Stepwise Combined Physical-Chemical Decellularization Method for Enhanced Extracellular Matrix Preservation in Cardiac Tissue Engineering

Abstract

The present invention relates to a stepwise physicochemical decellularization method for enhanced extracellular matrix preservation in cardiac tissue engineering. The decellularization method of the present invention comprises: 1) a physical treatment step of repeating freeze-thaw cycles; 2) a chemical treatment step of treatment with a low-concentration surfactant solution; and 3) an ultrasonic treatment step. The decellularization method of the present invention has the effect of reducing ECM loss by more than 50% compared to the use of the chemical method alone, removing more than 70% of residual DNA, and simultaneously securing biomechanical properties and economic efficiency.

Description

Technology Field

[0001] The present invention relates to a technology for decellularization and extracellular matrix (ECM) preservation to maximize the regeneration and restoration potential of heart tissue, comprising an innovative process that efficiently removes cellular components while maintaining the structural and functional integrity of the ECM by sequentially combining chemical and physical decellularization methods. Background Technology

[0002] Decellularization is a tissue engineering technique that allows for the acquisition of an extracellular matrix (ECM) from which cells have been removed, while preserving the existing vascular structure of a tissue or organ intact.

[0003] In particular, extracellular matrix preserved by decellularization technology possesses various advantages, such as reduced tissue immune rejection, high biocompatibility, applicability to various tissues, and excellent structural stability; therefore, it can be widely used for tissue regeneration, organ restoration, materials for artificial organ transplantation, wound healing, and skin substitutes.

[0004] Existing decellularization technologies include chemical and physical methods. Chemical methods primarily use chemical substances, such as surfactants, to remove cells; while they offer high cell removal efficiency, the use of strong surfactants can lead to the degradation of major components of the extracellular matrix (collagen, GAGs, etc.) and a decrease in biomechanical properties. Physical methods utilize temperature, force, and pressure to remove cells; although they minimize ECM damage, the presence of large amounts of cellular residue (such as DNA at 60-75 ng / mg) can impair the biocompatibility of the regenerated tissue.

[0005] Meanwhile, due to the dense extracellular matrix and complex microstructure of cardiac tissue, it was difficult to achieve a balance between cell removal and extracellular matrix preservation using conventional chemical or physical single methods.

[0006] Against this backdrop, the inventors have perfected a decellularization method that efficiently removes cellular components while maintaining the structural and functional integrity of the extracellular matrix by sequentially combining chemical and physical methods. Prior art literature

[0007] Korean Intellectual Property Office Registered Patent Publication No. 10-2181811 (2020.11.17) The problem to be solved

[0008] a) a physical processing step of repeating a freeze-thaw process in which separated heart tissue is frozen and then thawed at room temperature;

[0009] b) a chemical treatment step of immersing the physically treated heart tissue in a surfactant solution; and

[0010] c) a step of treating the chemically treated heart tissue with ultrasound; the present invention provides a decellularization method comprising the above steps.

[0011] Another objective of the present invention is to provide a decellularized tissue produced by the above-decomposed decellularization method. means of solving the problem

[0012] The advantages and features of the present invention and the methods for achieving them will become clear by referring to the embodiments described below in detail together with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below but may be implemented in various different forms. These embodiments are provided merely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the present invention, and the present invention is defined only by the scope of the claims.

[0013] The terms used in this specification are for describing the embodiments and are not intended to limit the invention. In this specification, the singular form includes the plural form unless specifically stated otherwise in the text. The terms "comprises" and / or "comprising" used in this specification do not exclude the presence or addition of one or more other components in addition to the components mentioned. Throughout the specification, the same reference numerals refer to the same components, and "and / or" includes each of the mentioned components and all combinations of one or more. Although terms such as "first," "second," etc., are used to describe various components, these components are not limited by these terms. These terms are used merely to distinguish one component from another. Therefore, the first component mentioned below may be the second component within the technical scope of the invention.

