WO2008088136A1

WO2008088136A1 - Long-term preservation method for skin tissue

Info

Publication number: WO2008088136A1
Application number: PCT/KR2007/006767
Authority: WO
Inventors: Jin-Young Kim; Jae-Hyoung Ahn; Seok-Beam Song; Ji-Hwa Chae; Seog-Jin Seo; Ke-Won Kang; Ho-Chan Hwang; Jung-Suk Lee
Original assignee: Hans Biomed Cor
Current assignee: Hans Biomed Cor
Priority date: 2007-01-15
Filing date: 2007-12-21
Publication date: 2008-07-24
Anticipated expiration: 2009-07-15
Also published as: KR100803384B1

Abstract

Provided is a long-term preservation method for a skin tissue and a dermal layer of a skin cell for transplantation, including: a first glycerol solution processing step of cleaning a skin tissue taken out of a skin tissue donor, immersing the skin tissue into a 45-55% glycerol solution at normal temperature, and performing stirring thereon; a second glycerol solution processing step of immersing and preserving the first glycerol solution processed skin tissue into a 65-75% glycerol solution at normal temperature! and a third glycerol solution processing step of immersing the second glycerol solution processed skin tissue into an 80-90% glycerol solution at normal temperature and continuously preserving the skin tissue at a temperature of 4°C to stabi lize the skin tissue. Therefore, any state of dehydrated skin tissue or an acellular dermal layer for transplantation preserved in the aforementioned method contains the glycerol solution instead of water. Accordingly, shapes and functions of various elements such as protein/collagen used to re-construct the skin for transplantation can be maintained, and a basement membrane for correcting re-attachment of a living endothelial cell or epithelial cell can be provided safely. In addition, a structure of an original tissue can be maintained without structural changes and damages.

Description

[DESCRIPTION] [Invention Title]

LONG-TERM PRESERVATION METHOD FOR SKIN TISSUE

[Technical Field]

The present invention relates to a method of stably preserving any state of skin tissue for a long time so as not to change a structure of an original tissue.

[Background Art]

Traditionally, a long-term preservation of the skin has been performed through quick freezing or freeze drying so that the skin is preserved in a state of a biological traumatic medicine material without activity. The quick freezing has limitations of storage and transportation and requires a high-cost freezing apparatus having physical and chemical characteristics. The freeze drying also requires a complex freeze drying apparatus. Freezing requires a freezing process (for example, at a rate of 0.5 to 5°C/min) that can be controlled and needs a cyroprotectant such as dimethyl sulfoxide (DMSO) when the donated skin tissue has to be processed as soon as possible.

The preserved skin tissue is in an inactive state, so that sterilization such as radiation and ethylene oxide gas can be applied to the preserved skin. However, radiation performed on an implant that has just taken out of a donor or an implant preserved in a frozen state may cause a breakdown of connective tissues of macromolecules or building of collagen matrices. In addition, the aforementioned processes cannot be easily controlled, so that a matrix structure of the skin may be hardened or weakened. Particularly, ethylene oxide gas is very toxic and generates toxic substances including chlorinated materials and water (see: ISO 10993-7, International Organization for Standardization, 1995). In addition, because of concern that the implant sterilized by the ethylene oxide gas may cause bad results for a patient and the ethylene oxide has potential toxicity, the use of the ethylene oxide gas is not recommended in a medical field.

In 1952, Billingham and Medawar show the fact that the skin can be preserved for a long time while maintaining an active state of the skin using a method of immersing the skin into a cyroprotectant and then slowly freezing the skin in a temperature range down to -196^°C. Since maintaining the cell activity of the skin is the most important object in the freezing preservation, the sterilization cannot be applied to the skin. Instead, the skin is disinfected by using an antibiotic or an antimicrobial. The antibiotic or the antimicrobial is effective for the bacteria removal, however, ineffective for the virus or spore removal. The skin in the active state which is frozen and preserved has been widely used for burn treatment as an implant along with an autotransplantation. However, it becomes a question whether maintaining the activity of the skin cell to be used as the implant is important, since the skin without the active state has been used for the burn treatment again. In addition, according to a record, 4.9% to 57% of the antibiotic is contaminated and disinfection is re-performed. Therefore, the sterilization or disinfection is not perfect to have a high removal rate.

For this reason, a development of skin implant without activity and factors for simple preservation, cheapness, and easy transportation are considered.

