KR20190131790A - Method for Decellularization of Tissue - Google Patents

Abstract

The present invention relates to decellularization of tissues for use in regenerative medicine. A tissue decellularization method according to an embodiment of the present invention may comprise: a step of preparing a tissue as a subject to be decellularized; a first cell removal step of primarily applying a first decellularization solution containing a surfactant to the tissue in order to remove cells contained in the tissue; and a second cell removal step of secondarily applying a second decellularization solution containing DNase and RNase to the tissue that has undergone the first cell removal step in order to remove DNA and RNA contained in the tissue.

Description

Method for Decellularization of Tissue

The present invention is for the decellularization of external tissues used in regenerative medicine, more specifically, using a surfactant and nucleic acid degrading enzymes, such as excellent decellularization effect, tissue damage that can minimize tissue damage during decellularization It relates to a decellularization method.

One method of forming a scaffold in which cells are cultured to artificially regenerate an organ is to remove cells from an animal or human tissue or organ, and then culture the cells to be transplanted into a patient. To this end, a method of removing cells from existing organs, seeding a patient's cells or immunomodulatory cells, and inducing the formation of the original organs has been proposed. The process of removing cells from existing organs is called decellularization, and reculturing the cells is called recellularization. The main purpose of the decellularization process is to obtain extracellular matrix from which cells have been removed while maintaining the original vascular structure of the organs.

On the other hand, when transplanting a graft derived from a living tissue of another person or a heterologous animal, the rejection reaction of the graft becomes a problem in the tissue of the recipient. As a solution to this problem, development of artificial tissue is expected. Although various polymers have been tried as materials, the compatibility between such materials and living tissues is low, so that dropouts or infections may occur at the junctions between the grafts and living tissues. Thus, in order to improve compatibility with living tissues, techniques have been developed to remove cells from living tissues and use decellularized living tissue, which is a remaining supportive tissue, as a graft.

Patent Publication No. 10-2017-0143465

On the other hand, in the case of using decellularized tissues in regenerative medicine, in addition to no rejection reaction, it is a question of whether tissue regeneration can be performed quickly. Decellularized tissue obtained from biological tissues derived from the same living body, even if decellularized tissue from which cells have been removed to the same extent, if the method or step of decellularization is different, adhesion of host cells to decellularized tissue, host cell There was a difference in the rate of attracting effect, the rate of infiltration of host cells into decellularized tissues, and the like, which greatly influenced the regeneration of tissues.

The problem to be solved by the present invention is to provide a method for decellularization of the tissues while excellent in decellularization effect, preventing damage to the tissues during decellularization.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

Decellularization method of a tissue according to an embodiment of the present invention for achieving the problem to be solved, preparing a tissue to be decellularized, and in order to remove the cells contained in the tissue, includes a surfactant In order to remove the DNA and RNA contained in the tissue subjected to the first cell removal step and the first cell removal step, the first cell removal step to the first decellular solution, DNA degrading enzyme and RNA degrading enzyme It may include a second cell removal step of treating the tissue second with the second decellularization liquid contained.

The surfactant is an amphoteric or nonionic surfactant.

The amphoteric surfactants include alkyldimethylamine oxide, alkylcarboxybetaine, alkylamidedopropylbetaine, alkylamidedopropylsulfobetaine, and alkylimidazolebetaine. alkylimidazoliumbetaine), 3-[(3-clamidepropyl) dimethylammonio] -2-hydroxy-1-propanesulfonate (3-[(3-cholamidopropyl) dimethylammonio] -2-hydroxy-1-propanesulfonate, CHAPSO) And 3-[(3-clamidepropyl) dimethylammonio] -1-propanesulfonate ((3-[(3-cholamidopropyl) dimethylammonio] -1-propanesulfonate, CHAPS).