[0014] Unless otherwise defined, all terms used herein (including technical and scientific terms) may be used in a meaning commonly understood by those skilled in the art to which the present invention pertains. Additionally, terms defined in commonly used dictionaries are not to be interpreted ideally or excessively unless explicitly and specifically defined otherwise.

[0015] One aspect of the present invention provides a decellularization method comprising: a) a physical treatment step of repeating a freeze-thaw process in which separated heart tissue is frozen and then thawed at room temperature; b) a chemical treatment step of immersing the physically treated heart tissue in a surfactant solution; and c) a step of treating the chemically treated heart tissue with ultrasound.

[0016] The above a) physical processing step may be a physical processing step that repeats freeze-thaw cycles, and a) a physical processing step that repeats a freeze-thaw process in which the separated heart tissue is frozen at -100 to -20 ℃ for 20 to 30 hours and then thawed at room temperature.

[0017] If the above freezing temperature is -20 ℃ or higher, the cells may not freeze well and cell destruction may not occur, and if the above freezing temperature is -100 ℃ or lower, damage to the extracellular matrix may occur. If the above freezing time is 20 hours or less, cell destruction may not occur completely, and if the above freezing time is 30 hours or more, damage to the extracellular matrix may occur.

[0018] The above a) physical treatment step may involve repeating the freeze-thaw process 2 to 5 times. If the freeze-thaw process is performed only once, cell destruction may not occur, and if the freeze-thaw process is repeated 5 or more times, damage to the extracellular matrix may occur.

[0019] The above physical treatment step a) may involve washing with a buffer solution 2 to 5 times after the freeze-thaw process is completed. Additionally, the washing may be performed using an ultrasonic cleaner utilizing ultrasound of 30 to 50 kHz, but is not limited thereto. If the washing is performed only once, cellular residues may remain in the tissue, which may hinder the subsequent decellularization process; if the washing is performed 5 or more times, major components of the extracellular matrix may be removed.

[0020] The above b) chemical treatment step may be a chemical treatment step in which the heart tissue is treated with a low concentration surfactant solution, and the chemical treatment step in which the physically treated heart tissue is immersed in the surfactant solution for 10 to 15 hours.

[0021] The surfactant solution in step b) of the chemical treatment above may be a nonionic surfactant or an anionic surfactant.

[0022] The above-mentioned nonionic surfactant is Triton X-100 (Polyethylene glycol tert -octylphenyl ether), glycerin fatty acid ester, sorbitan fatty acid ester, sucrose fatty acid ester, trihalose fatty acid ester, polyethylene glycol fatty acid ester, polyoxyethyleneglycerin fatty acid ester, polyoxyethylenesorbitan fatty acid ester, polyoxyethylenealkylether, polyoxyethylenealkylphenyl ether, polyoxyethylenepolyoxypropylene ether, alkyl (poly)glycerin ether, fatty acid alkanolamide, polyoxyethylene fatty acid alkanolamide, alkyl (poly)glycoside, alkylmaltoside, alkylthioglycoside, alkylmaltopyranoside, alkanoyl-N-methyl-D-glucosamine, N,N-bis(3-D-gluconamidepropyl)colamide (BIGCHAP) and It may be one or more selected from N,N-bis(3-D-gluconamidepropyl)deoxycolamide (Deoxy-BIGCHAP), but is not limited thereto.

[0023] The above-mentioned anionic surfactants are sodium dodecyl sulfate (SDS), fatty acid soap, alkoxycarboxylate, polyoxyethylenealkoxycarboxylate, alkylsulfonate, alkylbenzenesulfonate, alkylsulfuric acid ester, polyoxyethylenealkylsulfuric acid ester, alkyl phosphoric acid ester, α-sulfofatty acid ester, N-acylglutaminate, acyl-N-methyltaurate, N-alkylsarcosine, cholate, deoxycholate, It may be one or more selected from alpha olefin sulfonates (AOS), but is not limited thereto.

