• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    The present invention provides a skin care composition and a pharmaceutical combination thereof. The manufacturing process of the skin care composition is based on culturing cells on a serum-free medium for a certain number of days to obtain cytokines. The recovered cells are damaged by using multiple freeze-thaw cycles to obtain more polypeptide mixtures, which are further concentrated and desalted to obtain a composition having a certain molecular weight, which composition has a skin collagen production stimulating effect.
    公开号:CH710255A2
    申请号:CH01484/15
    申请日:2015-10-14
    公开日:2016-04-15
    发明作者:Tsai Cheng-Hsien；Sieber Martin
    申请人:Bionet Copr；
    IPC主号:
    专利说明:

    BACKGROUND OF THE INVENTION
    1. Field of the invention
    The present invention relates to a manufacturing method of a skin care composition, and more particularly to a manufacturing method for obtaining a composition containing certain components of the tissue culture of umbilical cord. The present invention also relates to a skin care composition having a skin collagen production stimulating activity.
    2. Description of Related Art
    Considering the conventional technologies, the growth factors such as Stern Cell Factor (SCF, Stem Cell Factor), Vascular Endothelial Growth Factor (VEGF), Epidermal Growth Factor (EGF, Epidermal Growth Factor) and Insulin-like Growth Factor (IGF, insulin-like growth factor) obtained by a method such as culturing epithelial cells and mesenchymal stem cells. The conventional methods require gradually decreasing temperatures for conventional freezing, which is time consuming. Furthermore, the salts contained in the media in a subsequent culture process can cause irritation of the skin and hair.
    SUMMARY OF THE INVENTION
    Without taking advantage of the use of the freeze-thaw cycle method and concurrent concentration and desalting, the production yield is low and the process is cumbersome and time consuming when using the conventional technologies. An object of the present invention is to provide a manufacturing method for a skin care composition, which method includes a step of using liquid nitrogen freeze-thaw cycles to damage cells with the purpose. To collect cell lysate resulting in an increased yield of a polypeptide mixture (or protein cocktail). A concentration and desalting process is used to obtain a composition having a specific molecular weight.
    In order to achieve the aforementioned object, the present invention provides a manufacturing method for a skin care composition, the method comprising: providing a porcine umbilical tissue; Washing the swine umbilical tissue with a wash solution to remove blood cells and body fluid from the swine umbilical tissue; Isolating cells from the porcine umbilical tissue for sub-culture to obtain cytokines; Subjecting the cells and the cytokines to at least two freeze-thaw cycles to obtain a polypeptide mixture; Concentrating and desalting the polypeptide mixture to obtain the composition, wherein the molecular weight of the composition is greater than 3000 Da (daltons).
    According to the present invention, "the washing solution" refers to an isotonic solution of the pig-hive 1-lacing tissue. Preferably, the wash solution is a 90% sodium chloride solution or phosphate buffered saline (PBS).
    According to the present invention, "cell isolation" refers to cutting the porcine umbilical cord into small pieces as umbilical cord fragments, and placing 4 to 6 umbilical cord fragments in a culture dish containing 10 ml growth medium (with α-MEM and 10%). The culture dish is then placed in a constant-temperature container to culture for 10 days with the addition of 3 ml of culture medium twice a week to obtain cells, and then the umbilical fragments are removed the container with constant temperature refers to a CO2 incubator (with 5% CO2).
    According to the present invention, "subculturing" refers to reducing cell density to maintain cell growth by collecting cells at the highest density of cell growth and then diluting the cells to inoculate a new culture dish. The dilution ratio depends on the cell types.
    According to the present invention, "freeze-thaw cycles" refer to placing the cells and the cytokines in liquid nitrogen for freezing, and then placing the cells at room temperature for thawing, in repetitive cycles, to determine the effect of the cells Damaging the cells to reach. As shown in the embodiments of the present invention, a cryogenic vial containing the cells is dipped directly into liquid nitrogen and then the cryogenic vial containing the cells is removed from the liquid nitrogen and placed in a 37 ° C water bath to thaw. The above steps are repeated for at least 2 cycles. The damaged cells are centrifuged for 15 to 20 minutes at 1000G.
    In accordance with the present invention, "concentration and desalting" refers to placing the polypeptide mixture in a filtration apparatus and then subjecting the filtration apparatus to centrifugation to remove supernatant to achieve concentration of the polypeptide mixture and removal of the salts. As shown in embodiments of the present invention, to reduce the volume of the polypeptide mixture, the polypeptide mixture is placed in an ultra-filtration apparatus or in a centrifugation apparatus to increase the concentration of the polypeptide mixture and to remove impurities (e.g., salts and the like). Subsequently, a composition is obtained which has a molecular weight of greater than 3000 Da (daltons).