The nonionic surfactants include glycerin fatty acid esters, sorbitan fatty acid esters, sucrose fatty acid esters, trihalose fatty acid esters, polyethylene glycol fatty acid esters, polyoxyethylene glycerin fatty acid esters, polyoxyethylene sorbitan fatty acid esters, and polyoxyethylene Alkyl ether (polyoxyethylene alkylether), polyoxyethylene alkyl phenyl ether, polyoxyethylene polyoxypropylene ether, alkyl (poly) glycerin ether, fatty acid alkanolamide, polyoxyethylene fatty acid alkanolamide, alkyl (poly) glycoside, alkyl mal Alkylmaltoside, alkylthioglycoside, alkylmaltopyranoside, alkanoyl-N-methyl-D-glucosamine, N, N-bis (3-D-gluconamidepropyl) collamide (N, N-bis (3-D-gluconamidepropyl) colamide (BIGCHAP) and N, N-bis (3-D-gl) Ruconamidepropyl) deoxycolamide (N, N-bis (3-D-gluconamidepropyl) deoxycolamide, Deoxy-BIGCHAP), or any combination thereof.

The first decellular solution is a proteolytic inhibitor selected from the group consisting of leupeptin, aprotinin, and combinations thereof, Tris NaCl MgCl2 buffer, Phosphate Buffered Saline buffer, Tris-Cl The first buffer solution may further include a buffer, a Saline Sodium Citrate buffer, a Hepes EDTA Neocuproine buffer, a Tris EDTA NaCl buffer, and a combination thereof.

The second decellularization solution is Tri NaCl MgCl 2 (TNM) buffer, Phosphate Buffered Saline (PBS) buffer, Tris-Cl buffer, Saline Sodium Citrate (SSC) buffer, Hepes EDTA Neocuproine (HEN) buffer, Tris EDTA NaCl (TEN) buffer And a second buffer liquid selected from the group consisting of a combination thereof.

In the second decellularization solution, 5-15 μg / ml of the DNA degrading enzyme may be included.

In the entire second decellular fluid, the RNA degrading enzyme may be included in the 150 to 250㎛ / ml.

Between the tissue preparation step and the first cell removal step, may further comprise a first washing step for washing the tissue with a wash solution.

The washing liquid may include distilled water and antibiotics.

Between the first cell removal step and the second cell removal step, may further comprise a second washing step of washing the tissue treated primarily with saline.

According to the present invention, there is provided a method for decellularization of tissues that is excellent in decellularizing effect and prevents damage to tissues during decellularization.

1 is a flow chart showing a method for decellularization of tissue according to an embodiment of the present invention.
2 to 4 are experimental results showing decellularization.
5 to 9 are experimental results showing whether the tissue is damaged in the decellularization process.

Advantages and features of the present invention and methods for achieving them will be apparent with reference to the embodiments described below in detail with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in various forms, and only the present embodiments are intended to complete the disclosure of the present invention, and the general knowledge in the art to which the present invention pertains. It is provided to fully convey the scope of the invention to those skilled in the art, and the present invention is defined only by the scope of the claims. DETAILED DESCRIPTION Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings. Regardless of the drawings, the same reference numbers refer to the same components, and “and / or” includes each and every combination of one or more of the items mentioned.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In this specification, the singular also includes the plural unless specifically stated otherwise in the phrase. As used herein, "comprises" and / or "comprising" does not exclude the presence or addition of one or more other components in addition to the mentioned components.

Unless otherwise defined, all terms (including technical and scientific terms) used in the present specification may be used in a sense that can be commonly understood by those skilled in the art. In addition, the terms defined in the commonly used dictionaries are not ideally or excessively interpreted unless they are specifically defined clearly.

Hereinafter, the decellularization method of the tissue according to an embodiment of the present invention.

1 is a flow chart showing a method for decellularization of tissue according to an embodiment of the present invention. Referring to Figure 1, the decellularization method of the tissue according to an embodiment of the present invention, the tissue preparation step, the first washing step, the first cell removal step, the second washing step, the second cell removal step It may include. Hereinafter, each step will be described in detail.