[0024] In the chemical treatment step b) above, the low-concentration surfactant solution may be a surfactant solution with a concentration of 0.3 to 0.7% w / v. If the concentration of the surfactant solution is 0.3% w / v or less, the chemical treatment of the heart tissue may occur incompletely, and if the concentration of the surfactant solution is 0.7% w / v or more, the loss rate of major components of the extracellular matrix (e.g., collagen, GAG, etc.) may increase.

[0025] According to a preferred embodiment of the present invention, the surfactant solution may be SDS at a concentration of 0.5% w / v or Triton X-100 at a concentration of 0.5% v / v.

[0026] If the immersion time of the surfactant solution in step b) above is 10 hours or less, cell residues may remain in the tissue or an incomplete decellularization reaction may occur, and if the immersion time is 15 hours or more, structural damage to the extracellular matrix may occur.

[0027] The above b) chemical treatment step may be performed under stirring conditions of 40 to 100 rpm.

[0028] In addition, the above b) chemical treatment step may further include a step of washing the heart tissue with a buffer solution 2 to 5 times after the chemical treatment is completed in order to remove residual surfactant solution.

[0029] The above c) ultrasonic treatment step is a step of treating the chemically treated heart tissue with ultrasound, and c) may treat the chemically treated heart tissue with ultrasound at 30 to 50 kHz for 10 to 20 minutes.

[0030] If the ultrasonic frequency is 30 kHz or less or the ultrasonic treatment time is 10 minutes or less, microcell fragments may not be properly separated, and if the ultrasonic frequency is 50 kHz or more or the ultrasonic treatment time is 20 minutes or more, collagen, which is a major component of the extracellular matrix, may be destroyed, and thereby the properties of the extracellular matrix (tensile strength, elasticity, etc.) may be degraded.

[0031] The above method may be d) to remove fine residues by additionally washing the ultrasonically treated heart tissue with a buffer solution 2 to 5 times.

[0032] The heart tissue may be cut into pieces of 0.5 to 2 cm³. If the heart tissue is cut into pieces of 0.5 cm³ or less, stability may be compromised, such as by loss of the tissue's shape or reduced tissue cohesion, and if the heart tissue is cut into pieces of 2 cm³ or more, chemical or ultrasonic treatment may occur unevenly, resulting in incomplete decellularization.

[0034] Another aspect of the present invention is to provide a decellularized tissue produced by the above-decellularization method.

[0035] The above decellularized tissue DNA residue may be 0 to 25 ng / mg.

[0036] The above decellularized tissue may retain 80 to 100% of the collagen compared to before decellularization. The collagen is one of the major components of the extracellular matrix, and is an essential element in forming the basic framework of the extracellular matrix and stably maintaining the three-dimensional structure of the tissue.

[0037] The above-mentioned decellularized tissue may retain 75 to 100% of glycosaminoglycans (GAGs) compared to before decellularization. The above-mentioned GAGs are one of the major components of the extracellular matrix, and in particular, they bind water in the extracellular matrix, swell upon water absorption, provide lubrication to the tissue, and thereby help the extracellular matrix maintain its structure even when subjected to external physical stimuli.

[0038] In addition, the decellularized tissue can improve the preservation of the extracellular matrix by minimizing the loss rate of collagen and GAGs, which are major components of the extracellular matrix.

[0040] In specific embodiments and experimental examples of the present invention, decellularized tissues were prepared according to the manufacturing method of the present invention, and a control group consisting of a chemical treatment alone, a physical treatment alone, and an ultrasonic treatment alone was prepared and the cell residues were analyzed. As a result, it was confirmed that the decellularized tissue of the present invention reduced the residual DNA from 60 to 75 ng / mg when the physical method was used alone to 20 ng / mg or less, thereby reducing the risk of immune rejection (Fig. 1).

