KR20190036882A - Manufacturing method of bio-graft or bio-implant comprising acellular dermal matrix derived from pig - Google Patents

Abstract

The present invention relates to a method for manufacturing acellular dermal matrix for bio-graft or bio-implant, which comprises the steps of collecting washed and disinfected skin; removing epidermis from the skin tissue of the collected skin; and manufacturing acellular dermal matrix by removing dermal cells in the skin tissue from which epidermis has been removed. The acellular dermal matrix for bio-graft or bio-implant manufactured according to the method of the present invention has a high tissue safety and can be effectively used as a substitute for autograft by maintaining an extracellular matrix structure and the basement membrane without damage.

Description

Technical Field [0001] The present invention relates to a method for manufacturing a dermal cell-free dermal epithelium,

The present invention relates to a method for producing an acellular dermis for living body implantation or insertion.

Skin is the largest organ covering the entire surface of the human body. It protects the body from physical and chemical stimulation by preventing the loss of body fluids and the inflow of harmful substances and microorganisms from the outside. In patients with severe skin loss due to severe burns, trauma, epithelial cancer resection, and skin disease, there is a protective barrier function to prevent infection and loss of body fluids in the damaged area, It should prevent serious contractions that may occur during the process. Methods for regenerating damaged skin tissue include autografts transplanted to the patient's own skin, allografts transplanted into other human skin, xenografts implanted in animal skin, . Of these methods, autologous transplantation is ideal, but in the case of a wide range of burns, there are limitations on the site where the tissue can be secured, and the harvesting site remains as a new wound site. Allogeneic transplantation plays a role in helping the cells move and heal around the wound rather than engage in permanent engraftment.

In particular, in case of third degree burns which are damaged to the epidermal layer, dermal layer and subcutaneous layer, skin graft treatment is indispensably required. The currently used skin transplantation therapy uses autologous transplantation method, which causes new trauma to a healthy area where skin has been removed, resulting in increased patient pain, time to cure, and economic burden. In addition, there is a problem in that, when a healthy body part such as a severe burn patient is not sufficiently left, the autotransplantation method can not be applied or the transplant operation must be performed several times. In order to solve the above problems, allografts using skin of another person, xenotransplantation using skin of other animals such as pigs, and the like have been tried, but they often cause other side effects as well as immune rejection.

On the other hand, in order to eliminate the immune rejection reaction, the acellular dermis substrate which removed the epidermis from the skin of the donated cadaver and remove the cells from the dermis is used mainly for freezing after storage for convenience of storage. There is a need for a method for producing an acellular dermis substrate having structural stability because of the problem of rapid decomposition after transplantation.

Accordingly, the inventors of the present invention confirmed that when the cells are removed using a surfactant, dermis for transplantation or insertion can be produced more effectively while studying a method for producing a cell-free dermis substrate.

Accordingly, it is an object of the present invention to provide a method for producing a cell-free dermis for in vivo transplantation or insertion for removing cells using a surfactant.

In order to accomplish the above object, the present invention provides a method of treating skin, comprising: Removing the skin layer from the skin tissue of the collected skin; And removing the cells of the dermal layer in the skin tissue from which the epidermal layer has been removed to produce a cell-free dermis; The present invention provides a method for manufacturing a cell-free dermis for vital implantation or insertion.

The non-cell dermis for vital implantation or insertion prepared according to the method of the present invention has a high tissue safety and can be effectively used as a substitute for autotransplantation by maintaining the extracellular matrix structure and the basement membrane without damage .

1 is a view showing a process of collecting pig skin.
FIG. 2 is a diagram showing a series of procedures for preparing a porcine skin-free acellular dermis substrate.
Fig. 3 is a diagram showing pig skin and acellular dermis tissues from which epidermis has been removed.
FIG. 4 is a graph showing the result of 100-fold and 200-fold magnification of an acellular porcine dermis substrate with an optical microscope after H & E staining.
FIG. 5 is a graph showing the resistance to collagenase according to the difference in sucrose concentration by treating sucrose at a final concentration of 10 to 40% by weight.

The present invention relates to a method of treating skin, comprising: collecting washed and disinfected skin; Removing the skin layer from the skin tissue of the collected skin; And removing the cells of the dermal layer in the skin tissue from which the epidermal layer has been removed to produce a cell-free dermis; The present invention provides a method for manufacturing a cell-free dermis for vital implantation or insertion.

