JP2016079177A - Composition which promotes growth of dermal papilla cell, method for manufacturing the same, and medicinal drug containing the composition - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a composition which promotes growth of a dermal papilla cell, a method for manufacturing the composition, and a medicinal drug containing the composition.SOLUTION: A method for manufacturing a composition has: a step of preparing an umbilical cord of swine; a step of cleaning the umbilical cord by cleaning fluid in order to completely remove hemocyte and biological fluid; a step of isolating a cell from the cleaned umbilical cord, performing passage culture, and obtaining cytokine; a step of repeating a freeze-thaw cycle at least for twice or more to the cytokine and the cell, and obtaining a polypeptide mixture; a step of conducting concentration and demineralization to the polypeptide mixture, and obtaining a composition having molecular weight larger than 3000 daltons.SELECTED DRAWING: Figure 1B

Description

The present invention relates to a method for producing a composition for promoting the growth of dermal papillary cells, which has a specific component obtained by culturing fibroblast-like cells derived from umbilical cord tissue.
Another aspect of the present invention relates to a composition that promotes the growth of dermal papilla cells.
Yet another aspect of the present invention relates to a pharmaceutical comprising an effective amount of the composition that promotes the growth of dermal papilla cells.

  Conventional techniques include stem cell factor (SCF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), culturing epithelial cells or mesenchymal stem cells. A growth factor such as insulin-like growth factor (IGF) is obtained.

However, the above prior art has the following problems.
Since the conventional freezing process is performed gradually, the time cost increases and the production amount per unit time decreases.
Moreover, since concentration and desalting have not been performed, a complex culture process has been performed, but the salts contained in the medium used in the culture process can irritate the skin and hair.

Therefore, the present invention has been filed and an object of the present invention is to provide a method for producing a composition that promotes the growth of dermal papilla cells.
The production method according to the present invention destroys cells in a freeze-thaw cycle using liquid nitrogen, collects a cell lysate, extracts a large amount of polypeptide or protein cocktail, A composition having a specific molecular weight is obtained by concentration and desalting.

  The invention of claim 1 of the present application includes a step of providing a swine umbilical cord tissue, a step of washing the umbilical cord with a washing solution to completely remove blood cells and bodily fluids, and separating cells from the washed cord. Performing subculture, obtaining cytokines, repeating freeze-thaw cycles for cytokines and cells at least twice to obtain a polypeptide mixture, and enriching and desalting the polypeptide mixture And obtaining a composition having a molecular weight greater than 3000 Daltons, and a method for producing a composition that promotes the growth of dermal papilla cells.

  In the invention of claim 2 of the present application, the composition obtained in the step of concentrating and desalting the polypeptide mixture to obtain a composition having a molecular weight greater than 3000 Daltons is derived from angiogenin, platelets The method for producing a composition for promoting dermal papilla cell growth according to claim 1, wherein the composition is selected from the group consisting of a growth factor, a fibroblast growth factor, and a combination thereof. provide.

  In the invention of claim 3 of the present application, in the step of separating cells from the washed umbilical cord, performing subculture, and obtaining cytokines, the cells separated from the umbilical cord in a serum-free medium for 3 days to The method for producing a composition for promoting the growth of dermal papilla cells according to claim 1, wherein cytokine is obtained by culturing for 18 days.

  In the invention of claim 4 of the present application, in the step of separating cells from the washed umbilical cord, performing subculture, and obtaining cytokines, the cells separated from the umbilical cord are cultured in a serum-free medium for 6 days. Thus, a method for producing a composition for promoting the growth of dermal papilla cells according to claim 1, characterized in that a cytokine is obtained.

  The invention according to claim 5 of the present application is the composition for promoting dermal papilla cell growth according to claim 1, wherein the umbilical cord tissue of the pig is an umbilical cord tissue derived from a pig free of a specific pathogen. A manufacturing method is provided.

  Invention of Claim 6 of this application is manufactured by the manufacturing method as described in any one of Claim 1 thru | or 5, and has an effect which accelerates | stimulates the growth of a dermal papilla cell, It is characterized by the above-mentioned. A composition that promotes the growth of

  The invention according to claim 7 of the present application is selected from the group consisting of angiogenin, platelet-derived growth factor, fibroblast growth factor, and combinations thereof, Compositions that promote papillary cell growth are provided.

  The invention of claim 8 of the present application has the effect of increasing the growth rate of dermal papilla cells to which the composition is applied by 5% to 50% compared to the growth rate of dermal papilla cells to which the composition is not applied. A composition for promoting the growth of dermal papilla cells according to claim 6, characterized by comprising:

  The invention according to claim 9 of the present application comprises an effective amount of the composition according to claim 7 or claim 8 and a pharmaceutically acceptable solvent and / or excipient, There is provided a pharmaceutical comprising a composition that promotes cell growth.