Particularly, a method of preserving the skin using a glycerol solution has been developed. This method has a simple and cheap process and can replace the preservation using the freeze drying. However, when a skin tissue taken out of a donor is immersed into 50% or more of the glycerol solution to be stabilized, the skin tissue shrinks and is damaged. In addition, at normal or higher temperature, the skin is damaged by activity of various proteases. In addition, when stirring is performed on the skin tissue for 24 hours or more, the skin tissue is physically damaged. Therefore, a development of a method of stably transporting and preserving the skin tissue without damage is required.

[Disclosure]

[Technical Problem]

The present invention provides a method of safely preserving any state of skin tissue taken out of a donor and an acellular dermal layer for transplantation in a glycerol solution for a long time so as not to change an original structure.

[Technical Solution]

According to an aspect of the present invention, there is provided a long-term preservation method for a skin tissue and a dermal layer of a skin cell for transplantation, including: a first glycerol solution processing step of cleaning a skin tissue taken out of a skin tissue donor, immersing the skin tissue into a 45-55% glycerol solution at normal temperature, and performing stirring thereon; a second glycerol solutionprocessingstepof immersing and preserving the first glycerol solution processed skin tissue into a 65-75% glycerol solution at normal temperature; and a third glycerol solution processing step of immersing the second glycerol solution processed skin tissue into a 80-90% glycerol solution at normal temperature and continuously preserving the skin tissue at a temperature of 4"C to stabilize the skin tissue.

Hereinafter, a preservation method for a skin tissue and an acellular dermal layer according to the present invention will be described in detail .

The preservation method according to the present invention includes transporting a skin tissue (step Sl) , cleaning the skin tissue (step S2), removing epidermis and cells (steps S3 and S4), and processing glycerol solutions through three steps.

<First Process> Immersion into 45-55% Glycerol Solution (Step S5)

A skin tissue including an epidermal layer and a dermal layer or including only a dermal layer is taken out of a donor and immersed into a 45-55% glycerol solution including an antibiotic and an antimicrobial at normal temperature.

Here, the donor is a person, a body, or an animal. The taken skin tissue may be managed by physical or biological factors. Therefore, in order to prevent the damage, the skin tissue is immersed into the 45-55% glycerol solution including the antibiotic and the antimicrobial at normal temperature and then stirring is performed thereon for 1 to 24 hours. When the skin tissue taken out of the donor is immersed into a 55% or more glycerol solution so as to be stabilized, the skin tissue shrinks and is damaged. In addition, above normal temperature, the skin tissue is damaged due to activity of various proteases. In addition, when the stirring is performed for 24 hours or more, the skin tissue may be physically damaged.

The 100% glycerol may be diluted with a 0.9% sodium chloride solution, a physiological saline solution being sold, a phosphate buffer solution, or water at a ratio of 50:50 v/v. The antibiotic is a material selected from the group consisting of penicillin, streptomycin, kanamycin, neomycin, bacitracin, gentamycin, and vancomycin. The antimicrobial is a material selected from the group consisting of amphotericin-B, nystatin, and polymyxin.

The aforementioned methods are also applied to the acellular dermal layer applying a technique for suppressing immunorejection. <Second Process> Immersion into 65-75% Glycerol Solution (Step S6)

After checking whether or not there is a problem with the skin tissue immersed into the 45-55% glycerol solution and the glycerol solution with the naked eye, the skin tissue is immersed into a 65-75% glycerol solution including an antibiotic and an antimicrobial at normal temperature. Here, for the glycerol solution, the 100% glycerol is diluted with a 0.9% sodium chloride solution, a physiological saline solution being sold, a phosphate buffer solution, or water at a ratio of 70:30 v/v. The skin tissue immersed into the 65-75% glycerol solution is processed at a temperature of 33^°C for three hours to guarantee proper contacts between the skin tissue and the glycerol solution. After the processing, the skin tissue and the glycerol solution are inspected with the naked eye, and stirring is performed on the skin tissue at normal temperature for 1 to 24 hours.

The antibiotic is a material selected from the group consisting of penicillin, streptomycin, kanamycin, neomycin, bacitracin, gentamycin, and vancomycin. The antimicrobial isamaterial selected from the group consisting of amphotericin-B, nystatin, and polymyxin. The aforementioned methods are also applied to the acellular dermal layer applying a technique for suppressing immunorejection. <Third Process> Immersion into 80-90% Glycerol Solution (Step S7)

After checking whether or not there is a problem with the skin tissue immersed into the 70% glycerol solution and the glycerol solution with the naked eye, the skin tissue is immersed into an 85% glycerol solution including an antibiotic and an antimicrobial at normal temperature. Here, for the glycerol solution, the 100% glycerol is diluted with a 0.9% sodium chloride solution, a physiological saline solution being sold, a phosphate buffer solution, or water at a ratio of 85:15 v/v. The skin tissue immersed into the 80-90% glycerol solution is processed at a temperature of 33°C for three hours to guarantee proper contacts between the skin tissue and the glycerol solution.

