KR20080075959A - Composition of the injectable agents for tissues repair including adipose derived stem cells and optimized culture media - Google Patents

Abstract

An injection composition comprising culture medium for optimizing efficacy of human adipose stem cells is provided to increase tissue engraftment efficiency of adipose stem cells, improve differentiation ability of fibroblasts and enhance tissue repair ability as compared to single injection of adipose stem cells. An injection composition for tissue repair comprises cultured medium of human adipose stem cells which is prepared by culturing human adipose stem cells in serum free medium containing adipose stem cell-activating compounds including antioxidant, vitamins or their derivatives, amino acids or their derivatives, inorganic matters, and optionally growth factor and collagen for more than 1 day. The skin tissue is repaired by injecting the composition for tissue repair together with the adipose stem cell into the skin tissue. Further, the amino acids or their derivatives are one or more selected from L-Alanine, L-Asparitic acid, L-Histdine, L-leucine or L- glutamine.

Description

Composition of the Injectable Agents for Tissues Repair including Adipose derived stem cells and optimized culture media}

Figure 1 shows the characteristics of the adipose stem cells isolated from adipose tissue, Figure 1a is a culture micrograph, Figure 1b is a flow cytometry photograph, Figure 1c is a tissue-specific multipotent photo,

Figure 2 is a diagram showing the effect of improving the viability of fat stem cells by the fat stem cell optimization constructs, Figure 2a is a micrograph of the fat stem cells engrafted on the plate, Figure 2b quantified the engrafted fat stem cells Is a graph,

3 is a diagram showing the fibroblast activation effect of the adipocyte stem cell optimization composition, Figure 3a shows the effect of promoting the mobility of fibroblasts, Figure 3b is a graph showing the quantification of the results of Figure 3a,

4 is a photograph showing an increase in dermis at the time of injection or transplantation alone or mixed with stem cells and adipose stem cell optimization components isolated from adipose tissue in an animal model using pigs,

FIG. 5 is a photograph showing the dermis changed when the stem cells are injected or transplanted from adipose tissue with Dermascan. FIG.

6A, 6B, 6C, and 6D show injection or implantation of a tissue regeneration injection containing adipocytes into or adjacent to the defect to correct cosmetic or degenerative defects in the skin, soft tissue, and bone of the individual. It is a picture confirming that wrinkle improvement is activated.

The present invention relates to an injectable composition for tissue regeneration for correcting cosmetic or degenerative defects of skin, soft tissue, bone of an individual.

More specifically, the present invention relates to an injectable composition for tissue regeneration comprising a fat stem cell optimization component, which is a culture medium in which human adipose stem cells are cultured for at least one day in a serum-free culture medium containing a cell activating compound, and a method for regenerating tissue using the same.

Stem cells are cells that have been differentiated into specific cells without progress and, if necessary, have the ability to differentiate into all kinds of cells constituting the body such as nerves, blood, and cartilage. Stem cells are divided into embryonic stem cells from embryos resulting from fertilized eggs and adult stem cells potentially retaining in some parts of the adult body.

Unlike embryonic stem cells, adult stem cells are extracted from autologous body tissues, which can avoid bioethical problems, and there is no fear of immune rejection. It also has the advantage of being differentiated from embryonic stem cells in that a large amount of cells for treatment can be obtained from autologous tissues. In addition, there is no possibility of developing cancer when transplanted into the body for actual organ regeneration, and in addition to producing cells needed immediately after transplantation, self-renewal is used to regenerate and store undifferentiated stem cells needed later. In addition, the various advantages of having tissue-specific differentiation that differentiates itself from the characteristics of surrounding tissues suggest the medical safety and high value of the adult stem cells. Therefore, studies on various diseases in vivo using this are being actively conducted.

Adult stem cells have the advantage that they can be obtained from autologous tissues as described above. Actual stem cells have been mainly taken from bone marrow. This is because the actual bone marrow harvesting itself is a high-risk procedure, there are a number of difficulties in obtaining sufficient cells for treatment due to the low yield of actual cells. Adipose tissue-derived stem cells used in the present invention have various superiority over conventional bone marrow-derived stem cells. In the past years, significant advances have been made in the field of biomass, but there have been no successful developments in the production of human fat or soft tissue. It is also true that various studies on the use of human adipose tissue have not been conducted. However, the recent identification of adult stem cells in adipose tissue (Zuk et al., Molecular Biology of Cell, 13: 4279-4295 (2002); Rodriguez et al., Biochimie, 87: 125-128 (2005)) Various studies on the application of fat stem cells have begun. Adipose stem cells have almost the same or better pluripotency and proliferation as compared to bone marrow-derived stem cells, and have the advantage of obtaining and culturing sufficient amounts of stem cells from small amounts of adipose tissue from actual clinical utility. The excellence is that the actual procedure is incomparably safer than bone marrow harvesting. Local anesthesia during the actual procedure not only reduces the risk of general anesthesia, but also allows the wound to heal within a week (Rei Ogaw., Current Stem cell research & therapy, 1: 13-20 (2006)). It is a factor that maximizes the clinical utilization and excellence of stem cells.

Despite the superiority of these adipocytes, the adipocyte stem cell research currently focuses only on methods that utilize the cell's multicellular differentiation ability, and little research has been done to maintain and enhance the autologous potential of the sugar cells.