Also, the present invention provides a method for producing a dermal cell for dermal implantation or insertion, wherein the dermal skin is collected using a dermatome.

Also, the present invention provides a method for producing a cell-free dermis for in vivo implantation or insertion, which comprises removing a dermal layer cell using a surfactant. Sodium Dodecyl Sulfate (SDS) may be used as the surfactant.

Also, the present invention provides a method for producing a cell-free dermis for living body implantation or insertion, wherein the skin is derived from a pig.

Hereinafter, the present invention will be described in more detail.

As used herein, the term " acellular dermis " refers to a dermal layer in which cells capable of causing an immune rejection reaction are removed by chemical treatment of the dermal layer separated from the skin tissue, and collagen and elastin are the main components.

The sucrose used in the present invention is a disaccharide composed of one molecule of D-glucopyranose and one molecule of D-fructofranose, and further contains glycerol, a phosphate buffer solution and an animal cell culture medium in the sucrose. Further treatment of the preservative also serves to protect and stabilize cell membranes and membrane proteins from freezing ice crystals. Therefore, the safety of the tissue of the frozen-preserved acellular dermal substrate prepared according to the present invention can be improved.

In the present invention, the composition for cryopreservation may be a solution prepared by mixing a cryopreservative agent such that the final concentration of sucrose is 20 to 40% by weight.

The composition for cryopreservation according to the present invention can improve the safety of the dermis tissue and maintain the structure of the extracellular matrix without damaging by using an ideal mixing ratio of glycerol, base solution and sucrose. When the sucrose is less than 20% by weight in the cryopreservative according to the present invention, the stability of the tissue may be weakened due to the ice crystal upon freezing. If the sucrose is contained in excess of 40% by weight, Sugar crystals can be generated and affect the safety of the tissue. In the present invention, the cryopreservative agent can be preferably prepared by dissolving sucrose in a solution in which the basic ingredients are mixed so that the final concentration is 25 to 35% by weight, more preferably 30% by weight.

As a basic component of the " cryopreservative " in the present invention, glycerol and a basic solution are used. In the present invention, the base solution refers to a solution which becomes a base when preparing the cryopreservative, and a buffer solution or an animal cell culture medium used for treating animal cells can be used. The buffer solution in the present invention can be used without any particular limitation as long as it is used in the art to treat animal cells. Examples of the buffer solution that can be used in the present invention include, but are not limited to, phosphate buffered saline (PBS), Tris-buffered saline (TBS), and citric acid buffer. The animal cell culture medium in the present invention may be any medium known in the art. The animal cell culture medium that can be used in the present invention may be selected from the group consisting of, for example, MEM (Minimum Essential Media), DMEM (Dulbecco's Modified Eagle Media), RPMI 1640, Iscove's Modified Dulbecco's Media, Defined Keratinocyte- But are not limited to, Keratinocyte-SFN (with BPE), KnockOut D-MEM, AmnioMAX-II Complete Medium, AmnioMAX-C100 Complete Medium.

In the present invention, the glycerol and the base solution are used in a mixing ratio of 0.5 to 3.5: 9, preferably 0.8 to 2: 9, more preferably 1: 9 on a weight basis. In the present invention, if the glycerol mixing ratio is less than 0.5, there may be a problem due to cold weather upon freezing, and if it exceeds 3.5, degeneration of tissue after freezing may be induced.

The cryopreservation agent may be a composition for cryoprotecting a cell-free dermis substrate, wherein the mixing ratio of the base solution composed of glycerol and a buffer solution to which an animal cell culture medium solution is added is 0.5: 8 to 3.5: 10 by weight.

The term " dermis " used in the present invention is one of three layers constituting the skin, occupying most of the skin, and located between the epidermis and subcutaneous tissue. The dermis is composed mainly of matrix proteins such as collagen fibers and elastic fibers, and the matrix proteins are known to be produced by fibroblasts. It also contains epidermal appendages originating from the nerves, blood vessels, and the epidermis, and plays an important role in the wound healing process.

In the present invention, the acellular dermis substrate refers to a substrate from which epidermal cells have been removed after the epidermis has been removed in order to eliminate the immune rejection reaction. Removal of epidermal and intradermal cells can be performed according to various methods known in the art, and there is no particular limitation thereto. Removal of the epidermis may be carried out by treatment with an enzyme such as trypsin, collagenase or dispase or a NaCl solution. Removal of the cells in the dermis can be performed, for example, by treating with Triton X100, Tween 20, Tween 40, Tween 60, Tween 80, or SDS (sodium dodecylsulfate). In the present invention, the acellular dermis may be a homogeneous or heterogeneous dermis, and the dermis may be a pig.