First, prepare a tissue to be decellularized (S10).

The biological tissues used for the decellularized tissues of the present invention are not particularly limited as long as the biological tissues used for the decellularized tissues of the present invention derived from vertebrates are biological tissues derived from vertebrates. Alternatively, a living tissue derived from algae is preferable, and a living animal derived from mammals, a livestock derived from birds, or a living tissue derived from humans is more preferable. For mammals, cattle, horses, camels, llama, donkeys, yaks, sheep, pigs, goats, deer, alpacas, dogs, raccoons, weasels, foxes, cats, rabbits, hamsters, guinea pigs, rats, mice, squirrels, america Raccoons, and the like. Moreover, as a livestock of a bird, a macaw, a parrot, a chicken, a duck, a turkey, a goose, a helmeted guinea fowl, a pheasant, an ostrich, a quail, an emu, etc. are mentioned. Among them, biological tissues of cattle, pigs, rabbits and humans are preferred from the viewpoint of safety.

As the site of the biological tissue, a site having an extracellular matrix structure is used, and as such a site, for example, the liver, kidney, ureter, bladder, urethra, tongue, tonsil, esophagus, stomach, small intestine, large intestine, anus, pancreas , Heart, blood vessel, spleen, lung, brain, bone, spinal cord, cartilage, testis, uterus, fallopian tube, ovary, placenta, cornea, skeletal muscle, tendon, kidney, skin and the like. As the site of biological tissue, cartilage, bone, liver, kidney, heart, pericardium, blood vessels, skin, small intestinal mucosa, lungs, brain, and spinal cord are preferable because of the high effect of tissue regeneration.

For example, the prepared tissue may be sciatic nerve tissue. Specifically, sciatic nerve tissue for decellularization can be obtained from neural tissue of the rat. Sciatic nerve tissue of SD (Sprague-Dawley) rats, which are easily supplied, is used, and sciatic nerve tissue can be obtained after dissecting the thigh skin and muscle of the rat.

Next, to wash the prepared tissue (S20). The prepared tissue is washed to remove blood or impurities remaining in the tissue. Since the washing is further performed afterwards, the washing step of the current step is referred to as a first washing step for convenience.

In the first washing step, the tissue may be washed with a washing solution including distilled water. More specifically, the first washing step may use a second distilled water (double distilled water). Here, the secondary distilled water has a lower osmotic pressure than the cytoplasm, thereby inducing cell hemolysis. And since the tissue of the animal is used as a raw material, the washing solution may contain antibiotics for sterilization and disinfection. For example, penicillin strapmycin (penicillin / streptomycin) may be used as an antibiotic. Antibiotics may be included at a concentration of 0.5% to 2.0% based on the entire wash solution. If the concentration of the antibiotic is less than 0.5%, the antibiotic may not be fully effective. If the concentration of the antibiotic exceeds 2.0%, the antibiotic effect on the excess may not be significant. In other words, there is no significant difference in antibiotic effect between the concentration of 2.0% and above.

The first washing step can be performed within about 7 hours. This process primarily removes blood or impurities remaining in the prepared tissue.

Subsequently, the washed tissue is first treated with a first decellularization solution containing a surfactant to remove the cells from the washed tissue. The primary treatment is referred to as a first cell removal step (S30) for convenience.

In the first cell removal step, the prepared tissue is treated with a first decellularization solution, which may include a surfactant, a proteolytic inhibitor, and a first buffer solution for dissolving them.

Since surfactants are amphiphilic, the cells can be removed by dissolving the cell and nuclear membranes of the tissue. Surfactants may include ionic and nonionic surfactants, and ionic surfactants may consist of anionic, cationic, amphoteric surfactants. Here, anionic surfactant is a case where a hydrophilic part is an anion atom group, and a cationic surfactant is surfactant which has both a cationic group and an anionic group in the same molecule. On the other hand, nonionic surfactants are surfactants which are dissolved without being ionized in water. In general, ionic surfactants have a functional group that exhibits a cation or an anion with a hydrophilic group, and thus may have a relatively strong surfactant action as compared to a nonionic surfactant. Among these, it is preferable that an amphoteric surfactant and a nonionic surfactant are used.

