KR20010092985A - Process for preparing and separating acellular dermal matrix for transplantation - Google Patents

Abstract

PURPOSE: Provided is a method for processing and separating dermal matrix for graft without any immune rejection, thereby making possible to graft skin into acute or chronic trauma patients. CONSTITUTION: Dermal matrix is separated from epidermal matrix without damaging basement membrane by controlling concentration, treatment conditions or ionic strength of enzymatic solution. The separated dermal matrix from epidermal matrix is processed by treating various surfactants or ultrasonicating to remove cells therefrom. Therefore, it can be grafted into a human body without immune rejection.

Description

Process for preparing and separating acellular dermal matrix for transplantation

The present invention relates to a cell-free dermal layer processing and separation method for transplantation, and more specifically, a method of treating acute or chronic trauma patients is used a self-transplantation method to remove the healthy skin tissue of the body and transplant it to the trauma site, A new trauma occurs in the healthy area where the skin is peeled off, which increases the pain of the patient and takes a long time to cure. In addition, if there are not enough healthy parts of the body, such as severe burn patients, it is difficult to perform multiple transplants. Recently, a reticulum method of processing healthy skin into reticular grafts and implanting them in a wider area has been carried out, but there are problems such as scarring and shrinkage in the graft site. Tagografts using other people's skin, xenografts using other animals' skin such as pigs, etc. have been attempted, but the immune rejection reactions can only provide a temporary therapeutic effect and eventually require autografts. Still hugging. In order to solve these problems, various studies are underway, and one of the efforts is to cultivate skin cells and transplant them into patients.

Since the development of epidermal cultivation methods (Rheinwald, JG, and Green, H., Cell 6: 331, 1975), the method of culturing autologous epidermal cells and transplanting them into patients has achieved significant results in improving patient survival. However, due to the lack of dermis, the long-term success rate was less than 50% (Rennekampff, HO, Kiessing, V. and Hansbrough, JF, J. of Surg. Res. 62: 288, 1996; Navsaria, HA, Myers, S. R). , Leigh, IM and Mckay IA, TIBTEC 13: 91,1995). In addition, even after engraftment, scars and contractures (contracture), etc. still have a problem that occurs.

In addition, attempts have been made to form skin-like structures by implanting skin cells derived from neonatal foreskin through three-dimensional culture, and commercial products are available in some countries (Wilkins, L. M, Watson, SR, Prosky, J., Meuniner, S. F and Parenteau, NL, Biotech & Bioeng. 43: 747, 1994; Sabolinski, ML, Alvarez, O., Auletta, M., Mulder, G. and Parenteau, NL Biomaterials 17: 311, 1996), which is also included in the category of tagi grafts, which do not engraft, and thus provide only a potential therapeutic or adjuvant effect.

Severe injuries that require skin transplantation include burns, traumatic or surgical skin insufficiency, and chronic ulcers. The most common cases of skin damage are burns: first-degree burns are injured in the epidermal layer, second-degree burns are injured in the epidermal and dermal layers, and third-degree burns are in the epidermal and dermal layers, and in severe cases, to the subdermal layer below the dermis. If it is damaged.

In the first degree burns, the apocrine gland, the eccrine gland sebaceous gland, and the epidermal cells around the hair roots multiply rapidly to recover the damaged area. In the case of second-degree burns, the wound is recovered in a similar manner, but apocrine gland, eccrine gland, sebaceous gland, and many parts of the hair root are damaged and only a few remain, which slows the recovery rate and increases the risk of infection and increases the burden of treatment costs. do.

In the case of third degree, all parts of apocrine gland, eccrine gland, sebaceous gland and hair root are damaged, and epidermal cell proliferation occurs only at the borderline with healthy skin. Therefore, the recovery rate is slower. Therefore, in most cases, a method of autografting by removing the skin of a healthy body part is used. This method still suffers from the disadvantages of wounds on healthy parts of the body, which are more painful for patients and a higher risk of infection. In addition, hospital stays are long and the cost of treatment is high. In addition, in case of severe burns, there is not enough healthy skin left to self-transplant.

In order to solve the above problems, the present invention has developed a method for removing an immune response component by processing a taga or heterogeneous dermis layer.

Tissues are obtained from donated bodies to prepare other cell-free dermal layers, and tissues such as pigs are processed to produce heterogeneous cell-free dermal layers. The cell-free dermal layer thus prepared has the following properties.