[0041] In addition, as a result of confirming the collagen and GAG loss of the decellularized tissue of the present invention and the control group, it was confirmed that the decellularized tissue of the present invention reduced collagen loss from 45% to 15% and GAG loss from 50% to 20% compared to the group treated with chemical treatment alone (Figs. 2a and 2b). Through this, it was confirmed that the decellularized tissue of the present invention can maintain the three-dimensional structure of cardiac tissue while excellently preserving biomechanical properties such as tensile strength and elasticity at over 85% (Figs. 3a and 3b). Effects of the invention

[0042] The decellularization method of the present invention significantly reduced collagen and GAG loss compared to chemical-only and physical-only decellularization methods while maintaining the three-dimensional structure and biomechanical properties of cardiac tissue, and reduced the risk of immune rejection by removing residual DNA to a high degree. Therefore, the decellularization method of the present invention has the effect of improving the preservation of the extracellular matrix and maximizing cell removal efficiency, and has high potential for clinical application. Furthermore, it increased cost efficiency by reducing the amount of chemical agents used and process time by half, and opened up the possibility of expansion to tissue engineering of other organs such as the lungs and liver through sequential process design. Brief explanation of the drawing

[0043] Figure 1 is a graph showing the amount of residual DNA after decellularization according to the decellularization method. Figure 2a is a graph showing the collagen loss rate after decellularization according to the decellularization method, and Figure 2b is a graph showing the GAG ​​loss rate after decellularization according to the decellularization method. Figure 3a is a graph showing the tensile strength loss rate after decellularization according to the decellularization method, and Figure 3b is a graph showing the elasticity loss rate after decellularization according to the decellularization method. Specific details for implementing the invention

[0044] The contents of the present invention will be explained in more detail below through the following examples and experimental examples. However, the scope of the present invention is not limited to the following examples and experimental examples, but includes variations of equivalent technical concepts.

[0046] Example 1. Stepwise physicochemical decellularization method

[0047] Porcine left ventricular tissue was used as the source tissue for the stepwise physicochemical decellularization of cardiac tissue. Prior to decellularization, the porcine left ventricular tissue was cut into 1 cm³ pieces and washed three times with PBS to remove blood and initial residues, thereby completing the pretreatment. The pretreated cardiac tissue was immersed in physiological saline and stored at 4°C for up to 24 hours.

[0048] Stepwise physicochemical decellularization of cardiac tissue first physically destroyed cells through a physical treatment step involving repeated freeze-thaw cycles. Specifically, the pretreated cardiac tissue was completely frozen in an ultra-low temperature freezer at -80°C for 24 hours, and then naturally thawed by leaving it at room temperature (22 to 25°C) for 2 hours. After repeating the above freeze-thaw process three times, a PBS (Phosphate Buffered Saline) wash was performed to remove residues and cellular debris from the tissue.

[0049] The above PBS washing was performed a total of three times. Specifically, the PBS washing process was conducted for 15 minutes per session, and the used PBS was replaced with fresh PBS at each washing step. In addition, the tissue was cleaned using an ultrasonic cleaner utilizing 40 kHz ultrasound, rather than a simple immersion washing method. To prevent residues released during each washing step from causing contamination in the next, a method was applied in which the PBS was replaced with fresh PBS after each washing (15 minutes) to proceed with the next wash. By performing this repeated washing a total of three times, the removal of impurities from the tissue and uniform decellularization were induced.

[0050] Subsequently, the cells were chemically lysed through a chemical treatment step involving treatment with a low-concentration surfactant solution. Specifically, a chemical treatment step was performed in which the physically treated heart tissue was immersed in a surfactant solution of 0.5% w / v SDS for 12 hours under stirring at 50 rpm. After the chemical treatment was completed, the tissue was washed a total of three times with PBS to remove drug residues.

[0051] Finally, to remove fine residues, ultrasonic treatment was performed. Specifically, microcellular fragments and drug residues were separated by treatment at a frequency of 40 kHz for 15 minutes. After the ultrasonic treatment was completed, residual contaminants were removed by washing with PBS a total of three times.