The present invention also relates to a method for treating skin, comprising: removing a skin layer from skin tissue; Removing the cells of the dermal layer in the skin tissue from which the skin layer has been removed to produce acellular dermis; Treating the acellular dermis with a composition for cryopreservation; And lyophilizing the acellular dermis; And a method for producing a cryopreserved acellular dermis substrate.

In the present invention, treating the composition for cryopreservation to skin tissue can be carried out according to various methods known in the art, and preferably infiltrates the skin with a composition for cryopreservation in a low temperature reactor. The time for penetration may vary depending on the size of the skin tissue, etc., and it may be infiltrated in a low-temperature reactor at, for example, 4 캜 for about 6 to 24 hours.

In the present invention, skin infiltrated with the composition for cryopreservation can be frozen using a controlled rate freezer capable of temperature control.

In the method for producing a cell-free dermis substrate, the skin can be frozen at a desired speed by using a freeze-drier capable of controlling the temperature during freezing. In the present invention, the rate at which the skin is frozen using a temperature-controlled controlled rate freezer is preferably from 0.1 ° C to -7 ° C per minute, more preferably from -0.5 ° C to -5 ° C, more preferably from -0.8 ° C -3 DEG C, and most preferably 1 DEG C per minute. In the present invention, when freezing is performed, the freezing rate is less than -0.1 ° C. per minute. If the freezing is performed too slowly, the solute in the tissue is larger than the tissue, so the freezing rate is lowered and the freezing temperature is slowly lowered. There is a problem of destroying the tissue and since the temperature of the skin permeated by the composition for cryopreservation and the temperature of the controlled rate freezer chamber can not be the same, Failure to regulate the latent heat of fusion by rapid freezing to -80 ° C at more than -7 ° C per minute may cause tissue destruction due to ice crystals.

The present invention also relates to a method for treating skin, comprising: removing a skin layer from skin tissue; Removing the cells of the dermal layer in the skin tissue from which the skin layer has been removed to produce acellular dermis; Treating the acellular dermis with a composition for cryopreservation; And lyophilizing the acellular dermis; The present invention provides a cryopreserved acellular dermis substrate prepared by the method of manufacturing a cryopreserved acellular dermis substrate.

Hereinafter, the present invention will be described in detail with reference to examples. These embodiments are for illustrative purposes only and do not limit the scope of the present invention.

Example  1. How to process pig skin material

1.1 Pig skin collection

After the pig skin was cleaned with physiological saline, the pig skin was depilated and disinfected with povidone to complete the pretreatment process for skin extraction. Then, the washed pig skin was easily cut into a size of 5 × 10 cm ² using a dermatome, and the above procedure is shown in FIG.

As shown in FIG. 1, it was confirmed that pig skin can be easily harvested by using skin segments.

1.2 Removal of the collected epidermis and Acellular cell  Production of dermal substrate

A treatment was performed to remove the epidermis from the harvested pig skin. The pork skin of 7/1000 inch thickness collected from 1.1 above was treated with 1 M NaCl (Sigma, USA) solution and then stirred in a 38 ° C reactor (P-039, Core Tech) for about 6 to 24 hours . Then, the epidermis was removed using a forceps. After removing the epidermis, the pig skin was washed with phosphate buffer solution (pH 7.2, GIBCO, USA). The washed dermis was added to 0.1% SDS, which is a surfactant, and the dermis cells were removed by stirring at room temperature for 1 hour. A series of the above experiments is shown in Fig.

Fig. 3 shows the result of examining pig skin and acellular dermis tissues from which epidermis has been removed.

As shown in FIG. 3, the pig skin having the epidermis and dermis was separated into epidermis and dermis through the epidermal process, and after removing the cells using the surfactant, only the dermis layer of the skin could be obtained Respectively.

The above process was established as an optimized raw material and processing step, and then the acellular dermis substrate processed through the present invention was used for the experiment.

Example  2. For living organ transplantation or insertion using cryopreservatives Acellular cell  Manufacture of dermal tissue

2.1 Preparation of cryopreservatives

Glycerol (Sigma, USA) and phosphate buffer solution containing animal cell culture medium were mixed at a weight ratio of 1: 9 to prepare a cryopreservative base solution. Sucrose (Sigma, USA) was added to the basic solution so as to have a final concentration of 30% and dissolved to prepare a cryopreservative.

