour, and reacted overnight with PBS containing the primary antibody. Washed three times with PBS, and reacted with a secondary antibody in the dark for 1 hour. After washing three times with PBS, it was mounted.

As a result, as shown in Figure 2, the multipotent stem cell sphere according to the present invention in the SH2 (CD105), SH3 / 4 (CD73), which are markers of Oct4 and mesenchymal stem cells, which can be said to be undifferentiated cell markers. It showed a positive response to.

Example  4: derived from fat Multiplicity  Differentiation of Stem Cells into Adipocytes

Adipocyte-derived multipotent adult stem cells obtained in Example 1 were prepared using α-MEM medium containing 5% FBS, 1 μM dexamethasone, 200 μM indomethacin, 10 μg / ml insulin, and 0.5 mM IBMX (3-isobutyl-1-methylxanthine). The cells were cultured for 2 weeks at to induce differentiation of multipotent stem cells into adipocytes, and analyzed using Oil red O staining. As a result, as shown in Figure 3, it was confirmed that the multipotent stem cells derived from human adipose tissue according to the present invention was differentiated into adipocytes.

Example  5: derived from fat Multiplicity  Animal experiments using stem cells

Using mice, fat volume preservation ability of adipose tissue-derived stem cells was confirmed. Mouse was used as a 12 12 test group and control group, the experimental group, the local 1ml as in Example 1 is derived from the adipose tissue isolated from multipotential subcutaneously in adult stem cells and fibroblasts, mouse ^{(5 × 10 5 / 200μl saline} ) fat It was injected under the head skin, which is not present, and the control group was injected with 1 ml of fat + vehicle (200ul saline).

After 15 days, the scalp of the mouse was incised to remove fat and the volume was measured. As a result, the injected fat volume was reduced to 31% in the control group, while the experimental group injected with the composition prepared in Example 1 maintained 45% of the injected fat volume. From these results, it was confirmed that the adipose tissue-derived stem cells and the fibroblasts according to the present invention have an effect of significantly reducing the fat volume decrease generated in the fat graft.

As described in detail above, the present invention has the effect of providing a composition for cosmetic or cosmetic skin containing adult stem cells, fibroblasts and fat or fat cells derived from human adipose tissue. Skin cosmetic or cosmetic composition according to the present invention to increase the skin elasticity, improve wrinkles and sagging skin, and to restore the depressions by molding as well as effective in beauty, the adult stem cells can be differentiated into fat cells It is also useful for forming breast tissue.

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, such a specific description is only a preferred embodiment, which is not limited by the scope of the present invention Will be obvious. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (6)

A composition for skin beauty or shaping, comprising (i) adult stem cells derived from human adipose tissue, (ii) fibroblasts, and (iii) fat or fat cells. The composition of claim 1, wherein the composition is for breast tissue formation or wrinkle removal. A method for preparing a composition for skin cosmetic or cosmetic composition comprising (i) adult stem cells derived from human adipose tissue, (ii) fibroblasts and (iii) fat or fat cells, comprising the following steps: (a) culturing the fat-containing suspension obtained by liposuction of human adipose tissue in a culture vessel, and then recovering the adipose-derived adult stem cells and fibroblast-containing cell layers adhered to the culture vessel surface; And (b) mixing the recovered adipose derived adult stem cells and fibroblast-containing cell layers with fat or adipocytes. The method of claim 3, wherein the adult stem cells are mesodermal derived cells. 4. The method of claim 3, wherein the adipose derived adult stem cells are cultured in α-MEM medium containing dexamethasone, indomethacin, insulin and IBMX to differentiate into adipocytes. A method for preparing a composition for cosmetic or cosmetic treatment comprising (i) adult stem cells derived from human adipose tissue, (ii) fibroblasts and (iii) fat or fat cells, comprising the following steps: (a) culturing human adipose tissue-derived pellets in NAC (N-acetyl-L-cysteine) -containing medium to prepare adult stem cells, and then recovering adipose derived stem cells and fibroblasts; And (b) mixing the recovered adipose derived adult stem cells and fibroblasts with fat or adipocytes.

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