In the studies for the clinical application of adult stem cells, there have been many studies on the possibility of tissue regeneration and treatment (Cecilia Roh and Stephen lyle., Pediatr. Res., 59: 100 ~ 103 (2006); H. Nakagawa et al., British Journal of Dermatology, 153: 29-36 (2005); Rei Ogawa., Current Stem Cell Research & Therapy, 1: 13--20 (2006)). Problems have been inherent in the fact that the viability is very low and the transplanted cells have various limitations, such as insufficient effect thereof. In order to optimize the tissue regeneration ability of adult stem cells, studies on techniques for increasing the survival rate of stem cell transplantation, optimizing the effects of grafting and activating them are still in the incomplete state.

The domestic research is more sluggish, and Korean Patent 2004-0024900 “Method for Inducing Differentiation of Human Stem Cells into Animal-Free Serum Culture Medium Composition and Hepatocytes” describes a method of differentiating human-derived stem cells using animal-free serum. In Korea Patent 2003-0004565, "Method for Producing Cells for Cell Transplantation", the use of stem cells for cell transplantation has been described, but there is no research on the activation method of fat stem cells.

Therefore, in the present invention, in order to optimize the tissue regeneration effect of the actual adipose stem cells, an injection for tissue regeneration containing a composition optimizing the viability and pharmacological action of fat stem cells and fat stem cells. The main composition of the injection according to the present invention is mainly composed of a culture solution of fat stem cells themselves and other optimized compositions in addition to the fat stem cells. This results in the effect of increasing the tissue viability and the fibroblast division ability of the transplantation of fat stem cells, maximizing the activity of the fat stem cells through such a composition to be used in the development of injections optimized for tissue regeneration effect do.

The present invention aims to optimize the composition of the injection for tissue regeneration containing adipose stem cells and increase the viability of the fat stem cells through the optimized composition to maximize their activity to utilize the tissue regeneration quickly and effectively.

The injection composition for tissue regeneration of the present invention includes its culture solution and culture conditions for optimizing the adipocyte stem cells themselves.

In order to achieve the above object, the present invention provides an injectable composition for tissue regeneration comprising a culture solution cultured for one or more days in a serum-free culture medium containing human adipose stem cells.

At this time, the cell activating compound includes an antioxidant, a vitamin or an analog thereof, an amino acid or an analog thereof, a growth factor or an analog thereof, collagen or an analog thereof and a mineral.

The antioxidant includes one or more substances selected from the group consisting of L-lipoic acid, coenzyme Q-10, C-MED 100, Trolox, GSH, and the like, and the vitamins or analogs thereof include vitamins A, B, C, D, E or one or more substances selected from the group consisting of vitamins, amino acids or analogs thereof include one or more substances selected from the group consisting of L-Alanine, L-Asparitic acid, L-Histdine, L-leucine or L-glutamine Growth factors or analogs thereof include vascular endothelial growth factor, hepatocyte growth factor, transforming growth factor-beta, platelet derived growth factor-A, keratin sulfate, basic fibroblast growth factor or cadherin (enveloped growth factor-2 and And one or more substances selected from the group consisting of: collagen or an analog thereof is one selected from the group consisting of collagen I-XIII, XVII, and fibronectin, etc. It comprises on the substance.

In another aspect, the present invention provides an actual treatment method for treating defects such as skin and soft tissues, teeth and bones by injecting the injection composition for tissue regeneration with adipose stem cells to the treatment site. The composition of the injection for tissue regeneration according to the present invention shows the effect of optimizing the actual treatment effect by increasing the viability of fat stem cells and optimizing fibroblast proliferation when the injection treatment according to the present invention.

Hereinafter, the present invention will be described in detail.

A first aspect of the present invention relates to an injectable composition for tissue regeneration comprising a culture medium in which the human adipose stem cells are cultured for at least one day in a serum-free culture medium containing a cell activating compound.

The injection composition for tissue regeneration according to the first aspect of the present invention contains a composition optimized to maximize the therapeutic effect by increasing the viability of the transplanted fat stem cells when administered to the treated tissue.

Cell viability refers to the survival of fat stem cells administered by subcutaneous injection to the skin tissue and engraftment, and through the expression of various extracellular matrix proteins secreted from the engrafted stem cells. Proliferation and differentiation of blasts is activated.

The injection composition for tissue regeneration of the first aspect of the present invention contains a fat stem cell optimization component, which refers to a culture medium obtained by culturing fat stem cells for at least one day in a serum-free culture medium containing a fat stem cell activating compound. .

In this case, the adipose stem cell activating compound includes an antioxidant, a vitamin or an analog thereof, an amino acid or an analog thereof, a growth factor or an analog thereof, collagen or an analog thereof, and a mineral. Among them, growth factors and collagen are secreted from fat stem cells when the stem cells are cultured in serum-free culture medium for at least one day, and thus, the growth factors and collagen do not need to be added to the initial culture medium.

Specifically, the adipose stem cells according to the present invention are adult stem cells in autologous fat, and can be obtained through a simple purification process among cells existing in adipose tissue. The process of acquiring adipose tissue uses adipose tissue that has been discarded in the process of liposuction commonly performed, and its usefulness is doubled because no additional invasive procedure is required.