2.2 For biotransplantation or insertion Acellular cell  Production of pig dermal tissue

The cell-free porcine dermis tissue prepared in Example 1.1 was washed with phosphate buffered saline. Thereafter, the cell-free dermal tissue was placed in a 4 ° C low-temperature reactor (P-039, Core Tech), the cryopreservative prepared in Example 2.1 was added, and the dermal tissue was infiltrated for 12 hours. The infiltrated cell-free porcine dermis tissue was placed in a polyamide bag (CryoBag, Origen, USA) and frozen to 150 ° C at a rate of -1 ° C per minute using a controlled rate freezer (14S-A, SY Lab, USA) To prepare final cell-free porcine dermal tissue for biotransplantation or insertion.

Experimental Example  1. Manufactured Dermal  Structural stability

In order to determine the dermal structural stability of the cell-free porcine dermis tissues, the porcine dermal tissue prepared according to the above example was subjected to H & E staining for histological examination.

The paraffin block was cut to a thickness of 4 탆 and dried to prepare a paraffin slice. For the deparaffinization, three times for 5 minutes in xylene, three times for 2 minutes in 100% ethanol, one minute for 1 minute in 90% ethanol, one for 1 minute in 80% ethanol and one for 1 minute in 70% After the reaction, it was washed in running water for 10 minutes. Hematoxylin staining solution was allowed to react for 10 minutes, washed in running water for 3 minutes, reacted with Eosin staining solution for 10 minutes, and washed until no eosin stain was observed in running water. 10 times for 1 second to 70% ethanol, 10 times for 1 second to 80% ethanol, 10 times for 1 second to 90% ethanol, 2 times for 1 minute to 100% ethanol, and 3 times for 3 minutes to xylene, The skin of the example was photographed with an optical microscope (Olympus BX51, H & E staining) and a scanning electron microscope (Hitachi S-4700, Japan) and is shown in Fig.

As shown in FIG. 3, in the case of the dermal tissue prepared by the manufacturing method of the example, the structural stability of the collagen structure constituting the dermis in the tissue was much better, and it was confirmed that the destruction of the tissue occurred during the freezing process was remarkably small.

Experimental Example  2. Collagenase resistance by treatment with sucrose concentration

In order to examine the stability of acellular dermis tissues according to the difference in sucrose concentration, sucrose was treated at a concentration of 10 to 40%, and the resistance test for collagenase was performed. 25 mg of the sample was added to 5 ml of 5 mM TES buffer to which 0.36 mM calcium chloride had been added and mixed. 0.1 ml of collagenase (0.1 mg / ml) was added to the sample and allowed to react at 37 ° C for one day while mixing well. After serial dilution of 4.0 mM L-leucine standard solution, the absorbance (VERSA max, Molecular Device, USA) was measured at 570 nm by treatment with ninhydrin color reagent to prepare a standard curve. The amount of L-leucine released from each sample was calculated using the standard curve of L-leucine, and the calculated L-leucine release was shown in FIG. As a positive control group, collagen powder was treated with collagenase and negative control group was treated with collagenase.

As shown in FIG. 4, in the group treated with 20 to 40% by weight of the final concentration of sucrose, the amount of L-leucine was reduced to 1/10 of that of the positive control, and the optimal concentration of 30% Group showed less release of L-leucine than the negative control without collagenase. As a result, the dermal tissue of the frozen-preserved acellular pig treated with the cryopreservative containing sucrose at a final concentration of 20 to 40% by weight had a high tissue stability, and the degradation rate by the collagenase was remarkably decreased, Also, it was confirmed that it decreased remarkably.

Claims (5)

(i) collecting washed and disinfected skin;
(ii) removing the skin layer from the skin tissue of the harvested skin; And
(iii) removing the cells of the dermal layer in the skin tissue from which the epidermal layer has been removed to produce an acellular dermis; The method of manufacturing a cell-free dermis for vital implantation or insertion according to claim 1, [2] The method of claim 1, wherein the harvesting in step (i) is performed using a dermatome. The method according to claim 1, wherein the step (iii) is performed using a surfactant. [4] The method according to claim 3, wherein the surfactant is sodium dodecyl sulfate (SDS). The method of claim 1, wherein the skin is derived from pigs.

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