After the processing, the skin tissue and the glycerol solution are inspected with the naked eye and preserved at a temperature of 4°C continuously. The solution is crystallized at a temperature lower than 4^°C and the skin may be physically damaged. In addition, when the tissue is exposed for a long time at a temperature higher than 4°C , the tissue may be damaged due to activity of various proteases. Therefore, the preservation is performed at the temperature of 4^°C continuously.

The aforementioned methods are also applied to the acellular dermal layer applying a technique for suppressing immunorejection. Any state of dehydrated skin tissue or an acellular dermal layer for transplantation preserved in the aforementioned method contains the glycerol solution instead of water. Therefore, shapes and functions of various elements such as protein/collagen used to re-construct the skin for transplantation can be maintained, and a basement membrane for correcting re-attachment of a living endothelial cell or epithelial cell can be provided safely. In addition, the antibiotic included in the glycerol solution removes the activity of cells that causes the immunorejection and does not allow bacterial contamination, so that the tissue can be easily applied to transplantation patients and easily preserved and transported. The preservation method using the glycerol solution through the three processes can also be applied to a skin tissue with epidermis and cells. In addition, only the first and second processes maybe performed among the three processes.

[Advantageous Effects]

Any state of dehydrated skin tissue or an acellular dermal layer for transplantation preserved in the aforementioned method contains the glycerol solution instead of water. Therefore, shapes and functions of various elements such as protein/collagen used to re-construct the skin for transplantation can be maintained, and a basement membrane for correcting re-attachment of a living endothelial cell or epithelial cell can be provided safely. In addition, a structure of an original tissue can be maintained without structural changes and damages. In addition, the antibiotic included in the glycerol solution removes the activity of cells that causes the immunorejection and does not allow bacterial contamination, so that the tissue can be easily applied to transplantation patients and easily preserved and transported. In addition, the antibiotic and the antimicrobial included in the glycerol solution removes the bacterial contamination. Therefore, the method can be used to preserve the skin tissue for a long time and guarantee safety for clinical uses, and the method can be used for a long-term preservation for a tissue without physiological activation of an animal or a person.

In addition, the preservation is maintained at the temperature of 4°C, so that structures having activity lose their activity, and an immune response does not occur.

[Description of Drawings]

FIG. 1 is a flowchart of a method of preserving a skin tissue by immersing the skin tissue into glycerol solutions through steps. FIG.2 is a picture of a structure of a skin tissue of an original material of FIG. 1. FIG.3 is a picture of a structure of the skin tissue preserved in glycerol of FIG. 1.

FIG. 4 is a picture of a structure of the skin tissue after freeze drying the original material of FIG. 2.

FIG. 5 is a picture of the skin tissue preserved in a 50% glycerol solution.

FIG. 6 is a picture of the skin tissue preserved in a 70% glycerol solution.

FIG. 7 is a picture of the skin tissue preserved in an 85% glycerol solution.

[Best Mode] Hereinafter, exemplary embodiments of the present invention will be described in detail.

A skin tissue of a body acquired from the Euro Skin Bank (ESB) or tissue banks authorized in the domestic and foreign countries is immersed into a 50% glycerol solution obtained by diluting the 100% glycerol with a 0.9% sodium chloride solution at a ratio of 50:50 v/v and adding 1,000,000 IU penicillin including lmg streptomycin sulfate and chemical sodium to the diluted solution at normal temperature, and stirring is performed thereon for 5 hours. Thereafter, the tissue and the solution are inspected with the naked eye, and the skin tissue is immersed into a 70% glycerol solution obtained by diluting the 100% glycerol with a 0.9% sodium chloride solution at a ratio of 70:30 v/v at a temperature of 33^°C for 3 hours.

Thereafter, stirring is performed on the immersed skin tissue at normal temperature for 13 hours. The stirred skin tissue and the glycerol solution are inspected with the naked eye, and the skin tissue is immersed into a 85% glycerol solution (referred to as Glycerolum >, Ph.Eur .Netherland) at a temperature of 33°C for 3 hours and is transported at normal temperature.