Human adipose tissue was obtained by liposuction under local anesthesia, and the extracellular matrix of adipose tissue was enzymatically treated with collagenase, followed by centrifugation and the separation of monocytes, erythrocytes, and various cell debris. Get

Normally, it can be obtained by collecting sterile adipose tissue, which is usually obtained in the process of liposuction performed in a hospital, and only pure adipose tissue is separated from the isolated liposuction.

The isolated stem cells are cultured in a non-inductive medium of Dulbecco's Modified Eagle's Medium (DMEM) containing serum to remove non-adhesive cells.

After culturing the adipose stem cells in a serum-free culture medium containing a cell activating compound for at least one day can obtain the fat stem cell optimization composition according to the present invention.

In order to increase the efficiency of the injection for tissue regeneration, the optimized composition of the present invention includes a cell activating compound which is effective for the viability of fibroblasts and fibroblast proliferation and anti-aging of adipose stem cells, and the cell activating compound includes an antioxidant, a vitamin or Analogs, amino acids or analogs thereof, growth factors or analogs thereof, collagen or analogs and minerals thereof.

Antioxidants or analogues thereof include L-lipoic acid, coenzyme Q-10, C-MED 100, retinoic acid, vinpocetin, piccamlon, quinic acid, quinate, adenine dinucleotide, acetyl-L-carnitine, dimethylanimo ethanol or One or more substances selected from the group consisting of L-hydroxy acids. Antioxidants include retinoic acid and its predecessor, vinpocetin, picamylone, which assist in this cycle, and quinic acid and quinate, which act as protein kinases. In addition, carbohydrate synthesis factors such as adenine dinucleotide and acetyl-L-carnitine are important nutrients to cells, dimethylanimo ethanol to stop apoptosis, and L- to be involved in cell proliferation. Lipoic acid, L-hydroxy acid, and other accelerating additives such as coenzyme Q-10 involved in amino acid production.

The vitamin or analog thereof includes one or more substances selected from the group consisting of vitamin A, B, C, D or vitamin E. Vitamin A is known to be involved in the primary immune response, associated cell developmental processes, and planned series of apoptosis reactions. Retinoic acid is known as a representative substance, which binds to and binds to the retinoic acid receptor (RAR). Regulate and activate related metabolic processes. Among them, the complexes bound to RAR-alpha, RXR-alpha, and RXR-beta were reported to promote or inhibit the expression of 128 genes involved in the development of T lymphocytes, which are major immune cells. In particular, the bcl2 family of genes, known as representative anti-apoptotic proteins, is significantly increased in apoptosis mechanisms (Rasooly, R. et al., J. Immunol., 175: 7916-7929 (2005); Spilianakis, CG et al. , Eur. J. Immunol., 35 (12): 3400-4 (2005); Evans T, Exp. Hematol., 33 (9): 1055-61 (2005)). This suggests that vitamin A may have an inhibitory effect on apoptosis.

Vitamin B, commonly known as riboflavin, plays an important role in maintaining human health, and a Swedish research team found that the treatment of this substance caused an increased primary immune response due to the effects of neutrophil migration. (Verdrengh, M. and Tarkowski, A., Inflamm. Res., 54 (9): 390-3 (2005)). This is expected to be able to expect an extended initial immune response effect due to the migration of primary immune cells.

The most important function of vitamin C is to promote collagen synthesis and synthesis of new fibroblasts, ascorbic acid. Co-treatment with other cytokines TGF-β and IFN-γ doubles the effect (Chung, J. H. et al., J. Dermatol. Sci., 15 (3): 188-200 (1997)). Vitamin D3, which has been widely used because it is known to affect cell development and differentiation more than other vitamins, is a cell growth and differentiation process, especially keratinocytes of the epidermis and osteoblasts, which are skeletal cells. And mediate signaling pathways that are important for the formation of osteoclasts. It also has inhibitory effects on various cytokines involved in the inflammatory response, such as IL-1α, IL-6, IL-8 (Alper, G. et al., Endocr. Rev., 23: 763 (2002)).

 Amino acids or analogs thereof include one or more substances selected from the group consisting of L-Alanine, L-Asparitic acid, L-Histdine, L-leucine or L-glutamine and the like. These substances contribute to nutrient sources and various metabolism (Kaori Shigemitsu. Et al., J. Biol. Chem., 274: 1058 (1999)).

Growth factor or an analog thereof is one selected from the group consisting of vascular endothelial cells, hepatocellular growth factor, transformation factor, platelet derived growth factor, keratin sulfate, fibroblast growth factor or cadherin (similar to epidermal growth factor-2) It contains the above substance. These growth factors bind to receptors on the cell surface and activate the proliferation and differentiation of cells as proteins (Connolly, DTJ Cell. Biochem, 47 (3): 219-23 (1991); Schott, RJ and Morrow) , LA Cardiovasc, Res., 27 (7): 1155-61 (1993); Neufeld, G. et al., Prog.Growth Factor Res., 5 (1): 89-97 (1994); Senger, DR et al., Cancer and Metastasis Reviews, 12 (3-4): 303-24 (1993)). The binding of growth factors to specific receptors activates the signal transduction chain, which transmits signals from the outside of the cell to the nucleus inside. As signal transduction continues, it acts like a large amount of other transporters, finally synthesizing new proteins. In this way, growth factors form important signaling pathways in the transmission, function, and control of intercellular interactions (Lawrence, DA Eur. Cytokine Netw, 7 (3): 363-74 (1996); Cox, DA and Maurer, T., Clin. Immunol.Immunopathol, 83 (1): 25-30 (1997); Alevizopoulos, A. and Mermod, N., Bioessays. 19 (7): 581-91 (1997)).