    Preferably, the composition comprises one or more ingredients selected from the group consisting of Basic Fibroblast Growth Factor (bFGF, basic fibroblast growth factor), Platelet-derived Growth Factor (PDGF, platelet derived growth factor), Transforming Growth Factor beta-1 ( TGF-β1, transforming growth factor beta-1) and a combination thereof.
    In the step of isolating cells from the porcine umbilical tissue for sub-culture to obtain cytokines, it is preferable to cultivate the isolated cells for 3 to 18 days on a serum-free medium to obtain the cytokines.
    In the step of isolating cells from the porcine umbilical tissue for sub-culture to obtain cytokines, it is preferable to cultivate the isolated cells for 12 days on a serum-free medium to obtain the cytokines.
    According to the present invention, "porcine umbilical tissue" refers to the umbilical tissue obtained from a specific pathogen-free (SPF) pig.
    The present invention further provides a composition obtained from the aforementioned production method, wherein the composition has a skin collagen production-stimulating effect.
    According to the present invention, "stimulating the production of skin collagen" refers to the presentation in the embodiments of the present invention, according to which the effect of the composition to an increased production of collagen by 0.5 to 3.5 times, compared with the control groups, leads.
    The present invention further provides a pharmaceutical combination comprising an effective amount of the aforementioned composition and the pharmaceutically acceptable solvent and / or carrier thereof.
    Preferably, the aforementioned effective amount of the composition is in a range of 0.05 ng / ml to 20 ng / ml.
    According to the present invention, "pharmaceutically acceptable solvents and / or carriers" refer to any solvent which can be used for oral or external human or animal use, such as alcohol-water co-solvents, water and physiological saline. Preferably, the pharmaceutically acceptable solvent and / or the carrier is water or physiological saline. The amount of solvent added is based on maintaining the effectiveness of the amount of composition.
    The production method of the present invention is based on culturing cells for a certain number of days on a serum-free medium to obtain cytokines. The cells are damaged by repeated use of freeze-thaw cycles with liquid nitrogen to obtain a greater amount of polypeptide mixtures, which are further concentrated and desalted to yield a composition having a specific molecular weight, the composition of which is the production of skin collagen has stimulating effect.
    BRIEF DESCRIPTION OF THE DRAWINGS
    [0020]<Tb> FIG. 1 <SEP> is a bar graph showing the results of using the composition of the present invention in a cell proliferation experiment.<Tb> FIG. 2 <SEP> is a bar graph showing the results of using the composition of the present invention in an MTS trial.Figs. 3 (a) to 3 (d) <SEP> show the results of using the composition of the present invention in a wound healing assay by taking pictures of cells. In Fig. 3 (a), DO stands for the day zero of the experiment, i. drawing a line in the culture dish containing the cells. In Fig. 3 (b), D3 stands for day 3 of the experiment. SFM stands for serum-free media. Figures 3 (c) and 3 (d) show photographs of the cells after addition of the composition of the invention to the culture media, at concentrations of 0.055 ng / ml and 0.11 ng / ml, respectively, for 3 days.<Tb> FIG. Figure 4 <SEP> is a bar graph showing the results of using the composition of the present invention in the attempt to increase the production of skin collagen, the X-axis showing the test dilution of bFGF.
    DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
    The following specific examples are used to illustrate the present invention. One skilled in the art can readily think of the other advantages and effects of the present invention. The present invention may also be adapted for other specific cases permitted for use. The details of the instructions may also be based on different perspectives and uses, with various modifications and changes that do not depart from the spirit of the present invention.
    EXAMPLE
    Production Example 1: Isolating cells from porcine umbilical cord
    A pig umbilical cord was purchased. The porcine umbilical cord was washed for 20 to 30 seconds with 75% ethanol and was subsequently washed with phosphate buffered saline (PBS). Then the swine umbilical cord was diced into 3 to 4 equal pieces and placed in a sterilized culture dish. The umbilical cord was opened by means of a scalpel or tweezers along the umbilical vein. Two tweezers were used to pull and open the umbilical cord. The arteries were gently removed from the umbilical cord without injecting blood onto the Wharton brawn (i.e., a gelatinous connective tissue within the umbilical cord). In the meantime, the veins were removed as well. The Wharton-Sulze block was removed from the cord amnion and placed in a fresh culture dish containing α-MEM to keep it moist. Subsequently, the porcine umbilical cord was diced by means of surgical scissors into small pieces in the form of umbilical cord fragments, and 4 to 6 umbilical cord fragments were placed in a culture dish containing 10 ml of growth medium (with α-MEM and 10% fetal calf serum (FBS)). The culture dish was then placed in a CO2 incubator (with 5% CO2), with 3 ml of culture media added twice a week. Subsequently, the umbilical fragments were removed after 10 days, and the dish was washed with PBS and refilled with fresh media. The cells were grown to reach 80% to 90% confluency and then sub-cultured for cell proliferation.