Anionic surfactants include fatty acid soaps, alkoxycarboxylates, polyoxyethylenealkoxycarboxylates, alkylsulfonates, alkylbenzenesulfonates, and alkylsulfonate esters. Salts (alkylsulfuric acid ester), polyoxyethylenealkylsulfuric acid ester, alkyl phosphoric acid ester, α-sulfofatty acid ester, N-acyl glutamate (N-acylglutaminate, acyl-N-methyltaurate, N-alkylsarcosine, N-alkylsarcosine, cholate, deoxycholate, or any one selected from these It can be a combination of.

Cationic surfactants include alkyldimethylamine, alkyldiethanolamine, alkyltriethylammonium salt, dialkyldimethylammonium salt, alkylpyridium salt, alkyl It may be any one selected from alkylbenzyldimethylammonium salt or a combination thereof.

Amphoteric surfactants include alkyldimethylamine oxide, alkylcarboxybetaine, alkylamidopropylbetaine, alkylamidopropylsulfobetaine, alkylimidazobetaine ), 3-[(3-clamidepropyl) dimethylammonio] -2-hydroxy-1-propanesulfonate (3-[(3-cholamidopropyl) dimethylammonio] -2-hydroxy-1-propanesulfonate, CHAPSO), It may be any one selected from 3-[(3-cramidepropyl) dimethylammonio] -1-propanesulfonate ((3-[(3-cholamidopropyl) dimethylammonio] -1-propanesulfonate, CHAPS) or a combination thereof. .

Nonionic surfactants include glycerin fatty acid ester, sorbitan fatty acid ester, sucrose fatty acid ester, trihalose fatty acid ester, polyethylene glycol fatty acid ester, polyoxyethylene glycerin fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene alkyl Ether (polyoxyethylene alkylether), polyoxyethylene alkyl phenyl ether, polyoxyethylene polyoxypropylene ether, alkyl (poly) glycerin ether, fatty acid alkanolamide, polyoxyethylene fatty acid alkanolamide, alkyl (poly) glycoside, alkyl malto Alkylmaltoside, alkylthioglycoside, alkylmaltopyranoside, alkanoyl-N-methyl-D-glucosamine, N, N-bis 3-D-gluconamide propyl) colamide (N, N-bis (3-D-gluconamidepropyl) colamide (BIGCHAP), N, N-bis (3-D-glucona) DE-propyl) oxy call to can be an amide (N, N-bis (3-D-gluconamidepropyl) deoxycolamide, Deoxy-BIGCHAP) any one or a combination thereof selected from the.

The proteolytic inhibitor included in the first decellularization solution may prevent the protein contained in the tissue from being degraded upon cell removal. For example, damage to various proteins and glycoproteins of the extracellular matrix can be minimized. Leupeptin or aprotinin may be used as a proteolytic inhibitor.

The first decellular solution may include a first buffer solution. The first buffer solution serves as a base for dissolving the surfactant and the proteolytic inhibitor. As the first buffer solution, for example, Tri NaCl MgCl 2 (TNM) buffer, Phosphate Buffered Saline (PBS) buffer, Tris-Cl buffer, Saline Sodium Citrate buffer, Hepes EDTA Neocuproine (HEN) buffer, and Tris EDTA (TEN) Any one selected from the group consisting of NaCl) buffer can be used.

When the first treatment is performed on the tissue prepared with the first decellularized cell, a second washing step (S40) is performed to remove the byproducts of the destroyed cells or impurities remaining in the tissue.