Collagen or an analog thereof includes one or more substances selected from the group consisting of collagen I to XIII, XVII, and fibronectin. Collagen is the most protein contained in the animal body. Occupies. Proteins in the cell and proteins in the blood are present in water, but collagen is present in the body as structures such as fibers and membranes (Bell, E. et al., Proc. Natl. Acad. Sci. USA). 76: 1274-1278 (1979)). Therefore, collagen's primary role is to shape and support the organs in the body to build the body's skeleton. The role of the second collagen is to fill between the cells of the body and the cells to act as an adhesive of the cells will increase the engraftment of fat stem cells by the collagen. In the body, the breakdown and synthesis of collagen are repeated repeatedly, but as aging progresses, the synthesis is slower than the breakdown, and the balance in the body is broken, which reduces the amount of collagen in the body. For example, in the skin, collagen in the dermis forms the basic structure, creating an intertwined net structure. This is the foundation of elasticity and elasticity, and when a force is applied, the net structure deforms according to the force. And when no force is applied, it is restored to its original form. However, as aging progresses, the amount of collagen decreases and the net structure is weakened, so that individual collagen becomes thinner or destroyed. The elasticity or elasticity of the skin is insufficient, leading to sagging or wrinkles of the skin (Leung, DY et al., Science., 191: 475-477 (1976); Stopak, D., and Harris, AK, Dev. Biol. 90: 383-398 (1982)) and the cell activating compounds will help to prevent this.

As described above, the adipose stem cell activating compounds include linoleic acid, quinic acid, quinate, coenzyme Q-10, L-lipoic acid. L-hydroxy acid, niacin, dimethylanimo ethanol (DMAE), α-tocopherol, ascorbyl palmitate, carotenoids, glycolic acid, dihydroeproandrosterone (DHEA), C-MED 100 (Optigene-X LLC Sherwsbury, NJ), carboxyalkyl esters, estriol, acetyl-L-carnitine, lycopene, nicotinamide adenine dinucleotide (NADH), piccamlon, vinpocetin, retinoic acid, antineoplastone, piccamlon, pro Cysteine, vitamins A, B, C, D or growth factors (vascular endothelial growth factor (VEGF), insulin-like growth factor-I (IGF-I), transforming growth factor-β (TGF-β), fiber Blast growth factor (FGF), human growth hormones).

Useful fortifying compounds that can cause stronger effects than the activating compounds include transcription factors, histone acetyl transferases, methyl binding proteins, cell membrane protective factors, differentiation factors, acetylcholine and precursors, estriols, acetylcholinesterases , Aminocaproic acid, and the like, and may include carotenoids, carboxyalkyl esters and calcium triphosphate, calcium monophosphate, which may induce the development of bone cells, which affect DNA repair. It belongs to the category of activating compounds.

Specifically, in the present invention, it is preferable to use a serum-free culture medium containing Ham's F-12 nutrient mixture containing an amino acid or an analog thereof and a vitamin or an analog thereof as a culture medium of adipose stem cells. Serum-free medium containing Ham's F-12 nutrient mixture according to the present invention is based on DMEM without addition of pH indicators such as phenol red and Ham's F-12 nutrient mixture is added at a ratio of approximately 1: 0.5-2. . At this time, it is possible to add oxidants such as L-glutamine, energy metabolites such as sodium pyruvate, and carbon regulators such as sodium bicarbonate.

The serum-free medium may further include an antioxidant or an analog thereof, a growth factor and its analogues, and collagen and an analogue thereof. Among these, the growth factor and its analogues and collagen and its analogues may be cultured with serum-free cells. If cultured in the medium for at least one day, it is secreted from the adipocytes, so it may not be added to the initial culture medium.

Subsequently, subcultures of autologous adipose stem cells in a serum-free medium containing the cell activating compound are serum-free before obtaining the culture solution. The culture medium contained useful growth factors and collagen derived from adipose stem cells, and thus was designated as an optimal stem cell optimizing construct.

The final concentration of the cell activating compound in the adipose stem cell optimization component according to the present invention is from 1uM to 20000uM of antioxidants, from 0.5uM to 100uM of vitamins or their analogues, from 10uM to 1000uM of amino acids or their analogues, from 10pg / ml to 100ng of growth factors or their analogs. / ml, collagen or analogue thereof 2.5ug / ml-100ug / ml and preferably 0.001uM-150mM of minerals.

The utility of serum-free media comprising the adipose stem cell activating compounds established in the present invention can be found in Ham's F-12 nutrient mixtures. This mixed solution is designed to support the growth and homeostasis of cells, and to increase the stability of various minerals and amino acids and growth factors secreted from adipose stem cells. Vitamin-based nutrients and other factors that can promote production are formed by mixing in proportions. Fat stem cell culture and growth factor production in the serum-free medium according to the present invention, when compared with the conditions of serum medium containing a general animal serum, no reduced or negative effects, and some growth factors In contrast to serum media that show higher productivity in serum-free media and have unknown components by serum, it is possible to verify all component contents of the culture medium. This suggests that various variables that can come from animal serum included in serum medium can be minimized, and the present invention can achieve the desired effect at a cost of about 50%.