The transported skin tissue is taken out and placed on an analysis glass of 24.5x24.5 cm so that a dermis layer is a lower portion. The skin tissue is cut into pieces having a size of 4^χl2 cm to 5x8 cm². The cut skin tissue is immersed in a bottle containing an epidermis removal solution and then stirring is performed thereon at normal temperature for 15 hours. The stirred skin tissue is taken out of the bottle and placed on a new analysis plate of 24.5x24.5 cm², and an epidermal layer is slightly held and pulled out of a dermal layer by using tweezers, and the remaining dermal layer is cleaned and the solution remaining on the dermal layer is removed.

Thereafter, the dermal layer is immersed into a bottle containing a cell removal solution and then shaking is performed thereon at normal temperature at a speed of 40±5 rpm for 15 hours. Thereafter, the solution is removed, and a 50 ml of DuIbecco's phosphate buffered saline (DPBS) from Sigma chemical company, USA is added. Shaking is then performed thereon at normal temperature at a speed of 60±5 rpm for 10 minutes, and the buffered saline is removed to produce an acellular dermal layer for transplantation. The produced acellular dermal layer is immersed into a 50% glycerol solution obtained by diluting the 100% glycerol with a 0.9% sodium chloride solution at a ratio of 50:50 v/v and adding 1,000,000 IU of penici 11 in including lmg streptomycin sulfate and chemical sodium to the diluted solution at normal temperature, and stirring is performed thereon for 24 hours. Thereafter, the tissue and the solution are inspected with the naked eye, and the acellular dermal layer for transplantation is immersed into a 70% glycerol solution obtained by diluting the 100% glycerol with a 0.9% sodium chloride solution at a ratio of 70:30 v/v at a temperature of 33°C for 3 hours. Thereafter, stirring is performed on the immersed acellular dermal layer for transplantation at normal temperature for 20 hours. The stirred acellular dermal layer and the glycerol solution are inspected with the naked eye, and the acellular dermal layer is immersed and preserved in a 85% glycerol solution (referred to as Glycerolum 85%, Ph.Eur.Netherland) at a temperature of 4^°C.

FIG.2 is a picture of a skin tissue of an original material, and FIG.3 is a picture of the skin tissue preserved in the glycerol solution. As shown in the pictures, a collagen tissue of the skin tissue preserved in the glycerol solution does not change substantially. On the other hand, FIG.4 is a picture of the freeze-dried skin tissue. As compared with FIGS. 2 and 3, it can be seen that the skin tissue preserved in the glycerol solution is in a better state than the freeze-dried skin tissue.

FIGS. 5, 6, and 7 are pictures of the skin tissues preserved in the 50%, 70%, and 85% glycerol solutions according to the aforementioned embodiments, respectively. It can be seen that the collagen structure is not significantly affected even if a concentration of the glycerol is increased.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the present invention as defined by the appended claims.

Claims

[CLAIMS] [Claim 1]

A long-term preservation method for a skin tissue comprising: a first glycerol solution processing step of cleaning a skin tissue taken out of a skin tissue donor , immersing the skin tissue into a 45-55% glycerol solution at normal temperature, and performing stirring thereon; and a second glycerol solution processing step of immersing and preserving the first glycerol solution processed skin tissue into a 65-75% glycerol solution at normal temperature.

[Claim 2]

The method of claim 1, further comprising a third glycerol solution processing step of immersing and preserving the second glycerol solution processed skin tissue in a 80-90% glycerol solution at normal temperature.

[Claim 3]

A long-term preservation method for a skin tissue comprising: a step of cleaning a skin tissue taken out of a skin tissue donor; a step of removing epidermis from the cleaned skin tissue; a step of removing cells from the skin tissue from which the epidermis is removed; a first glycerol solution processing step of immersing the skin tissue from which the cells are removed into a 45-55% glycerol solution at normal temperature and performing stirring thereon! a second glycerol solution processing step of immersing the first glycerol solution processed skin tissue into a 65-75% glycerol solution at normal temperature and performing stirring thereon; and a third glycerol solution processing step of immersing and preserving the second glycerol solution processed skin tissue in a 80-90% glycerol solution at normal temperature.

[Claim 4]

The method of any one of claims 1 to 3, wherein the first and second glycerol solutions contain an antibiotics and an antimicrobial .

[Claim 5]

The method of any one of claims 1 to 3, wherein the stirring is performed on the glycerol solutions for 1 to 24 hours.

[Claim 6]

The method of any one of claims 1 to 3, wherein the glycerol solution processed skin tissue is continuously preserved at a temperature of 4°C to stabilize the tissue.