(a) Provide a protein / collagen template of cells that can be reconstituted and treated by the transplant patient itself.

(b) provide an intact basement membrane that ensures reattachment of living endothelial cells or epithelial cells.

(c) Does not cause immune rejection of transplant patients.

(d) Tissue hardening does not occur.

(e) Lyophilization allows easy storage and transport at room temperature.

The acellular dermal layer manufacturing process is as follows.

Organization

When the tissue is separated from the body and transported, there is a risk of tissue damage due to hypoxia, degradation by autolytic enzymes, and damage to extracellular epilepsy by proteolytic enzymes. In addition, depending on the osmotic pressure of the carrier solution may be physically damaged. In addition, there is always a risk of contamination by microorganisms such as bacteria and fungi. Therefore, the solution used for transporting tissue should be added with substances that can prevent degradation by hypoxia, degradation by autolytic enzymes, degradation by proteins and enzymes, and antibiotics and antibacterial agents to prevent microbial contamination. shall.

Appropriate buffers should be included to prevent tissue damage from osmotic pressure. The osmotic pressure of the tissue carrier solution should have an osmotic pressure of about 260-320mOsm / kg, the plasma osmotic pressure.

In the case of commercial medium, which is widely used for animal cell culture, the osmotic pressure is about 260-320mOsm / kg, which is similar to the osmotic pressure of plasma. Therefore, commercial medium is used as a basic solution, and various components are added thereto.

To prevent contamination of bacteria and fungi, antibiotics such as penicillin, streptomycin, kanamycin, neomycin, bacitracin, gentamycin, vancomycin, etc. may be added alone or in combination to make amphotericin-B, nystatin, polymyxin Antibacterial agents such as the like are added alone or in combination. In addition, enzyme inhibitors should be added to prevent tissue damage caused by various enzymes.

Enzyme inhibitors include n-ethylmaleimide (NEM), phenylmethylsulfonyl fluoride (PMSF), ethylenediaminetetraacetic acid (EDTA), ethylene glycol-bis (2-aminoethyl) -N, N, N ', N Chelating materials such as' -tetraacetic acid (EGTA) and the like, and protease inhibitors such as lupetin and apoprotinine are used.

In addition, tissues should be transported in a way that minimizes physical damage.

Most enzymatic reactions are affected by temperature and become the most active around the human body temperature of 37 ℃, thus transporting the tissue at low temperature of 4 ℃.

Generally, the ice box is filled with ice. Transporting the tissue at a temperature that is too low for the carrier solution to ice may cause damage to the tissue and should be avoided.

Epidermal layer removal

Generally, various proteolytic enzymes are used to separate the dermal and epidermal layers. In the case of using an enzyme, if the concentration is low or the processing time is too short, separation is not performed well. If the concentration is high or the processing time is too long, damage to cells or tissues occurs. Therefore, the treatment should be done according to appropriate concentration and time. Enzymes used to separate the dermal and epidermal layers include neutralases such as dispases, termolysine, trypsin, and the like. The dermal and epidermal layers can be separated by treatment with 1.0 units / ml of dispase at 37 ° C for 60 to 120 minutes. However, enzyme concentration and treatment conditions should be well controlled to prevent damage to the basement membrane. The dermal layer and the epidermal layer can be separated by treating termolysin at a concentration of 200 µg / ml for 30 minutes at 37 ° C. Termolysin lowers the risk of damaging the basement membrane than dispase. Another method is to separate the two layers of tissue by changing the ionic strength of the solution, which also depends on conditions such as ionic strength, treatment time, and treatment temperature. Treatment with at least 1 mole of sodium chloride solution at 37 ° C. for 14 to 32 hours can separate the dermal and epidermal layers. In one or more moles of sodium chloride solution, bacteria and fungi cannot grow, which reduces the risk of microbial contamination. The dermal and epidermal layers can also be separated by treatment with 20 mM ethylenediaminetetraacetic acid (EDTA) solution at 37 ° C. for 14 to 32 hours. EDTA can reduce tissue damage caused by proteases because EDTA acts as a protease inhibitor. Therefore, when 1 ~ 5mM EDTA is added to 1 mol of sodium chloride solution, the dermal and epidermal layers can be separated while minimizing microbial contamination or enzyme damage.