When the injection or transplantation of the fat stem cell optimization component according to the present invention is mixed with the passaged and subcultured fat stem cells, the fat stem cells are activated to have a good effect on the surrounding environment, thereby cosmeticting the skin, soft tissue, and bone of the individual. It is possible to quickly correct enemy or degenerative defects.

According to a second aspect of the present invention, an injection composition for regenerating tissue containing a culture solution of adipose stem cells cultured in a serum-free culture medium containing a cell activating compound for at least one day is injected with adipose stem cells to a treatment site, and the skin is soft. A method of treating tissue, tooth and bone defects.

Finally, 1uM to 20000uM of antioxidants, 0.5uM to 100uM of vitamins or their analogues, 10uM to 1000uM of amino acids or their analogues, 10pg / ml to 100ng / ml of growth factors or their analogs, 2.5ug / ml ~ 100ug of collagen or their analogues / ml and Mineral 0.001uM ~ 150mM In the case of the medium containing, it can be used as an injection composition for tissue regeneration without going through the step of culturing fat stem cells.

Defects in the skin, bones, soft tissue, and dental tissue can be caused by causes such as wounds, disease, and aging. Therefore, there is a need for a method for restoring and strengthening the growth of defective tissues while avoiding scars and wounds.

The method of the present invention involves the injection or transplantation of adipose stem cells and the injectable composition for tissue regeneration according to the present invention into the tissue adjacent or at the defect site. Defects that can be corrected in this way include wrinkles, stretch marks, fine scars, non-traumatic skin depression, acne scars, lip insufficiency, periodontal defects, soft tissue defects, bone defects, burns, skin ulcers, and the like. Since the cells to be injected as presented herein are autologous, they are histocompatibility with the subject and can be expanded to subculture in a cell culture system.

In addition, adipose stem cells and the injectable composition for tissue regeneration according to the present invention may be administered with a biodegradable acellular matrix, which obtains an environment similar to a living tissue even in vitro by a substrate in which the administered stem cells play a structural role. Therefore, it enhances the engraftment with the surrounding tissue during injection or transplantation and provides stability for normal cell function.

Absorbable cell-free substrates are divided into gels, beads, sponges, sheets, and skeletal forms depending on the use of the cell support. Commercially available substrates such as collagen sponges, chitosan alginate sponges, chitosan beads, and hyaluronic acid sponges are used. Take advantage.

The present invention will be described in more detail based on the following examples, which are not limited by the embodiments of the present invention.

Example 1 Isolation and Confirmation of Adipose Stem Cells

Human adipose tissue was obtained by liposuction under local anesthesia and treated with liposuction to obtain adipose stem cell population. This can be obtained by aseptic collection of adipose tissue, which is usually obtained in the course of liposuction performed in a hospital.

Human liposuction obtained from a clinic (leaders dermatology) is washed with an equal volume of phosphate buffered saline (PBS) and only adipose tissue separated.

Extracellular matrix of adipose tissue was enzymatically treated with 0.075 ~ 0.125% collagenase for 45 minutes in a 2 ~ 5% CO ₂ incubator at 30 ~ 37 ℃ and centrifuged for 5 minutes at 1200 ~ 1,500g for optimal enzyme treatment. To obtain a stromal vascular fraction containing dense stem cells. Thereafter, the cells were washed with PBS, other tissues were removed through a 70 μm nylon cell filter, and cell debris and mononuclear cells including red blood cells were separated by Histopaque-1077.

The isolated mononuclear cells were incubated with Dulbecco's Modified Eagle's Medium (DMEM), 5 ~ 10% fetal bovine serum (FBS), 1 ~ 2% penicillin / streptomycin, and 0.17 ~ 0.34% Sodium bicarbonate. Stem cells are isolated by removing non-adherent cells after incubation for 24 to 48 hours in a 2-5% CO ₂ incubator (see FIG. 1A).

Subcultures are performed when 80% confluence of the cells in culture in the flask in the composition and culture environment as described above. In subculture, the cells from which the culture medium was removed were washed with PBS, dropped into 0.25 ~ 0.5% Trypsin-EDTA, and the cell suspension was centrifuged. The cell pellet was suspended in 3 ml of non-inductive medium, and the cell number was measured and viability was tested. Incubate 1 ml of cell suspension in each of three T75 flasks containing 9 ml of medium.

For cell number measurement and viability test, 0.1 ml of cell suspension was mixed with 0.2% trypan blue in the same amount, and the stained cells were counted in the microscope field using a hemocytometer to determine the percentage of total cells.

Subcultures were performed from one to several times to culture sufficient fat stem cell numbers to obtain adipose stem cell optimization constructs.

Specifically, the non-inducible medium is used in combination with Dulbecco's Modified Eagle's Medium (DMEM), Ham's F-12 nutrient mixture of Table 1 below.