  The composition for promoting dermal papilla cell growth according to claim 9, wherein the effective amount of the composition is 0.05 ng / ml to 20 ng / ml. Pharmaceutical products containing products.

The “cleaning solution” as used in the present invention is a solution that is isotonic with the umbilical cord of pigs.
The washing solution referred to in the present invention may be, for example, a 90% sodium chloride solution or a phosphate buffered saline (PBS).

In the present invention, “cell separation” means, for example, 4 to 6 pieces of umbilical cord slices cut from a pig's umbilical cord, for example, 10 ml of growth medium composed of alpha MEM medium and 10% fetal bovine serum. After cultivating the culture dish, the culture dish containing the umbilical cord strip is cultured in a constant temperature incubator. On the 10th day of the culture, the umbilical cord strip is removed from the growth medium of the culture dish, for example, 3 To obtain cultured cells by adding milliliters of fresh growth medium.
In addition, the constant temperature incubator is preferably an incubator having, for example, a 5% carbon dioxide atmosphere.

In the present invention, “passaging” means that when cultured cells become dense, the cells are collected and diluted, and then the diluted cells are continuously cultured from a low density to a culture dish having a new medium. It is to be.
The dilution ratio varies depending on the cell type.

The term “freeze-thaw cycle” as used in the present invention refers to exerting an effect of destroying a cell membrane by repeating freeze-thaw of cytokines and cells with liquid nitrogen and then thawing at room temperature a plurality of times.
For example, a vial holding cells is immersed in liquid nitrogen and frozen, and then the frozen vial is thawed at least twice in a 37 ° C. water bath, and the broken cells are further removed at 1000 G. You may centrifuge for 15 to 20 minutes.

The term “concentration and desalting” as used in the present invention means that the solution containing the polypeptide mixture is filtered and the supernatant is removed, thereby reducing the volume of the solution containing the polypeptide mixture and increasing the concentration. At the same time, it is to remove salts.
For example, the solution containing the polypeptide mixture is filtered with an ultrafiltration device or a centrifugal filtration device, and the volume of the solution containing the polypeptide mixture is reduced, the concentration is increased, and impurities such as salts are removed. Compositions having a molecular weight greater than Dalton (Da) can be obtained.

  As used herein, “pig umbilical cord tissue” refers to umbilical cord tissue derived from a specific pathogen free (SPF) pig.

  The “effect of promoting the growth of dermal papilla cells” as used in the present invention refers to the dermis to which the composition according to the present invention is applied compared to the growth rate of dermal papilla cells to which the composition according to the present invention is not applied. The growth rate of papillary cells is increased by 5% to 50%.

The “pharmaceutically acceptable solvent and / or excipient” as used in the present invention is a solvent / excipient suitable for internal use or external use for humans or animals. For example, the pharmaceutically acceptable solvent and / or excipient may be an aqueous ethanol solution, water, physiological saline.
Further, the pharmaceutically acceptable solvent and / or excipient is water or physiological saline, and the addition amount of the solvent and / or excipient maintains an effective amount of the composition according to the present invention. This is something that can be done.

In the method for producing a composition for promoting the growth of dermal papilla cells according to the present invention, cytokines can be recovered by culturing cells for a predetermined number of days using a serum-free medium.
Further, by repeating freeze-thaw cycles, cells can be destroyed to obtain a polypeptide mixture, and a composition having a specific molecular weight can be obtained by concentration and desalting.
The obtained composition has an effect of promoting the growth of dermal papilla cells. Specifically, the growth rate of dermal papilla cells to which the composition of the present invention is applied is increased by 5% to 50% compared to the growth rate of dermal papilla cells to which the composition of the present invention is not applied. .

It is a photograph which shows the state of the first day in the experiment in which the composition which concerns on this invention is given to a test subject's forehead and hairline. It is a photograph which shows the state which continued the experiment for which the composition which concerns on this invention was given to the test subject's forehead and the hairline of hair for two months.

  Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the drawings.

[Production Example 1]
This production example relates to separating cells from pig umbilical cord.