Cell removal of the dermis layer

Immune responses are mainly caused by membrane proteins present in the cell membrane. Therefore, removing the cells can minimize the immune response. By using the difference in physical and chemical properties between cells and extracellular epilepsy, only cells are selectively removed without damage to tissues. The main component of the cell membrane is phospholipids, and various surfactants can be used to remove the cells without damaging the tissue. For this purpose, an ionic surfactant such as sodium dodecyl sulfate (SDS), or Triton X-100, Tween 20, Tween 40, Tween 60, Tween 80, Nonidet P-10 (NP-10), Nonidet P- Nonionic surfactant, such as 40, (NP-40), etc. are used.

The dermal layer is treated with SDS solution at 0.2 to 1% concentration at room temperature for 30 to 120 minutes at room temperature to remove cells without damaging the tissue. In addition, treatment with Tween 20 solution at 0.1-2.0% concentration at room temperature for 30-180 minutes or treatment with 0.2-2% concentration of Triton X-100 or nonidetpi-40 solution at 22-37 ° C for 30-180 minutes is also required. Cells can be removed without damaging the tissue.

In addition to the above chemical methods, cells can also be removed by physical methods. Cells can be removed by treating the dermis with 10-100 Hz ultrasound for 5 to 60 minutes.

In addition, by using a combination of surfactant and ultrasonic can also remove the cells without damaging the tissue.

Freeze Storage

The freezing solution consists of a buffer solution that maintains the ionic strength or osmotic pressure of the solution, a freeze protection agent that prevents physical and chemical damage of the dermal layer tissue when frozen, and a dry protection agent that prevents structural changes of the dermal layer tissue when dried. Cryoprotectants increase the glass transition temperature to increase the stability of frozen tissue. As the glass transition temperature increases, the drying rate can be increased by increasing the specific gravity of the glass or square ice, which is less stable than hexagonal ice. In addition, glassy ice and square ice have a smaller ice size, which causes less damage. Therefore, cryoprotectant must contain cryoprotectant. Currently used cryoprotectants include dimethyl sulfoxide (DMSO), dextran, sugar, propylene glycol, glycerol, mannitol, sorbitol, fructose, trehalose, raffinose, 2.3-butanediol, ethyl starch (HES), polyethylene glycol, Polyvinylpyrrolidone (PVP), proline, hetastarch, serum albumin and the like. A combination of these components and various base components is used to prepare a freezing solution. Among them, dextran, glycerol, hetastarch, mannitol, and ethyl hydroxide starch are used as serum substitutes, and polyethylene glycol is also used as a stabilizer for protein injections. As such, a freezing solution is prepared based on substances known to have little harm to the human body. The dermal layer is immersed in the prepared freezing solution, and the freezing solution can penetrate well by using an appropriate method. The dermal layer in which the freezing solution has penetrated is stored in a cryogenic freezer below -70 ° C.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the present invention, and the contents of the present invention are not limited to the following examples.

Example

How to prepare human dermal dermal layer

The skin tissue of the body was obtained from Korea Tissue Bank. Skin tissue was taken and placed in RPMI 1640 medium (Life Technology, USA) to which antibiotics and 2 mM EDTA (Sigma, USA) were added and transported using an ice-filled ice box to maintain a temperature below 4 ° C. Upon arrival, we checked to see if the ice was completely melted and still there, and used only the skin tissue that was transported without the ice completely melting. Transfer the container with skin tissue to a sterile box. After removal from the container, the dermal layer is placed down and placed on a 24.5 x 24.5 cm 2 assay plate (Nalge Nunc, USA). Cut it to the appropriate size so that it is rectangular. The size is set to 4 × 4 cm 2 to 6 × 10 cm 2. The skin fragments are placed in a Petri dish (Nalge Nunc, USA) containing 40 ml of 1.0 unit / ml dispase (Life Technology, USA) with the dermis layer down. This petri dish is placed at 37 ° C for 90 minutes. After 90 minutes, transfer to a sterile operation box and add 0.1 mole of EDTA solution to stop the dispase activity. Gently hold the epidermal layer with tweezers and peel off from the dermal layer to remove the epidermal layer. Remove the remaining enzyme solution. Add 40 ml of Dulbecco's phosphate buffered saline to the same Petri dish. The petri dish is slowly shaken at room temperature for 5 minutes at a speed of 30 ± 5 revolutions per minute.