    Production Example 2: Subculturing of cells
    A vacuum was used to remove the media. The cells in the dish were washed with 5 ml PBS. The PBS was then removed. Subsequently, 3 ml of a 0.25% trypsin-EDTA solution was added to each culture dish. The culture dishes were incubated at 37 ° C for 5 minutes. The isolated cells were collected in a 15 ml centrifuge tube to be centrifuged for 5 minutes at 400 G, and then the supernatant was removed. Subsequently, 2 ml of medium was added to the tube and the cells were resuspended by means of a pipette. 10 μl of cell suspension were removed and mixed with 10 μl of trypan blue to count the number of cells by means of an automatic cell counter.
    Production Example 3: Production of cytokines
    To obtain cytokines, the cells prepared in Preparation Example 2 were inoculated in a culture dish to grow the cells to a density of 3 × 10 5 to 7 × 10 5 cells per dish. The cells were cultured at 37 ° C in a CO2 incubator with changing media (twice a week, i.e., approximately every 3-4 days) until cell growth reached 90% confluency.
    When cell growth reached 90% confluence, the cells were washed twice with 5 ml PBS. Then, 8 ml of serum-free medium was added to each culture dish. The dishes were incubated at 37 ° C in a CO2 incubator for 12 days to obtain cytokines.
    Preparation Example 4: Collection of Polypeptide Mixtures
    Some cytokines were released into the culture media and others remained in the cells. The culture media were collected. The adherent cells were treated with 3 ml of 0.2% trypsin-EDTA solution at 37 ° C for 5 minutes and then collected for centrifugation at 40 G for 5 minutes. The supernatant was removed. The cells precipitated by centrifugation were washed once with 10 ml of PBS. The cells were recentrifuged to remove the supernatant and were then resuspended in 3 ml of PBS. The cell suspensions were transferred to cryogenic vials. The cryogenic vials containing the cells were immersed directly in liquid nitrogen and then thawed in a 37 ° C water bath. The aforementioned freeze-thaw cycle was repeated twice to destroy the cells. The disrupted cells were centrifuged at 1000 G for 15-20 minutes. The collected supernatants and cell lysates were mixed to obtain the mixtures of polypeptide considered for overall yield. The concentrations of the supernatants or the polypeptide-containing mixtures were measured by ELISA (Enzyme Linked Immunosorbent Assay).
    Production Example 5: Measurement of the concentration of the polypeptide mixture and desalting
    It was necessary to concentrate the polypeptide mixtures to obtain the polypeptide in certain concentrations. To obtain the concentrated polypeptide mixtures, the resulting supernatants and cell lysates from Preparation Example 4 were placed in an ultra-filtration apparatus (model number: Amicon Stir Cell) or a centrifugation apparatus (Amicon Filter Centrifugation Device) to reduce the volumes of the polypeptide mixtures and their concentrations enlarge. The concentrated polypeptide mixture was transferred to a collection tube. 2 ml of concentrated mixture was taken to determine the concentration of the polypeptide mixture.
    Production Example 6: Freeze-drying of polypeptide mixtures
    50 ml of the concentrated polypeptide mixtures obtained from Preparation Example 5 were placed in a zippered bag at -80 ° C overnight (12 to 16 hours). To obtain the lyophilized powder of the polypeptide mixture, the concentrated polypeptide mixture bag was placed in the freeze dryer for 4-5 days to lyophilize the polypeptide mixtures. The lyophilized polypeptide mixture powder was resuspended with sterilized water to obtain a bFGF (Basic Fibroblast Growth Factor) solution at the final concentration of 10 ng / ml. The bFGF solution was filtered through a 0.22 μm filter and stored at -80 ° C. The concentration was adjusted on the basis of the bFGF concentration. TGF-β1 is at a concentration of 168 pg / ml. PDGF is at a concentration of 6 ng / ml.
    Production Example 7: The Measurement of MTS Cell Proliferation
    The MTS (3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium) reagent was obtained from Promega (CellTiter 96 <® AQueous One Solution Cell Proliferation Assay). The measuring steps involved adding 20 μl of the above reagent to 100 μl of medium used for culturing the cells for 4 days. The mixed solution was placed in a 5% CO2 incubator at 37 ° C for 1.5 hours, and the absorbance was measured at 450 nm by means of a spectrophotometer to examine the proliferation of the living cells.
    Preparation Example 8: Stimulating collagen production
    The reagent used was taken from the Procollagen Type 1 C Peptide (PIP) EIA Kit. A 100 μl antibody-POD conjugated solution was added to a 96-well immunoassay microtiter plate, followed by addition of the samples to be tested into each respective well of the 96-well immunoassay microtiter plate for reaction at 37 ° C for 3 hours. Subsequently, the 96-well immunoassay microtiter plate was washed four times with 400 μl of PBS and a 100 μl substrate solution was added to the reaction for 15 minutes. Then 100 μl of a stop solution was added and the absorbance was measured at 450 nm.
