CH710157A2 - Composition for increasing the proliferation of dermal papilla cells and the pharmaceutical combination and the manufacturing method thereof. - Google Patents

Abstract

The present invention provides a composition for increasing the proliferation of dermal papilla cells. The composition is obtained based on culturing cells for a certain number of days on a serum-free medium to obtain cytokines. The recovered cells are damaged using multiple freeze-thaw cycles to obtain a greater amount of polypeptide mixtures which are further concentrated and desalted to yield a composition having specific molecular weights, the composition containing one, skin papillary cell proliferation, at an increased rate of about 5% to 50%, has an enlarging effect.

Description

BACKGROUND OF THE INVENTION

1. Field of the invention

The present invention relates to a preparation method of a composition for increasing the proliferation of dermal papilla cells, and more particularly to a production method for obtaining a composition containing certain components of tissue culture of fibroblast-like cells of an umbilical cord tissue. The present invention also relates to a composition for increasing the proliferation of dermal papilla cells, wherein the composition has an enlarging effect on the proliferation of dermal papilla cells. The present invention also relates to a pharmaceutical combination which comprises an effective amount of the aforesaid composition to achieve the effect of increasing skin papillary cell proliferation.

2. Description of Related Art

Considering conventional technologies, growth factors such as Stern Cell Factor (SCF, Stem Cell Factor), Vascular Endothelial Growth Factor (VEGF, Epidermal Growth Factor) and Insulin-like Growth Factor (IGF, insulin-like growth factor), 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 preparation method for a composition for increasing the proliferation of dermal papilla cells, which method results in a process using liquid nitrogen freeze-thaw cycles to damage cells for the purpose of collecting cell lysate 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 composition for increasing the proliferation of dermal papilla cells, the method comprising: providing a porcine umbilical tissue; Washing the umbilical cord tissue with a washing solution to remove blood cells and body fluid from the pig-umbilical cord tissue; Isolating cells from the porcine umbilical tissue for subculturing 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 porcine umbilical 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 of growth medium (with α-MEM and 10% fetal Calf serum (FBS)). 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, with the constant temperature container placed on a CO2 incubator (with 5% CO2) refers.

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 cell membrane to reach. As shown in the embodiments of the present invention, a cryogenic vial containing the cells is dipped directly into liquid nitrogen, 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.

According to 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, concentrate the polypeptide mixture and remove the salts. As shown in the embodiments of the present invention, 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 includes one or more ingredients selected from the group consisting of angiogenin, platelet-derived growth factor (PDGF, platelet derived growth factor), fibroblast growth factor 7 (FGF7, fibroblast growth factor 7) 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 on a serum-free medium for 3 to 18 days 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 on a serum-free medium for 6 days 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 an enlarging effect on the proliferation of dermal papilla cells.

According to the present invention, "enlargement of dermal papillary cell proliferation" refers to the presentation in the embodiments of the present invention that the effect of the composition results in an increased growth rate of dermal papilla cells by 5% to 50% compared to the beginning of the trial or the control group.

The present invention further provides a pharmaceutical combination which comprises an effective amount. the aforementioned composition and a pharmaceutically acceptable solvent and / or a 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 solution. Preferably, the pharmaceutically acceptable solvent and / or the carrier is water or physiological saline. The amount of the added solvent is based on obtaining the effect of the amount of the composition.

The production method of the present invention is based on culturing cells for a certain number of days in a serum-free medium to obtain cytokines. The cells are damaged by repeatedly using freeze-thaw cycles to obtain a larger amount of polypeptide mixtures, which are further concentrated and desalted to obtain a composition having a specific molecular weight, which composition has an enlarging effect on dermal papilla cell proliferation , with an increased growth rate of dermal papilla cells by 5% to 50% compared to the beginning of the experiment.

BRIEF DESCRIPTION OF THE FIGURES

[0020] <Tb> FIG. 1A <SEP> shows images of the transition between forehead and hairline of a subject on day 0 of attempting to increase the proliferation of dermal papilla cells by applying the composition of the present invention. <Tb> FIG. Figure 1B shows images of the forehead and hairline transition of a subject about 2 months (i.e., sixtieth day) of attempting to increase the proliferation of dermal papilla cells by applying the composition of the present invention.

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 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 splashing blood on the Wharton's spleen (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 was diced into small pieces in the form of umbilical fragments by using surgical scissors, and 4 to 6 umbilical 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 medium 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 culture dishes 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 6 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 tubes. The cryogenic tubes 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 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). In order to obtain the freeze-dried powder of the polypeptide mixture, the bag of concentrated polypeptide mixture was placed in the freeze-dryer for 4-5 days for freeze-drying of the polypeptide mixtures. The lyophilized polypeptide mixture powder was resuspended with sterilized water to obtain the polypeptide mixture. The polypeptide solution was filtered through a 0.22 pm filter to obtain a composition containing 55 ng / ml angiogenin, 6 ng / ml platelet-derived growth factor (PDGF, platelet-derived growth factor), and 4 ng / ml fibroblast growth factor 7 (FGF7 , Fibroblast growth factor 7), stored at -80 ° C.

Embodiment 1: Hair growth experiment

A flying needle was used to create a minimally invasive wound (whose depth was about 0.2 mm to 2.5 mm) in a desired field of application of a subject. The composition obtained from Preparation Example 6 was applied in the desired range of application. 1 ml was applied for each application, 7 applications long. The frequency of application was once a week until every 2 weeks. The results of the application of the composition obtained from Preparation Example 6 to the transition between the forehead and the hairline of a subject are shown in FIG. 1A. As shown in FIG. 1B, the new hair grew because of the increase in proliferation of dermal papilla cells after the composition of the present invention was applied for about 2 months, since dermal papilla cells can form follicles and are one of the critical cells for hair growth. Also, the newly grown hair is thicker.

Claims (10)

A method of producing a composition for increasing the proliferation of a dermal papilla cell 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 the porcine umbilical tissue for subculturing to obtain cytokines; Subjecting the cells and the cytokines to at least two freeze-thaw cycles to obtain a polypeptide mixture; and Concentrate and desalt the polypeptide mixture to obtain the composition. The production process of claim 1, wherein the molecular weight of the composition is greater than 3000 Da (Daltons). The production 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 components selected from the group consisting of angiogenin, platelet-derived growth factor (PDGF, growth factor platelet derived), fibroblast growth factor 7 (FGF7, fibroblast growth factor 7) and a combination thereof. 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. 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 6 days, to get cytokines. 6. The manufacturing method of claim 1, wherein the porcine umbilical tissue is obtained from a specific pathogen-free (SPF) pig. A composition obtained from any one of the manufacturing methods of claims 1-6, wherein the composition has an enlarging effect on skin papillary cell proliferation. 8. The composition of claim 7, wherein the composition increases the proliferation of dermal papilla cells, with an increased growth rate of dermal papilla cells by 5% to 50%. A pharmaceutical combination for increasing the proliferation of dermal papilla cells comprising an effective amount of the composition of claim 7 or 8 and a pharmaceutically acceptable solvent and / or carrier thereof. The pharmaceutical combination of claim 9, wherein the effective amount of the composition is in a range of 0.05 ng / ml to 20 ng / ml.