Ham's F-12 Nutrient Mixture Composition and Concentration Components Concentration (mg / L) Morality (mM) Components Concentration (mg / L) Morality (mM) Amino acid Vitamins D-Pantothenic Acis 2.24 0.00895 Biotin 0.0035 0.0000143 Glycine 18.75 0.25 Choline chloride 8.98 0.0641 L-Alanine 4.45 0.05 D-Calcium pantothenate 2.24 0.0047 L-Arginine hydrochloride 147.5 0.699 Folic acid 2.65 0.00601 L-Asparagine-H ₂ O 7.5 0.05 i-Inositol 12.6 0.07 L-Aspartic acid 6.65 0.05 Niacinamide 2.02 0.0166 L-Cysteine hydrochloride-H ₂ O 17.56 0.0998 Pyridoxine hydrochloride 2.031 0.00986 L-Cystine 2HCl 31.29 0.1 Riboflavin 0.219 0.000582 L-Glutamic Acid 7.35 0.05 Thiamine hydrochloride 2.17 0.00644 L-Glutamine 365 2.5 Vitamin b12 0.68 0.000502 L-Histidine hydrochloride-H ₂ O 31.48 0.15 Inorganic Salts L-Isoleucine 54.47 0.416 Calcium Chloride (CaCl ₂ ) (anhyd.) 116.6 1.05 L-Leucine 59.05 0.451 Cupric sulfate (CuSO ₄ -5H ₂ O) 0.0013 0.0000052 L-Lysine hydrochloride 91.25 0.499 Ferric Nitrate (Fe (NO ₃ ) ₃ "9H ₂ O) 0.05 0.000124 L-Methionine 17.24 0.116 Ferric sulfate (FeSO ₄ -7H ₂ O) 0.417 0.0015 L-Phenylalanine 35.48 0.215 Magnesium Chloride (anhydrous) 28.64 0.301 L-Proline 17.25 0.15 Magnesium Sulfate (MgSO ₄ ) (anhyd.) 48.84 0.407 L-Serine 26.25 0.25 Potassium Chloride (KCl) 311.8 4.16 L-Threonine 53.45 0.449 Sodium Chloride (NaCl) 6995.5 120.61 L-Tryptophan 9.02 0.0442 Sodium Phosphate dibasic (Na ₂ HPO ₄ ) anhydrous 71.02 0.5 L-Tyrosine disodium salt dihydrate 55.79 0.214 Sodium Phosphate monobasic (NaH ₂ PO ₄ ) anhydrous 54.3 0.45257 L-Valine 52.85 0.452 Zinc sulfate (ZnSO ₄ -7H ₂ O) 0.432 0.0015

When the adipose stem cells are cultured using the serum-free culture medium containing the cell activating compound as described above, growth factors and collagen are secreted from the stem cells to obtain the adipose stem cell optimization constructs according to the present invention. In Example 2 below, the components of the adipose stem cell optimization constructs were analyzed.

In addition, the present invention performs flow cytometry for the identification of fat stem cells. Stem cells express a number of adhesion and surface proteins. These proteins SH-2, SH-3 and SH-4 monoclonal antibodies recognize cell surface epitopes of human mesenchymal stem cells. Peptide sequencing and absorbance analysis showed that SH-3 and SH-4 were identified as CD73 (ecto-5'-nucleotidase) and SH-2 as CD105 (Endoglin). These cell surface markers are shared by adipose stem cells (Barry F et al., Biochem Biophys Res Commun, 289 (2): 519-24 (2001)).

Cultured fat stem cells are analyzed by passage using Fluorescence Activated Cell Sorter (Beckman Coulter). Specifically, the method is described, cells are harvested with 0.25% trypsin-EDTA, washed with PBS and 10 ⁵ cells per ml are reacted with CD73-PE, CD105-FITC antibodies, specific markers of mesenchymal stem cells. Analyzes with argon laser at 488 nm.

As a result, PLA cells isolated from adipose tissue showed 5.27% of stem cell homology (see FIG. 1B), after two passages 92.32% for CD73-PE and 90.67% for mesenchymal stem for CD105-FITC Cell homology was shown (see FIG. 1B).

In addition, to observe the tissue specific differentiation ability of the isolated adipose stem cells was performed as follows.

Passage the cultured cells to about 90% of the bottom area of the 6 well plate and differentiate into osteoblasts and fat cells. Osteoblast differentiation was carried out by adding differentiation medium (10mM β-glycerophosphate, 50ug / ml L-ascorbic acid, 100nM dexamethason) every 3 days to differentiate for 10 days and staining with Alizarin red S. Add differentiation medium (0.5mM isobutyl-1-methyl-3-xanthine, 100uM indomethasin, 1uM dexamethason, 10ug / ml insulin) and incubate for 3 days, then incubate for 10 days with 10ug / ml Insulin. This was repeated three times and observed by staining with Oil red O.

As a result, it was confirmed that the photo of the right column differentiated into adipocytes compared to the control photo of the left column (see FIG. 1C).

Example 2 Determination of the amount of activated protein in a composition capable of optimizing fat stem cells

The amount of various cytokines in the adipose stem cell optimization constructs obtained by <Example 1> was measured using an ELISA kit of R & D, and is shown in Table 2 below.