The washing solution used for washing in the second washing step may be a solution containing NaCl in distilled water. NaCl is, for example, at a concentration of 1M. The wash solution may also contain antibiotics for sterilization and disinfection. For example, penicillin strapmycin (penicillin / streptomycin) may be used as an antibiotic.

Subsequently, in order to remove DNA and RNA contained in the tissues subjected to the first cell removal step, the first cell removal is performed by treating the tissues secondly with a second decellularization solution containing DNA degrading enzyme and RNA degrading enzyme. Proceed to step S50.

The second decellularization solution used in the second cell removal step includes a second buffer solution and a DNA degrading enzyme and an RNA degrading enzyme.

The second buffer solution serves as a base for dissolving DNA and RNA degrading enzymes. As the buffer solution, for example, Tri NaCl MgCl 2 (TNM) buffer, Phosphate Buffered Saline (PBS) buffer, Tris-Cl buffer, Saline Sodium Citrate (SSC) buffer, Hepes EDTA Neocuproine (HEN) buffer, and Tris EDTA NaCl ) Any one selected from the group consisting of buffers can be used.

DNA degrading enzymes and RNA degrading enzymes decompose DNA and RNA components, respectively, to aid their elimination. DNA degrading enzyme may include 5 to 15 µg / ml in the second decellularization solution. If the content of the DNAase is less than 5 µg / ml, it may be difficult to sufficiently degrade the DNA component in the tissue. When the content of the DNAase exceeds 15 µg / ml, the effect of decomposing the DNA component more than the excess content may not be expressed.

On the other hand, RNA degrading enzyme may be included in the second total decellular solution 150 to 250㎛ / ml. If the content of RNAase is less than 150 μg / ml, it may be difficult to sufficiently degrade the DNA component in the tissue. If the content of the RNA degrading enzyme exceeds 250 μg / ml, the effect of degrading the RNA component more than the excess content may not be expressed.

If necessary, the first or second decellular solution may further include a chelating agent and / or an organic solvent.

By inactivating calcium ions and magnesium ions in decellularized tissues, the chelating agent can prevent calcification when the granulated decellularized tissues of the invention are applied to diseased sites.

Chelating agents include ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), diethylenetriaminepentaacetic acid (DTPA), hydroxyethylethylenediamine triacetic acid (HEDTA) , Triethylenetetramine hexaacetic acid (TTHA), 1,3-propanediamine tetraacetic acid (PDTA), 1,3-diamino-2-hydroxypropane tetraacetic acid ( 1,3-diamino-2-hydroxypropane tetraacetic acid (DPTA-OH), hydroxyethyleneimino diacetic acid (HIDA), dihydroxyethylglycine (DHEG), glycol etherdiamine tetraacetic acid (glycol ether diamine tetraacetic acid (GEDTA), dicarboxyethyl-glutamic acid (CMGA), 3-hydroxy-2,2'-iminodisuccinic acid (3-hydroxy-2,2 ' iminocarboxylic acid chelating agents or salts thereof, such as -iminodisuccinic acid (HIDA) and dicarboxylethylaspartic acid (ASDA); Hydroxycarboxylic acid chelating agents such as citric acid, tartaric acid, malic acid, lactic acid, or salts thereof may be used.

The organic solvent is highly effective in removing lipids, and an organic solvent in aqueous solution is preferable, and ethanol, isopropanol, acetone, and dimethyl sulfoxide are preferable.

For the tissue that has undergone the second cell removal step, the tissue may be washed with a washing solution to remove impurities remaining in the tissue after cell removal. Similar to the first washing step described above, the washing may be performed by using distilled water containing antibiotics.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example: Neural Tissue Preparation and Decellularization Progress

In order to proceed with decellularization of neural tissues, neural tissues were obtained from rats. Sciatic nerve tissue of SD (Sprague-Dawley) rats was easily obtained, and the sciatic nerve tissue was obtained by dissecting the thigh skin and muscle of the rat. Obtained nerve tissue was prepared in a size of about 1mm * 20mm.