[0053] Comparative Example 1. Chemical treatment performed alone 1

[0054] The preparation of the raw material tissue (pretreatment) of Example 1 was performed in the same manner, but chemical treatment was carried out alone without physical treatment or ultrasonic treatment. In the chemical treatment, a 1% SDS solution was used as the surfactant solution.

[0056] Comparative Example 2. Chemical treatment performed alone 2

[0057] The preparation of the raw material tissue (pretreatment) of Example 1 was performed in the same manner, but chemical treatment was carried out alone without physical treatment or ultrasonic treatment. For the chemical treatment, a 1% Triton X-100 solution was used as the surfactant solution.

[0059] Comparative Example 3. Physical processing performed alone

[0060] The preparation of the raw material tissue (pretreatment) of Example 1 was performed in the same manner, but physical treatment was carried out alone without chemical treatment or ultrasonic treatment. The physical treatment method was performed in the same manner as the physical treatment method of Example 1.

[0062] Comparative Example 4. Ultrasonic treatment method alone

[0063] The preparation of the raw material tissue (pretreatment) of Example 1 was performed in the same manner, but ultrasonic treatment was carried out alone without chemical or physical treatment. The ultrasonic treatment method was performed in the same manner as the physical treatment method of Example 1.

[0065] Experimental Example 1. Analysis of Cell Residues

[0066] The cell residues of the decellularized tissue (Example 1) and its control group (Comparative Examples 1 to 4) prepared by the decellularization method of the present invention were analyzed to confirm whether cell components were efficiently removed. The cell residues were analyzed by measuring the DNA contained in the decellularized tissues of Example 1 and Comparative Examples 1 to 4 using a NanoDrop spectrophotometer.

[0067] As a result of analyzing the cell residues, Comparative Example 4, which was treated with ultrasound alone, showed the highest residual DNA concentration at approximately 74.5±4.43 ng / mg; Comparative Example 3, which underwent physical treatment alone, showed a residual DNA concentration of approximately 60±4.39 ng / mg; and Comparative Examples 1 and 2, which underwent chemical treatment alone, recorded approximately 12±1.82 ng / mg and 25.5±3.11 ng / mg, respectively. Meanwhile, Example 1, the decellularized tissue of the present invention, showed the lowest residual DNA concentration at approximately 9.75±2.21 ng / mg (Fig. 1).

[0068] Accordingly, the decellularization method of the present invention contains residual DNA of 10 ng / mg or less, and compared to the ultrasonic-only treatment group with the highest residual DNA concentration, the decellularization method of the present invention can have an excellent cell removal effect of removing more than 85% of residual DNA.

[0070] Experimental Example 2. Analysis of Extracellular Matrix Components - Collagen and GAG

[0071] The components of the decellularized tissue preserved by the decellularization method of the present invention (Example 1) and its control group (Comparative Examples 1 to 4) were analyzed to confirm whether the major components of the extracellular matrix were well preserved. Collagen, one of the major components of the extracellular matrix, was quantified using a hydroxyproline assay, and GAG was analyzed using a DMMB assay.

[0072] As a result of analyzing the collagen loss rate of the extracellular matrix, Comparative Example 1, which underwent chemical treatment with 1% SDS alone, recorded a collagen loss rate of 44.75±4.57%, followed by Comparative Example 2, which underwent chemical treatment with 1% Triton X-100 alone, recording a collagen loss rate of 31±3.65%; thus, it was confirmed that the chemical-alone treatment group is the decellularization method exhibiting the highest collagen loss rate. Additionally, Comparative Example 4, the ultrasound-alone treatment group, recorded a collagen loss rate of approximately 20.25±1.70%, while Comparative Example 3, which underwent physical treatment alone, recorded the lowest collagen loss rate of approximately 11±1.82%. Meanwhile, the decellularized tissue of the present invention (Example 1) recorded a collagen loss rate of approximately 15±2.16% (Fig. 2a).