This Petri dish is transferred back to the Aseptic Working Box to remove Dulbecco's phosphate buffer solution. In Dulbecco's phosphate buffer solution, 40 ml of 1% Tween 20 (Sigma, USA) was added to the same petri dish. This petri dish is slowly shaken at room temperature for 120 minutes at a speed of 30 ± 5 revolutions per minute. Transfer this Petri dish back to the Aseptic Working Box to remove Dulbecco's phosphate buffer solution containing 1% Tween20. Place 40 ml of Dulbecco's phosphate buffer in the same petri dish. The petri dish is slowly shaken at room temperature for 5 minutes at a speed of 30 ± 5 revolutions per minute. Transfer this Petri dish back to the Aseptic Working Box to remove Dulbecco's phosphate buffer. Dulbecco's phosphate buffer solution contains 6% sugar (Sigma, USA), 3.5% sorbitol (Sigma, USA), 1.5% mannitol (Sigma, USA), and 5% glycerol (Sigma, USA) 40 ml of the solution is placed in the same petri dish. The petri dish is slowly shaken at room temperature for 15 minutes at a speed of 30 ± 5 revolutions per minute. The petri dish was transferred back to a sterile box and 6% sugar (Sigma, USA), 3.5% sorbitol (Sigma, USA), 1.5% mannitol (Sigma, USA), 5% glycerol (Sigma, USA) Remove the added Dulbecco's phosphate buffer. This petri dish is stored in a cryogenic freezer at -70 ° C.

As described above, the acellular dermal layer for transplantation can be processed and separated according to the method of the present invention, thereby bringing the effect of transplanting healthy skin to acute or chronic trauma patients.

Claims (14)

Tissue transport solution to inhibit the activity of metalloprotease by adding ethylenediaminetetraacetic acid (EDTA) at a concentration of 1 to 5 mM in the basal medium The group according to claim 1, wherein the basal medium comprises MEM, DMEM, IMDM, RPMI 1640, MCDB 151, MCDB 153 medium, medium 199, DMEM / F12 1: 1 mixed medium, DMEM / F 12 1: 3 mixed medium. Animal cell culture medium composition, characterized in that selected from Soaking tissue in 1 ~ 1.2M sodium chloride solution and maintaining it at 22 ~ 37 ℃ for 12 ~ 36 hours, then separating dermal layer and epidermal layer Method of separating dermis and epidermal layer after immersing the tissue in the solution of 20-25mM EDTA added to 260-320mOsm buffer solution of osmotic pressure and maintaining it at 22-37 ℃ for 12-36 hours Method of separating dermal and epidermal layers by adding 1-5 mM EDTA to the solution of claim 3 to inhibit the activity of metallic proteolytic enzymes Method of separating dermal and epidermal layers by adding 1-5 mM EDTA to the solution of claim 4 to inhibit the activity of metallic proteolytic enzymes Method of separating dermal and epidermal layers after soaking tissue in the distaase enzyme solution at 0.6-2.4 unit / ml concentration after 20-120 minutes at 22-37 ℃ or 6-18 hours at 2-8 ℃ Method of separating dermal and epidermal layers after soaking the tissue in 200-500 ㎍ / ml concentration of termolysin enzyme solution for 20 to 120 minutes at 22 to 37 ℃ or for 6 to 18 hours at 2 to 8 ℃ Soaking tissue in 0.05 ~ 0.25% trypsin enzyme solution and keeping it for 22 hours at 22 ~ 37 ℃ to separate dermal layer and epidermal layer Method using a solution containing 0.2% (0.53mM) ethylenediaminetetraacetic acid (EDTA) in the solution of claim 10 Soaking tissue in SDS solution of 0.2 ~ 1% concentration and removing cells in tissue by stirring at 22 ~ 37 ℃ for 30 ~ 180 minutes Soaking tissue in Triton X-100 solution at 0.2 ~ 2% concentration and removing cells in tissue by stirring at 22 ~ 37 ℃ for 30 ~ 180 minutes Soaking tissue in Tween 20 solution with 0.2 ~ 2% concentration and removing cells in tissue by stirring at 22 ~ 37 ℃ for 30 ~ 180 minutes Method of removing cells in tissues by treating dermis layer with ultrasonic wave of 10-100∼ for 5 ~ 60 minutes