Based on the prepared neural tissues, the experiment was performed under the conditions and steps shown in Table 1 below. Reagents were used commercially available from sigma.

TABLE 1

First wash: run at room temperature for 7 hours

* First cell removal: proceed at room temperature for 24 hours using a surfactant

* Second wash: proceed at room temperature for 15 hours with 1M NaCl solution

Second cell removal: Example-proceeded at 37 ° C. for 24 hours with DNA / RNA degrading enzyme

               Comparative Example-24 hours using a surfactant (at room temperature)

* Storage: Store after washing with PBS (phosphate buffered saline) (room temperature)

Experimental Example 1: Determination of Decellularization

The degree of decellularization of neural tissues advanced by the above examples was measured. DAPI (4 ′, 6-diamidino-2-phenylindole) staining was performed on the tissues of Examples 1 to 2 and Comparative Examples 1 to 3 that were subjected to decellularization. The results are shown in FIGS. 2 and 3, respectively.

Referring to Figure 2, in the decellularized tissue according to an embodiment of the present invention, almost no cell nuclei stained with DAPI. On the other hand, when confirming the Figure 3, the tissue treated by the comparative example was confirmed that a large number of cell nuclei stained by DAPI compared to the example.

On the other hand, referring to Figure 4, it can be seen that in the tissues treated by the example, the DNA content and the number of nuclei per area were significantly smaller than those of the tissues treated by the comparative example. That is, according to the embodiment of the present invention, it was found that the decellularization in the tissue is remarkable.

Experimental Example 2: Determination of tissue retention during decellularization

After decellularization progressed by the above examples, the maintenance of neural tissue was measured. To this end, the tissues of Examples 1 to 2 and Comparative Examples 1 to 3 subjected to decellularization were stained using known H & E staining. The results are shown in FIGS. 5 and 6, respectively.

Referring to Figure 5, the decellularized tissue according to an embodiment of the present invention was found that the decellularization proceeded with almost no tissue breakage. On the other hand, when confirming the Figure 6, the tissues treated by the comparison it was found that the breakage of the tissues was found to be significantly compared to the embodiment.

On the other hand, in order to determine whether the extracellular matrix in the neural tissue is preserved, the tissues of Examples 1 and 2 and Comparative Examples 1 to 3 were stained using a known laminin antibody. The results are shown in FIGS. 7 and 8, respectively.

Referring to Figure 7, in the decellularized tissue according to an embodiment of the present invention was confirmed that the extracellular matrix in the tissue is relatively well preserved. On the other hand, as shown in Figure 8, compared to the embodiment in the tissue treated by the comparative example was not better preserved state of the extracellular matrix in the tissue.

On the other hand, referring to Figure 9, in the tissue treated by the embodiment it can be seen that the collagen content and tissue completeness is higher than the tissue treated by the comparative example. That is, according to the embodiment of the present invention, it was found that tissue is maintained and decellularization proceeds.

In conclusion, according to one embodiment of the present invention, it was found that the decellularization effect is excellent, and tissue damage is also minimized during decellularization.

If decellularized tissue is used in regenerative medicine, there should be no rejection reaction, so decellularization of regenerative tissue is essential. In addition, in order to perform the function of the tissue smoothly when using the regenerated tissue, the deformation or loss of the tissue should be minimized during decellularization. The present invention has a marked decellularization effect and minimizes damage to tissues during decellularization, and therefore, the present invention is optimized for the production of regenerative tissues necessary for regenerative medicine.