Fat Cell Activating Protein Content in Adipose Stem Cell Optimization Constructs Name of protein Concentration (pg / ml) PDGF AA (Platelet-Derived Growth Factor A-chain) PIGF (placenta growth factor) KGF (Keratan sulfate proteoglycan lumican) TGF beta 1 (Transforming Growth Factor beta 1) TGF beta 2 (Transforming Growth Factor beta 2) HGF (Hepatocyte Growth Factor) Vascular Endothelial Growth Factor (VEGF) bFGF (Fibroblast Growth Factor basic) Collagen type I Fibronectin 44.407 (± 2.563) 37.873 (± 1.695) 86.28 (± 20.330) 103.332 (± 1.697) 75.418 (± 95.982) 670.94 (± 86.917) 809.53 (± 95.982) 131.353 (± 30.310) * 921.47 (± 49.653) * 1466.48 (± 460.213)

In Table 2, the content of Collagen type I and Fibronectin is ng / ml.

Example 3 Confirmation of Viability Enhancement of Adipose Stem Cells

Some of the adipose stem cells (1 × 10 ⁵ to 1 × 10 ⁷ cells) isolated by Example 1 were stored in a cell freezing solution containing no serum, and other portions (1 × ^{10 5} to 1 × 10 ⁷ cells) were stored as cell activation compounds. A serum-free culture medium was included and cultured to separate the adipocyte stem cell optimization constructs containing various growth factors and large amounts of collagen.

Uncultured fat stem cells (1X10 ⁵ ~ 1X10 ⁷ cells) stored in the cell freezing solution were put in a 37 degree water bath for 10-30 seconds to thaw the frozen cells, and then suspended using 1-10 ml of serum-free medium. Cell pellets obtained from the isolation were cultured on a plate in an adipocyte-optimizing construct 100 times or more, and the survival rate of each fat stem cell was measured by culturing on the same plate using serum-free medium at the same ratio as the control group.

The measurement method was confirmed to settle the cells through real-time photography (see Fig. 2a) and when measuring the number of cells attached to the plate using the MTT assay after 7 days culture (see Fig. 2b), the experimental group was settled compared to the control group to survive One cell was found to be about 1.5 times more.

This can be achieved by the expression of various extracellular matrix proteins secreted from the seated cells based on the stable survival of the transplanted cells upon injection or transplantation of adipocytes uncultivated into or near the defect. Means proliferation and differentiation are activated.

Example 4 Migration of Fibroblasts Using Compositions That Can Optimize Adipose Stem Cells

In order to make a random wound, 5x10 ⁵ fibroblasts were placed in a 24 well plate with 95% humidity using medium containing DMEM, 5-10% fetal bovine serum, 1-10% penicillin / streptomycin, and 0.17-0.34% sodium bicarbonate. , 30 ~ 37 ℃, 2 ~ 5% CO ₂ After 100% confluent incubator, make wound using plastic micropipette and remove previous media and fibroblast debris using 0.5ml ~ 2ml PBS. At the bottom of the destroyed dermis, 0.5 ml to 2 ml of the adipose stem cell optimization component including the adipose stem cells obtained in <Example 1> and the fibroblast mixture solution were added to the test group, and fibroblasts were added as a control. Photographs are taken every 24 hours (see Figure 3a) to measure the distance of the cells and then quantify them (see Figure 3b).

As a result, when only fibroblasts were inserted, the effects of fibroblasts had an indirect effect on the proliferation and migration of keratinocytes in the upper dermis. Migration of fibroblasts inserted into the Dermis portion by branch growth factors and collagen is made, which directly affects the proliferation and migration of keratinocytes (see FIG. 3B).

The above experiments confirmed that various growth factors and collagen secreted from adipose stem cells promote the migration of fibroblasts, which further affects the proliferation and migration of dermis keratinocytes.

When injection or transplantation of adipose stem cells containing a composition for tissue regeneration at a site adjacent to a defect or a defect portion of an individual, the viability of the adipose stem cells is increased (see FIGS. 2A and 2B), whereby secretions are separated from the composition for tissue regeneration. In combination with various growth factors and collagen, it will be effectively used for tissue regeneration by promoting the proliferation and migration of keratinocytes (see FIGS. 3A and 3B) of the individual's fibroblasts.

Example 5 Animal Experiment by Injection or Transplantation of Adipose Stem Cells

Male micropigs weighing 9.8 kg were subjected to general anesthesia, and the back was cut with an electric hair clipper, and then partitioned into 2 cm x 2 cm wide tiles. A control group containing only adipose stem cells, a composition for optimizing fat stem cells, and a test group containing fat stem cells infiltrated 1 × 10 ⁶ cells per compartment with a 1cc insulin syringe in vivo. Adipose stem cells were isolated from a twenty-two year old female thigh and used within five passages. The depth of injection was aimed at the interdermis between the dermis and epidermis, and the experiment was conducted by a current dermatologist.

Four weeks after the injection and eight weeks after the injection, a total of three injections were made at the same site, and the same experiment was conducted with two dogs for error checking. Twelve weeks later, micropigs were anaesthetized and slaughtered to collect subcutaneous cutaneous skin tissue. After cutting the skin at the injection site, 10 μm frozen tissue sections were prepared by a conventional method, and the prepared tissue sections were fixed in 4% paraformaldehyde for more than 24 hours, followed by hematoxylin-eosin staining and Masson`. After staining with s trichrome staining was observed by light microscopy.

