Water-soluble decellularization substrate for supercritical carbon dioxide process

Abstract

According to one embodiment of the method, the content and physiological activity of physiologically active substances within the decellularized substrate can be maintained, and a low osmotic pressure can be maintained during the dissolution process of the decellularized substrate through supercritical carbon dioxide treatment. Therefore, the water-soluble decellularized substrate composition produced by one embodiment of the method and the decellularized substrate material containing it not only have low biotoxicity and excellent tissue regeneration ability, but can also be efficiently compounded with other substances, and can be effectively used as a cosmetic or medical material.

Description

Technical Field

[0001] The present invention relates to a method for producing a water-soluble decellularized matrix having improved physiological activity performance.

Background Art

[0002] The extracellular matrix (ECM) is a non-cellular structure of a tissue or organ composed of various macromolecules secreted from cells. The extracellular matrix can be used as a natural scaffold material that is structurally complete and can be used according to biological purposes. These specific compositions and functions are variously manifested depending on the form of the specific source tissue.

[0003] The decellularization process is a process of separating extracellular matrix components from a biological tissue through chemical treatment. Through the decellularization process, components having immunogenicity such as cell components are removed from the biological tissue, while physiological active substances involved in the embodiment of the environment and function of the biological tissue, such as extracellular matrix and some growth factor proteins, can be preserved. Therefore, the decellularized matrix containing various physiological active substances thus preserved, such as collagen, glycosaminoglycan, various cytokines, etc., promotes tissue regeneration and provides a more natural biomimetic microenvironment for cell growth and differentiation. Such a decellularized matrix can be used alone or in a composite state with other substances for tissue regeneration and the like.

[0004] On the other hand, during the decellularization process or the dissolution process of the decellularized extracellular matrix produced through it, physiologically active substances within the decellularized matrix are removed or denatured depending on reaction conditions such as solvent, degrading enzymes, temperature conditions, and pH conditions, resulting in a decrease in physiological activity. Furthermore, methods utilizing existing surfactants result in the decellularized matrix containing residual surfactants, which can be toxic when applied to the body. In addition, the osmotic pressure of the composition containing the decellularized matrix increases depending on the reaction conditions, and this increase in osmotic pressure reduces the mixing level with other substances, leading to a decrease in the efficiency of compounding with other cosmetic or medical materials or components.

[0005] To address the aforementioned problems, the inventors have developed a technology for producing a decellularized substrate composition that maintains the content and physiological activity of physiologically active substances within the decellularized substrate, while simultaneously maintaining a low osmotic pressure and facilitating complexation with other substances. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] One embodiment provides a method for dissolving a decellularizing substrate, comprising the step of treating a solution containing a decellularizing substrate powder with supercritical carbon dioxide, wherein the step of treating with supercritical carbon dioxide is performed at 31 to 60°C.

[0007] Another embodiment provides a method for producing a water-soluble decellularizing substrate composition, comprising the step of treating a solution containing a decellularizing substrate powder with supercritical carbon dioxide, wherein the step of treating with supercritical carbon dioxide is carried out at 31 to 60°C.

[0008] Another embodiment provides a water-soluble decellularized substrate composition produced by the method for producing the water-soluble decellularized substrate composition described above.

[0009] Other objects and advantages of the present invention will become even clearer from the following detailed description, along with the attached claims and drawings. Any matters not described herein are readily apparent and can be inferred by any skilled person in the art of the present invention or a similar art, and are therefore omitted from this description. [Means for solving the problem]

[0010] Throughout this specification, when a part "includes" a component, unless otherwise specified, this means that it includes other components, rather than excluding them. Furthermore, unless the context explicitly states otherwise, each step may be performed in a different order than that specified. That is, each step may be performed in the same order as specified, substantially simultaneously, or in reverse order.

[0011] One embodiment provides a method for dissolving a decellularizing substrate, comprising the step of treating a solution containing a decellularizing substrate powder with supercritical carbon dioxide, wherein the step of treating with supercritical carbon dioxide is performed at 31 to 60°C.

[0012] The term "decellularized substrate" as used herein may be used interchangeably with "decellularized tissue," "decellularized extracellular matrix," or "decellularized material." The decellularized substrate refers to tissues and organs of humans or animals such as pigs and cattle that have undergone decellularization, removing the extracellular matrix and other cellular components, such as the nucleus, cell membrane, and nucleic acids. On the other hand, a "decellularized organ" refers to an organ, such as a heart or kidney, that has been decellularized while maintaining the overall structure of the organ. It is a concept that is clearly distinguished from the "decellularized substrate" as defined herein because it is impossible to formulate the extracellular matrix into a water-soluble dosage form, as the purpose is to re-transplant cells or to use the organ while maintaining its overall physical structure.