With a pig umbilical cord, the umbilical cord is washed with 75% ethanol for 20-30 seconds and further washed with PBS to completely remove blood cells and fluids. The washed umbilical cord is divided into three or four equal parts and placed on a sterilizer dish.
Furthermore, after the umbilical cord is incised along the vein with a scalpel or tweezers, the arteries and veins are removed so that blood is not applied to the Howton jelly while the incised umbilical cord is spread with two tweezers. .
The Wharton jelly is transferred from the umbilical amniotic membrane to fresh alpha MEM medium so that the wettability of the Wharton jelly is maintained.
An umbilical strip is obtained by cutting and tearing the umbilical cord from which the artery, vein and Wharton's jelly have been removed with a surgical scissors.
The 4 to 6 umbilical cord strips are seeded on a culture dish containing 10 ml of a growth medium composed of alpha MEM medium and 10% fetal calf serum, and then the culture dish containing the umbilical cord strips is divided into 5 Cells are cultured in the culture dish so that they are stored in an incubator with a% carbon dioxide atmosphere and 3 ml of the growth medium is added to the culture dish twice a week. On day 10 of culture, the umbilical cord strips are removed from the culture medium in the culture dish, the cells in the culture dish are washed out with PBS, then fresh growth medium is added, and the cells are cultured to a state of 80% to 90% confluence. .

[Production Example 2]
This production example relates to subculture of cells.

The growth medium is removed from the culture dish holding the cells cultured to 80% to 90% confluence with an aspirator, and then the cells held in the culture dish are washed away with 5 ml of PBS.
PBS is removed and trypsin-EDTA at a concentration of 0.25% is added to the culture dish.
The culture dish to which the trypsin-EDTA has been added is allowed to stand at 37 degrees Celsius for 5 minutes, and then the separated cells are transferred to a 15-ml centrifuge tube.
After centrifugation at 400 G for 5 minutes, the supernatant is removed, 2 ml of growth medium is added and the cells are resuspended to prepare a cell suspension.
Cells are counted with an automatic cell counter with respect to a mixed solution obtained by mixing 10 microliters of the cell suspension and 10 microliters of trypan blue.

[Production Example 3]
This production example relates to the production of cytokines.

To produce cytokines, the cells obtained in Preparation Example 2, was inoculated for cytokine production culture dish, each cytokine-producing culture dish, from the cultured until cell numbers is 3x10 ⁵ to 7x10 ⁵ In a carbon dioxide atmosphere incubator, change the growth medium at 37 degrees Celsius twice a week, ie, every third or fourth day, and culture until 90% confluent.

  The cells cultured to 90% confluence are washed twice with 5 ml of PBS, and 8 ml of serum-free medium is added to each cytokine-producing culture dish. Incubate for 6 days in an atmospheric incubator to obtain cytokines.

[Production Example 4]
This production example relates to the recovery of a polypeptide mixture.

Some cytokines are released into the medium, while other cytokines remain in the cells without being released into the medium.
The medium is collected, and cells attached to the surface of the culture dish for cytokine production are treated with trypsin-EDTA having a concentration of 0.2% added to the culture dish at 37 degrees Celsius for 5 minutes. The cells detached by trypsin-EDTA treatment are collected, centrifuged at 40 G for 5 minutes, the supernatant is removed, and the precipitated cells are resuspended in 10 ml of PBS. Then re-centrifuge and remove the supernatant, then resuspend the cells in 3 ml PBS.
The resuspended cells are transferred to a freezing vial and immersed in liquid nitrogen and frozen.
Thaw frozen vials in a 37 ° C water bath.
Cells are destroyed by repeating the freeze-thaw cycle twice.
The disrupted cells are centrifuged at 1000 G for 15 to 20 minutes, and then the supernatant is collected as a solution containing the polypeptide mixture, and the cell lysate suspended in the supernatant is recovered to obtain the total output. Improve.
In addition, the concentration of the polypeptide mixture in the supernatant is measured by enzyme-linked immunosorbent assay (ELISA), for example, for FGF7 or PDGF.

[Production Example 5]
This production example relates to the concentration and desalting of polypeptide mixtures.

By concentrating the solution containing the polypeptide mixture, a specific concentration of the polypeptide can be obtained. The supernatant liquid described in Production Example 4 and the cell lysate suspended in the supernatant liquid are subjected to ultrafiltration equipment (stirred cell, Amicon, USA) or centrifugal filtration device (filter centrifugation device, USA, Amicon). Is used to reduce the volume and increase the concentration of the solution containing the polypeptide mixture to obtain a polypeptide mixture concentrate.
The polypeptide mixture concentrate is transferred from the ultrafiltration device or centrifugal filtration device to a collection tube and stored. Two milliliters of the polypeptide mixture concentrate may be taken and the concentration measured.

[Production Example 6]
This production example relates to lyophilization of a polypeptide concentrate.