    Embodiment 1: Cell proliferation experiment
    5 × 10 4 human fibroblasts were cultured in a 6-well cell culture dish for 4 days. Then, the medium was replaced by the bFGF-containing medium obtained in Preparation Example 6 at the dilution concentrations of 0.055 ng / ml and 0.11 ng / ml. On the seventh day of cell cultivation, the cells were treated with trypsin to dissolve them from the culture dish. Cell numbers were counted and the results are shown in FIG. When the 0.11 ng / ml composition was used, the proliferation of human fibroblasts increased by 31.8% compared to the control group (indicated by "0").
    Embodiment 2: MTS trial
    2 × 10 3 human fibroblasts were cultured in a 96-well cell culture dish for 4 days. Then, the medium was replaced by the bFGF-containing medium obtained in Example 6. On the fourth day of cell culture, the cells were assayed for living cell proliferation based on the MTS cell proliferation measurement described in Preparation Example 7. The results are shown in FIG. When the 0.11 ng / ml composition was used, the proliferation of live human fibroblasts increased by 35.9% compared to the control group.
    Embodiment 3: Wound healing trial
    Cell motions were examined by means of an in vitro scratch test. 5 × 10 4 human fibroblasts were cultured in a 6-well cell culture dish for 7 days. Then, the medium was replaced by the bFGF-containing medium obtained from Production Example 6, and a line was drawn in the culture dish. Images of the cells were taken on the tenth day to document cell movement. The results are shown in Figs. 3 (a) to 3 (d). In comparison of the 0.055 ng / ml and 0.11 ng / ml composition with the control group, the human fibroblasts migrated to cover the vacancy caused by the scratch on the third day after the scratch was generated. Therefore, the bFGF solutions have a concentration of 0.055 ng / ml to 0.11 ng / ml, which increases the growth and movement of the fibroblasts.
    Embodiment 4: Collagen production experiment
    According to Preparation Example 8, the effect of increasing collagen production was tested using bFGF-containing media obtained from Preparation Example 6. The higher concentration compositions have a better effect, the preferred amount being in the range of 0.0625 ng / ml to 1 ng / ml.
    权利要求:
    Claims (10)
    [1]
    A manufacturing method for a skin care composition comprising the steps of:Providing a porcine umbilical tissue;Washing the swine umbilical tissue with a wash solution to remove blood cells and body fluid from the swine umbilical tissue;Isolating cells from porcine umbilical tissue for subculturing to obtain cytokines;Subjecting the cells and the cytokines to at least two freeze-thaw cycles using liquid nitrogen to obtain a polypeptide mixture;and concentrating and desalting the polypeptide mixture to obtain the composition.
    [2]
    The production process of claim 1, wherein the molecular weight of the composition is greater than 3000 Da (Daltons).
    [3]
    The manufacturing method of claim 1, wherein in the step of concentrating and desalting the polypeptide mixture to obtain a composition, the resulting composition comprises one or more ingredients selected from the group consisting of Basic Fibroblast Growth Factor (bFGF, basic fibroblast growth factor), Platelet Derived Growth Factor (PDGF, platelet derived growth factor), transforming growth factor beta-1 (TGF-β1, transforming growth factor beta-1) and a combination thereof.
    [4]
    The production method according to claim 1, wherein in the step of isolating cells from the porcine umbilical tissue for sub-culture to obtain cytokines, the isolated cells are placed in a serum-free medium to be allowed to grow for 3-18 days cultivate to obtain cytokines.
    [5]
    The production method according to claim 1, wherein in the step of isolating cells from the porcine umbilical tissue for sub-culture to obtain cytokines, the isolated cells are placed in a serum-free medium to culture for 12 days, to get cytokines.
    [6]
    6. The manufacturing method according to claim 1, wherein the porcine umbilical tissue is obtained from a specific pathogen-free (SPF) pig.
    [7]
    A composition obtained from any of the manufacturing processes of claims 1-6, wherein the composition has a skin collagen production stimulating activity.
    [8]
    A pharmaceutical combination comprising an effective amount of the composition of claim 7 and a pharmaceutically acceptable solvent and / or carrier thereof.
    [9]
    The pharmaceutical combination of claim 8, wherein the effective amount of the composition is in a range of 0.05 ng / ml to 20 ng / ml.
    [10]
    The pharmaceutical combination of claim 9, wherein the effective amount of the composition is in a range of 0.0625 ng / ml to 1 ng / ml.
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    同族专利:
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    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    TW103135488A|TW201613622A|2014-10-14|2014-10-14|Composition for skincare and pharmaceutical composition and preparation method thereof|
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