JP6188249B2 - Wound healing composition derived from porcine umbilical cord tissue, method for producing the same, and pharmaceutical product for wound healing containing the composition - Google Patents

Description

The present invention relates to a process for the preparation of umbilical (umbilical Cords) a wound healing composition having specific components obtained by culturing tissue-derived fibroblast-like cells, especially.
Another aspect of the present invention relates to a wound healing composition that stimulates an increase in skin collagen.

  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 thereof is to provide a method for producing a composition for skin care.
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 comprises a step of providing 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 umbilical cord, Cells separated from the umbilical cord are subcultured in serum-free medium for 12 days to obtain cytokines, and freeze-thaw cycles are repeated at least twice for cytokines and cells to obtain a polypeptide mixture And concentrating and desalting the polypeptide mixture to have a molecular weight greater than 3000 Daltons, basic fibroblast growth factor, platelet-derived growth factor, and transforming growth factor-β1. and having the steps of: obtaining a composition having, sets for wound healing from porcine umbilical tissue Method for producing an object to provide.

The invention according to claim 2 of the present application is characterized in that the umbilical cord tissue of the pig is an umbilical cord tissue derived from a pig that is free of a specific pathogen, and the composition for wound healing derived from a umbilical cord tissue of a pig according to claim 1 A manufacturing method is provided.

The invention of claim 3, produced by the production method according to claim 1 or 2, that have a stimulatory effect on the increase in skin collagen, that an active ingredient component derived from porcine umbilical tissue A wound healing composition is provided.

The invention of claim 4 of the present application is derived from porcine umbilical cord tissue, comprising an effective amount of the composition of claim 3 and a pharmaceutically acceptable solvent and / or excipient. A wound healing pharmaceutical comprising a wound healing composition is provided.

The invention according to claim 5 of the present application is for wound healing derived from pig umbilical cord tissue according to claim 4, wherein the effective amount of the composition is 0.05 ng / ml to 20 ng / ml. A wound healing medicament comprising the composition is provided.

The “cleaning solution” as used in the present invention is a solution that is isotonic with the swine umbilical cord.
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 seeding the culture dish, the culture dish containing the umbilical cord strip is cultured in a constant temperature incubator.
On the 10th day of culturing, the umbilical cord strips are removed from the growth medium of the culture dish and, for example, cells are obtained by culturing by adding 3 ml of fresh growth medium each time twice a week.
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 stimulating the increase in skin collagen” in the present invention means that the amount of increase in skin collagen by the composition according to the present invention is 0.5% compared to the control to which the composition according to the present invention is not applied. It increases by a factor of 3.5 to 3.5.

The “pharmaceutically acceptable solvent and / or excipient” as used in the present invention is a solvent / excipient suitable for human or animal internal or external use.
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 skin care 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 stimulating an increase in skin collagen.

It is a columnar graph which shows the result of the cell growth experiment to which the composition concerning the present invention is applied. It is a columnar graph which shows the result of the MTS test to which the composition concerning the present invention is applied. It is a photograph which shows the state of the cell of the wound healing experiment to which the composition which concerns on this invention is applied. The first photo labeled “D0” shows the scratch on the culture dish holding the cells on the first day of the experiment. The second to fourth photographs labeled “D3” show the situation on the third day of the experiment, and “SFM” displayed on the second photograph is a serum-free medium. That means. In addition, what is displayed on the third photograph shows the situation in which the composition according to the present invention is added to a concentration of 0.055 ng / ml, and is displayed on the fourth photograph. This shows the situation where the composition according to the present invention is added so that the concentration becomes 0.11 ng / ml. It is a columnar graph which shows the result of the collagen increase experiment to which the composition concerning the present invention is applied. The concentration of bFGF displayed on the horizontal axis is used as a reference for the dilution test.

  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 12 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 bFGF 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 10 ng / ml basic fibroblast growth factor (bFGF) solution.
The bFGF solution as the composition according to the present invention is filtered through a 0.22 μm membrane filter and stored at −80 degrees Celsius.
Further, based on the bFGF concentration, the bFGF solution contains 168 pg / ml transforming growth factor-β1 (Transforming growth factor-β1, TGF-β1) and 6 ng / ml platelet-derived growth factor (platelet-derived). growth factor (PDGF).

[Production Example 7]
This production example relates to a method for measuring cell proliferation in an MTS test.