50 ml of the concentrated polypeptide mixture prepared in Preparation Example 5 is placed in a chuck bag and frozen at −80 ° C. overnight (12 to 16 hours).
The freeze-dried apparatus is subjected to a freeze-drying process for 4 to 5 days with respect to the frozen polypeptide mixture concentrate held in the chuck bag by a freeze-drying apparatus.
The lyophilized polypeptide mixture concentrate is resuspended in sterile water to obtain a polypeptide solution.
The polypeptide solution can be filtered through a 0.22 μm membrane filter to obtain the composition according to the present invention.
The composition is stored at -80 degrees Celsius.
The composition also comprises 55 ng / ml angiogenin, 6 ng / ml platelet-derived growth factor (PDGF) and 4 ng / ml fibroblast growth factor-7 (FGF7). Have

  This example relates to promoting hair growth.

A microneedle is used to create a fine wound having a depth of 0.2 mm to 0.25 mm at the site of administration of the subject's skin, and the composition obtained in Production Example 6 is applied to the administration site at a single dose. Make 1 milliliter and give a total of 7 doses. The frequency of administration is to administer once every one to two weeks.
As shown in FIG. 1A, when the composition obtained in Production Example 6 is administered to the heel portion of the subject's forehead, as shown in FIG. 1B, the dermal papilla cells constituting the hair follicle are the main growth factor of hair. Since it is a cell, two months after administration of the composition according to the present invention, the growth of the dermal papilla cells not only promotes new hair growth, but also increases the thickness of the newly grown hair. be able to.

In the method for producing a composition for promoting the growth of dermal papilla cells according to the present invention, cytokines can be recovered by culturing the cells for a predetermined number of days using a serum-free medium.
Further, by repeating freeze-thaw cycles, cells can be destroyed to obtain a polypeptide mixture, and a composition having a specific molecular weight can be obtained by concentration and desalting.
The obtained composition has an effect of promoting the growth rate of dermal papilla cells. Therefore, the present invention has industrial applicability.

Claims (10)

Providing porcine umbilical cord tissue;
Washing the umbilical cord with a washing solution to completely remove blood cells and body fluids;
Separating the cells from the washed umbilical cord, performing subculture, and obtaining cytokines;
Repeating a freeze-thaw cycle for cytokines and cells at least twice to obtain a polypeptide mixture;
Producing a composition that promotes the growth of dermal papilla cells, comprising: concentrating and desalting the polypeptide mixture to obtain a composition having a molecular weight greater than 3000 Daltons. Method.   The composition obtained in the step of subjecting the polypeptide mixture to concentration and desalting to obtain a composition having a molecular weight greater than 3000 Daltons comprises angiogenin, platelet derived growth factor, fibroblast growth factor, The method for producing a composition for promoting dermal papilla cell growth according to claim 1, wherein the composition is selected from the group consisting of a combination thereof and a combination thereof.   In the step of separating cells from the washed umbilical cord, performing subculture and obtaining cytokines, the cells separated from the umbilical cord are cultured in a serum-free medium for 3 to 18 days, thereby producing cytokines. The method for producing a composition for promoting dermal papilla cell growth according to claim 1, wherein   In the step of separating cells from the washed umbilical cord, performing subculture and obtaining cytokines, culturing the cells separated from the umbilical cord in a serum-free medium for 6 days to obtain cytokines The method for producing a composition for promoting the growth of dermal papilla cells according to claim 1, wherein   The method for producing a composition for promoting the growth of dermal papilla cells according to claim 1, wherein the umbilical cord tissue of pig is umbilical cord tissue derived from a specific pathogen-free pig.   A composition for promoting the growth of dermal papilla cells, which is produced by the production method according to any one of claims 1 to 5 and has an effect of promoting the growth of dermal papilla cells.   The composition for promoting the growth of dermal papilla cells according to claim 6, wherein the composition is selected from the group consisting of angiogenin, platelet-derived growth factor, fibroblast growth factor, and combinations thereof. .   The growth rate of dermal papilla cells to which the composition is applied is increased by 5% to 50% compared to the growth rate of dermal papilla cells to which the composition is not applied. The composition according to 6, which promotes the growth of dermal papilla cells.   A composition for promoting the growth of dermal papilla cells, comprising an effective amount of the composition according to claim 7 or claim 8 and a pharmaceutically acceptable solvent and / or excipient. Contains pharmaceuticals.   The pharmaceutical composition containing the composition for promoting the growth of dermal papilla cells according to claim 9, wherein an effective amount of the composition is 0.05 ng / ml to 20 ng / ml.