Hematoxyline and eosin staining showed that the skin dermal layer thickness was increased by 10% compared to the control group when constituents for optimizing fat stem cells were added. In addition, collagen accumulation is increased in the upper dermis and fibrils are in close contact with each other, indicating that the Glenz zone is thickened. In the dermal layer, scattered collagen fibers are thickened and thickened, indicating that they are newly produced and undergoing remodeling. Masson's trichrome staining was also found to be rich in collagen fibers and ground substance in the densely packed dermis with no gaps in the dermis as compared to the control group when the constituents for optimizing fat stem cells were added (see FIG. 4).

Example 6 Skin Improvement Effect by Injection or Transplantation of Autologous Adipose Stem Cells

Using ultrasound technology, the effect on the skin can be analyzed qualitatively and quantitatively. This technique can measure changes in skin thickness and can help determine the effectiveness of treatments for lasers or other skin. Ultrasound can be imaged using a high frequency sound wave. When a high frequency signal is emitted from the emitting source to the skin, the ultrasound signal emits an echo signal. An echo signal is generated at each layer of the tissue. Depending on the size and amplitude of these echoes and the time it takes to return, two-dimensional images of the skin are displayed.

When such a two-dimensional image is displayed, the thickness of the epidermis, the dermis, and the subcutaneous fat layer can be measured while identifying the structure of the skin. In measuring the thickness of the skin, a computer can be used to calculate the distance between two points and measure the distance (thickness) with an accuracy of 0.01 mm.

The autologous adipose stem cells isolated in <Example 1> were treated by injecting the stem cell optimization constructs into the wound site. As a result of measuring with a skin thickness gauge, the thickness increased from 2.054 to 2.317 mm, and all showed similar effects in ten subjects (see FIG. 5).

Example 7 Wrinkle Improvement Effect by Injection or Transplantation of Autologous Adipose Stem Cells

In order to verify the antiwrinkle effect of the adult stem cells in women aged 30 to 50 years, 50 subjects were selected from each experimental group.

Autologous adipose stem cells were injected with the stem cell optimizing composition around the right eye of a woman for 8 weeks at a frequency of twice a day, and the other site was visually observed after 4 weeks and 8 weeks as a comparative example. (See FIGS. 6A, 6B, 6C, and 6D) and the results are summarized as follows.

A noticeable effect Mitigating effect no effect Example 18 32 0 Comparative example 9 17 24

As discussed above, adipose stem cell optimizing constructs include various growth factors and adipose stem cell activating compounds such as collagen to increase the in vivo engraftment rate of adipose stem cells and optimize their activity. This is expected to increase its efficiency as a composition for tissue regeneration using adipose stem cells for correcting cosmetic and degenerative defects in skin, bone, soft tissue, dental tissue, etc. of an individual. Medicines such as wrinkle improvement and treatment, wound healing, burn, scar, skin improvement and treatment through various growth factors included in the composition for tissue regeneration using adipose stem cells and fat stem cells having tissue specific differentiation, It is expected to be usefully applied to quasi-drugs and cosmetics.

Claims (12)

An injection composition for tissue regeneration comprising a culture medium in which human adipose stem cells are cultured for at least one day in a serum-free medium containing adipose stem cell activating compounds. The composition of claim 1, wherein the adipose stem cell activating compound comprises an antioxidant, a vitamin or an analog thereof, an amino acid or an analog thereof and a mineral. The composition of claim 2, wherein the adipose stem cell activating compound further comprises a growth factor or an analog thereof and collagen or an analog thereof. The method of claim 2, wherein the antioxidant is L-lipoic acid, coenzyme Q-10, C-MED 100, retinoic acid, vinpocetin, piccamlon, quinic acid, quinate, adenine dinucleotide, acetyl-L-carnitine, dimethylani A composition comprising one or more substances selected from the group consisting of parent ethanol or L-hydroxy acids. 3. The composition of claim 2, wherein the vitamin or analog thereof comprises one or more substances selected from the group consisting of A, B, C, D or Vitamin E. The composition of claim 2, wherein the amino acid or analog thereof comprises at least one substance selected from the group consisting of L-Alanine, L-Asparitic acid, L-Histdine, L-leucine, L-glutamine and the like. The method of claim 3, wherein the growth factor or analog thereof comprises one or more substances selected from the group consisting of vascular endothelial cells, hepatocellular growth factors, transformation factors, platelet derived growth factors, keratin sulfate, fibroblast growth factor, cadherin, and the like. A composition, characterized in that. The composition of claim 3, wherein the collagen or analogue thereof comprises at least one substance selected from the group consisting of I to XIII, XVII and the like and fibronectin. The composition of claim 1, wherein the serum-free medium containing the cell activating compound is a serum-free medium containing Ham's F-12 nutrient mixture. The composition of claim 9, wherein the medium further contains an antioxidant or an analog thereof, a growth factor or an analog thereof, and collagen or an analog thereof. A method of regenerating skin tissue by injecting a tissue composition with an adipose stem cells into a skin tissue with an injection composition for regeneration of tissue containing a culture solution of human adipose stem cells in serum-free medium containing a cell activating compound. Antioxidants, vitamins or analogs thereof, amino acids or analogues thereof, growth factors or analogues thereof, collagen or analogues thereof, and Injection composition containing minerals A method of regenerating skin tissue by injecting it into the skin tissue along with fat stem cells.

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