As a reagent for the MTS test, Promega's CellTiter (registered trademark) AQueous One Solution Cell Proliferation Assay is used.
In the MTS test, 20 microliters of the reagent was added to 100 microliters of the cell culture medium on the fourth day, and the medium to which the reagent was added was added at 37 degrees Celsius and 5% carbon dioxide atmosphere. The sample is allowed to stand in an incubator for 1.5 hours, and a spectrophotometer is used to measure the proliferation rate of viable cells by measuring absorbance at a wavelength of 450 nm.

[Production Example 8]
This production example relates to a collagen increase experiment.

Procollagen Type I C-peptide (PIP) EIA kit is used as a reagent for collagen increase experiments.
First, 100 microliters of antibody-POD conjugate solution is added to each 96-well immunoassay plate.
Add 20 microliters of test per well, react at 37 degrees Celsius for 3 hours, and then wash the 96-well immunoassay plate 4 times with 400 microliters of PBS once.
To the washed 96-well immunoassay plate, 100 microliters of substrate solution is added per well, allowed to react for 15 minutes, and then 100 microliters of stop solution is added. The absorbance is measured using a meter at a wavelength of 450 nm.

  This example relates to cell proliferation experiments.

Human fibroblasts are cultured in 6-well plates at a density of 5 × 10 ⁴ / well. On the fourth day of culture, the medium is replaced with the medium obtained in Production Example 6 and containing a bFGF solution with a bFGF concentration of 0.055 ng / ml or 0.11 ng / ml, respectively.
On day 7 of culture, cells are detached from the 6-well plate by trypsin treatment, and the number of cells is counted.
As shown in FIG. 1, the proliferation rate of human fibroblasts cultured with bFGF at a concentration of 0.11 ng / ml is 31.8%, as compared with the control.

  This example relates to the MTS test.

Human fibroblasts are cultured in 96-well plates at a density of 2 × 10 ³ / well.
On the fourth day of culture, the medium is replaced with a medium containing the bFGF solution obtained in Production Example 6.
After further culturing for 4 days, cell proliferation is measured by the method described in Production Example 7 above.
As shown in FIG. 2, the growth rate of viable human fibroblasts cultured in bFGF at a concentration of 0.11 ng / ml is 35.9% compared to the control.

  This example relates to wound healing experiments.

Cell migration is measured in an in vitro scratch test. Human fibroblasts are cultured in 6-well plates at a density of 5 × 10 ⁴ / well.
On the seventh day of culture, the medium is replaced with the medium containing the bFGF solution obtained in Production Example 6 and scratches are made in the wells.
On the 10th day of culture, the state of cell migration is recorded with photographs.
As shown in FIG. 3, compared to the control, human fibroblasts cultured with bFGF at a concentration of 0.11 ng / ml on the third day after scratch can migrate and fill wounds caused by scratch.
That is, bFGF at a concentration of 0.055 ng / ml to 0.11 ng / ml has an effect of promoting fibroblast growth and migration.

  This example relates to a collagen increase experiment.

When the effect of stimulating the increase in skin collagen was measured in the medium containing the bFGF solution obtained in Production Example 6 by the method described in Production Example 8, the effect was proportional to the concentration of the bFGF solution. I understand that.
In particular, the concentration of bFGF is preferably 0.0625 ng / ml to 1 ng / ml.

In the method for producing a composition for skin care 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 stimulating an increase in skin collagen.

Claims (5)

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, subculturing the cells separated from the umbilical cord in a serum-free medium for 12 days, and obtaining cytokines;
Repeating a freeze-thaw cycle for cytokines and cells at least twice to obtain a polypeptide mixture;
The polypeptide mixture is concentrated and desalted, has a molecular weight greater than 3000 Daltons, and has a basic fibroblast growth factor, platelet-derived growth factor, and transforming growth factor-β1 A method for producing a wound healing composition derived from porcine umbilical cord tissue. The method for producing a wound healing composition derived from porcine umbilical cord tissue according to claim 1, wherein the umbilical cord tissue of the porcine is umbilical cord tissue derived from a specific pathogen-free pig. Produced by the production method according to claim 1 or 2, that have a stimulatory effect on the increase in skin collagen, characterized in that an active ingredient component derived from porcine umbilical tissue, wound healing compositions . Wounds containing the composition of claim 3 in an effective amount, a pharmaceutically acceptable and having a solvent and / or excipients, a wound healing composition derived from porcine umbilical tissue Healing medicine. Effective amount of the composition, 0.05 ng / ml to no, characterized in that a 20 ng / ml, according to claim 4, pharmaceuticals for wound healing containing the wound healing composition derived from porcine umbilical tissue .