Although the embodiments of the present invention have been described above, the present invention is not limited to the above embodiments and can be manufactured in various forms, and a person of ordinary skill in the art to which the present invention pertains has the technical idea of the present invention. However, it will be understood that other specific forms may be practiced without changing the essential features. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

Claims (11)

Preparing a tissue to be decellularized;
A first cell removal step of first treating the tissue with a first decellularization solution containing a surfactant to remove cells contained in the tissue; And
A second cell removal step of secondly treating the tissue with a second decellularizing fluid including DNA degrading enzyme and RNA degrading enzyme to remove DNA and RNA contained in the tissue which has been subjected to the first cell removing step; Including, method for decellularization of tissue. According to claim 1,
Wherein said surfactant is an ionic, amphoteric or nonionic surfactant. The method of claim 2,
The amphoteric surfactants include alkyldimethylamine oxide, alkylcarboxybetaine, alkylamidedopropylbetaine, alkylamidedopropylsulfobetaine, and alkylimidazolebetaine. alkylimidazoliumbetaine), 3-[(3-clamidepropyl) dimethylammonio] -2-hydroxy-1-propanesulfonate (3-[(3-cholamidopropyl) dimethylammonio] -2-hydroxy-1-propanesulfonate, CHAPSO) And 3-[(3-cramidepropyl) dimethylammonio] -1-propanesulfonate ((3-[(3-cholamidopropyl) dimethylammonio] -1-propanesulfonate, CHAPS) tissues of any one or combination thereof Method of decellularization. The method of claim 2,
The nonionic surfactants include glycerin fatty acid esters, sorbitan fatty acid esters, sucrose fatty acid esters, trihalose fatty acid esters, polyethylene glycol fatty acid esters, polyoxyethylene glycerin fatty acid esters, polyoxyethylene sorbitan fatty acid esters, and polyoxyethylene Alkyl ether (polyoxyethylene alkylether), polyoxyethylene alkyl phenyl ether, polyoxyethylene polyoxypropylene ether, alkyl (poly) glycerin ether, fatty acid alkanolamide, polyoxyethylene fatty acid alkanolamide, alkyl (poly) glycoside, alkyl mal Alkylmaltoside, alkylthioglycoside, alkylmaltopyranoside, alkanoyl-N-methyl-D-glucosamine, N, N-bis (3-D-gluconamidepropyl) collamide (N, N-bis (3-D-gluconamidepropyl) colamide (BIGCHAP) and N, N-bis (3-D-gl) Cone amide propyl) deoxy call amides (N, N-bis (3-D-gluconamidepropyl) deoxycolamide, Deoxy-BIGCHAP) any one or bingbeop evasion saturation of a combination thereof in selected tissues. According to claim 1,
The first decellular solution is
Proteolytic inhibitors selected from the group consisting of leupeptin, aprotinin, and combinations thereof; And
Tri NaCl MgCl2 (TNM) buffer, Phosphate Buffered Saline (PBS) buffer, Tris-Cl buffer, Saline Sodium Citrate (SSC) buffer, Hepes EDTA Neocuproine (HEN) buffer, Tris EDTA NaCl (TEN) buffer, and combinations thereof Decellularization method of the tissue further comprising a first buffer solution selected from the group. According to claim 1,
The second decellularization solution is Tri NaCl MgCl 2 (TNM) buffer, Phosphate Buffered Saline (PBS) buffer, Tris-Cl buffer, Saline Sodium Citrate (SSC) buffer, Hepes EDTA Neocuproine (HEN) buffer, Tris EDTA NaCl (TEN) buffer And a second buffer solution selected from the group consisting of a combination thereof. According to claim 1,
Decellularization method of the tissue containing the 5 to 15 ㎍ / ml of the DNA degrading enzyme in the entire second decellular solution. According to claim 1,
Decellularization method of the tissue containing the entire RNA decomposing enzyme 150 to 250㎛ / ml in the second decellularization solution. According to claim 1,
Between the tissue preparation step and the first cell removal step, the decellularization method of the tissue further comprises a first washing step of washing the tissue with a wash solution. The method of claim 9,
The washing solution is a cell decellularization method comprising distilled water and antibiotics. According to claim 1,
Between the first cell removal step and the second cell removal step, decellularization method of the tissue further comprises a second washing step of washing the tissue treated primarily with saline.

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