[0073] In addition, analysis of the GAG ​​loss rate of the extracellular matrix revealed that Comparative Example 1, which underwent chemical treatment with 1% SDS alone, recorded a GAG loss rate of 48.25±4.5%, followed by Comparative Example 2, which underwent chemical treatment with 1% Triton X-100 alone, recording a GAG loss rate of 36±3.65%; thus, it was confirmed that the chemical-alone treatment group exhibited the highest GAG loss rate among decellularization methods. Furthermore, Comparative Example 4, the ultrasonic-alone treatment group, recorded a GAG loss rate of approximately 24.25±2.75%, while Comparative Example 3, which underwent physical treatment alone, recorded the lowest GAG loss rate of approximately 14.5±2.08%. Meanwhile, the decellularized tissue of the present invention (Example 1) recorded a GAG loss rate of approximately 20±1.63% (Fig. 2b).

[0074] Therefore, through the above results, it was confirmed that the decellularized tissue produced by the decellularization method according to the present invention can preserve the major components of the extracellular matrix (collagen and GAGs) well, and thereby maintain the three-dimensional structure of the heart tissue well.

[0076] Experimental Example 3. Evaluation of Biomechanical Properties - Tensile Strength and Elastic Modulus

[0077] 3-1. Tensile Strength Analysis

[0078] Tensile strength among the biomechanical properties of the decellularized tissue of the present invention (Example 1) and its control group (Comparative Examples 1 to 4) was evaluated to confirm whether the biomechanical properties of the extracellular matrix are well preserved.

[0079] For the analysis of tensile strength, Example 1 and Comparative Examples 1 to 4 were cut into uniform sizes, and at least three samples were prepared for each sample to ensure statistical significance. All samples were stored in PBS to maintain moisture until measurement, and the exact thickness and width of the samples were measured at least three times with calipers immediately before measurement, and the average value was calculated. Tensile strength was analyzed using an Instron mechanical testing machine (load cell 10 N, crosshead speed 50 mm / min).

[0080] The ultimate tensile strength (UTS) for each sample was calculated using the following Equation 1, and the final tensile strength loss rate (%) was calculated using the following Equation 2 by comparing the UTS of each treatment group with the UTS of the tissue before decellularization.

[0081] [Mathematical Formula 1]

[0082] UTS (Maximum Tensile Strength) = Maximum Load (N) / Initial Cross-sectional Area (mm²) 2 )

[0083] (Here, the initial cross-sectional area is average thickness x average width.)

[0084] [Mathematical Formula 2]

[0085] Tensile strength loss rate (%) = [1 - ((UTS by treatment group) / (UTS of pre-decellularized tissue))] X 100

[0086] In Experimental Example 3, biomechanical properties were analyzed using Instron equipment.

[0087] As a result of analyzing the tensile strength loss rate of the extracellular matrix, the tensile strength loss rate of Comparative Example 1, which was chemically treated alone with 1% SDS, was approximately 100±4.39%, and the tensile strength loss rate of Comparative Example 2, which was chemically treated alone with 1% Triton X-100, was approximately 51±4.54%, confirming that the greatest tensile strength loss occurred in the group treated alone with chemicals. In addition, Comparative Example 4, which was treated alone with ultrasound, recorded a tensile strength loss rate of approximately 29.25±3.09%, and Comparative Example 4, which was treated alone with physical treatment, recorded the lowest tensile strength loss rate of approximately 9.5±2.64%. Meanwhile, the decellularized tissue of the present invention (Example 1) recorded a tensile strength loss rate of approximately 15±3.65% (Fig. 3a).

[0088] 3-2. Elasticity Modulus Analysis

[0089] The elastic modulus among the biomechanical properties of the decellularized tissue of the present invention (Example 1) and its control group (Comparative Examples 1 to 4) was evaluated to confirm whether the biomechanical properties of the extracellular matrix are well preserved.

[0090] For the analysis of elastic modulus, Example 1 and Comparative Examples 1 to 4 were cut into uniform sizes, and at least three samples were prepared for each sample to ensure statistical significance. All samples were stored in PBS to maintain moisture until measurement, and the exact thickness and width of the samples were measured at least three times with calipers immediately before measurement to calculate the average value. The elastic modulus was analyzed using an Instron mechanical testing machine (load cell 10 N, crosshead speed 1 mm / min).

[0091] The Young's modulus for each sample was calculated using the slope of the linear region (generally the 0-5% strain range) in the stress-strain curve as shown in Equation 3, and the final modulus was calculated as the average of three repeated measurements of each sample.

[0092] [Mathematical Formula 3]

[0093] Young's modulus (E) = Δσ / Δε (slope in the linear region)

[0094] (Here, stress (σ) is load (N) / initial cross-sectional area (mm²), and strain (ε) is displacement (mm) / initial length (mm).)

[0095] Next, the elasticity modulus (E) of each treatment group was finally compared with the elasticity modulus of the tissue before decellularization, and the final elasticity loss rate (%) was calculated using the following mathematical formula 4.

[0096] [Mathematical Formula 4]

[0097] Elasticity loss rate (%) = [1 - ((E by treatment group) / (E of tissue before decellularization))] X 100

[0098] As a result of analyzing the elastic loss rate of the extracellular matrix, the elastic loss rate of Comparative Example 1, which was chemically treated alone with 1% SDS, was approximately 96.25±3.40%, and the elastic loss rate of Comparative Example 2, which was chemically treated alone with 1% Triton X-100, was approximately 48±4.24%, confirming that the greatest loss in tensile strength occurred in the group treated alone with chemicals. In addition, Comparative Example 4, which was treated only with ultrasound, recorded an elastic loss rate of approximately 29.5±1.29%, and Comparative Example 4, which was treated only with physical treatment, recorded the lowest elastic loss rate of approximately 9.4±2.32%. Meanwhile, the decellularized tissue of the present invention (Example 1) recorded an elastic loss rate of approximately 15.1±4.69% (Fig. 3b).

[0099] Therefore, through the above results, it was confirmed that the decellularization method according to the present invention can maintain biomechanical properties, such as tensile strength and elasticity, at least 85%.

[0101] Although the embodiments and experimental examples of the present invention have been described above, the present invention is not limited to the above embodiments and experimental examples and can be manufactured in various different forms. A person skilled in the art will understand that the present invention can be implemented in other specific forms without changing the technical concept or essential features of the invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

Claim 1 a) a physical treatment step of repeating a freeze-thaw process of freezing separated heart tissue and then thawing it at room temperature; b) a chemical treatment step of immersing the physically treated heart tissue in a surfactant solution; and c) a step of treating the chemically treated heart tissue with ultrasound; comprising a decellularization method. Claim 2 A decellularization method according to claim 1, wherein the physical treatment step a) involves repeating the freeze-thaw process 2 to 5 times. Claim 3 A decellularization method according to paragraph 2, wherein the physical treatment step a) involves washing 2 to 5 times with a buffer solution after the freeze-thaw process is completed. Claim 4 A decellularization method according to claim 1, characterized in that the surfactant solution in the chemical treatment step b) is a nonionic surfactant or an anionic surfactant. Claim 5 A decellularization method according to claim 1, characterized in that the chemical treatment step b) is performed under stirring conditions of 40 to 100 rpm. Claim 6 A decellularization method according to claim 1, wherein the method is characterized by d) additionally washing the ultrasonically treated heart tissue with a buffer solution 2 to 5 times to remove fine residues. Claim 7 A decellularization method according to claim 1, characterized in that the heart tissue is cut into pieces of a size of 0.5 to 2 cm³. Claim 8 Decellularized tissue produced by the decellularization method of claim 1. Claim 9 In claim 8, the decellularized tissue is characterized by having a DNA residue of 0 to 25 ng / mg. Claim 10 In claim 8, the decellularized tissue is characterized by preserving 80 to 100% of collagen compared to before decellularization. Claim 11 In claim 8, the decellularized tissue is characterized by preserving 75 to 100% of glycosaminoglycans (GAGs) compared to before decellularization.

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