[0013] In one specific example, the decellularized matrix is ​​derived from human or animal tissue such as pigs or cattle, including skin tissue, adipose tissue, heart tissue, corneal tissue, or cartilage tissue, but is not limited to these.

[0014] The extracellular matrix (ECM) refers to a complex collection of biomolecules that fill the space inside or outside tissues. The extracellular matrix consists of a variety of molecules synthesized by cells and secreted and accumulated outside the cell, such as fibrous proteins, complex proteins like proteoglycans, and cell-adhering proteins like fibronectin and laminin. Therefore, the composition of the extracellular matrix differs depending on the type of cell from which it originates or the degree of cell differentiation.

[0015] On the other hand, the reaction conditions of the decellularization process, such as the solvent used, the degrading enzyme, the temperature conditions, and the pH conditions, actually affect the content of physiologically active substances in the decellularized substrate, the degree of their physiological activity, the residue of toxic substances (e.g., surfactants), and the osmotic pressure.

[0016] As used herein, the term "solution" means a state in which two substances are mixed, for example, a state in which a solute and a solvent are mixed. In one specific example, the solution containing the decellularized substrate powder means a mixture, suspension, or aqueous solution in which the decellularized substrate powder is contained in distilled water. In this specification, the term "solution" may be used interchangeably with mixture, suspension, aqueous solution, composition, dissolve, aqueous solution, etc.

[0017] As used herein, the term "dissolution" means the phenomenon in which a solute is mixed with a solvent, for example, an increase in the solubility of the solute in the solvent compared to its existing state. In one specific example, the method for dissolving the decellularized substrate means increasing the solubility of the decellularized substrate powder in distilled water with a mixture containing the powder. In another specific example, the method for dissolving the decellularized substrate can increase the solubility of the powder by lowering the osmotic pressure of the solution compared to methods that treat with acidic or alkaline solvents. In another specific example, the method for dissolving the decellularized substrate can increase the solubility while maintaining the pH of the solution at a neutral level. In another specific example, the method for dissolving the decellularized substrate may increase the solubility while maintaining the physiological activity of the physiologically active substances within the decellularized substrate, or it may increase the solubility while maintaining the biocompatibility of the solution.

[0018] The decellularized substrate powder is also a dried powder of the extracellular matrix that has undergone the decellularization process, which is obtained by drying the decellularized extracellular matrix and then pulverizing it. For example, it is obtained by drying the decellularized extracellular matrix that has undergone the decellularization process and then pulverizing it. The drying method for producing the extracellular matrix in a dry state is not particularly limited and can be any method commonly used in the industry. Non-limiting examples of the drying method include air drying, natural drying, spray drying, freeze-drying, and vacuum drying. These methods can be used individually or at least two methods can be used together. In one specific example, the decellularized substrate dried powder is a freeze-dried powder of the decellularized substrate.

[0019] In one specific example, the method for dissolving the decellularized substrate powder may be a method of dissolving the decellularized substrate powder in distilled water, and may not further include a step of treating with an acidic or alkaline solvent.

[0020] The solution containing the decellularizing substrate powder comprises the decellularizing substrate powder and distilled water. In one specific example, the decellularizing substrate powder is contained in distilled water in an amount of 0.01 to 5% by weight, for example, 0.05 to 5% by weight, 0.1 to 5% by weight, 0.2 to 5% by weight, 0.3 to 5% by weight, 0.4 to 5% by weight, 0.5 to 5% by weight, 0.6 to 5% by weight, 0.7 to 5% by weight, 0.8 to 5% by weight, 0.9 to 5% by weight, 1.0 to 5% by weight, 0.05 to 4% by weight, 0.1 to 4% by weight, 0.2 to 4% by weight, 0.3 to 4% by weight. , 0.4 to 4% by weight, 0.5 to 4% by weight, 0.6 to 4% by weight, 0.7 to 4% by weight, 0.8 to 4% by weight, 0.9 to 4% by weight, 1.0 to 4% by weight, 0.05 to 3% by weight, 0.1 to 3% by weight, 0.2 to 3% by weight, 0.3 to 3% by weight, 0.4 to 3% by weight, 0.5 to 3% by weight, 0.6 to 3% by weight, 0.7 to 3% by weight, 0.8 to 3% by weight, 0.9 to 3% by weight, or 1.0 to 3% by weight, but not limited to these.

[0021] The step of treating with supercritical carbon dioxide includes injecting supercritical carbon dioxide into a reactor containing decellularization substrate powder and distilled water, which serves to dissolve the decellularization substrate powder in the distilled water. The composition containing the thus dissolved decellularization substrate can subsequently be formed into a dosage form with desired properties or compounded with other substances and used as an efficient cosmetic or medical material. The steps of injecting the decellularization substrate powder and distilled water into the reactor and injecting supercritical carbon dioxide can be performed simultaneously or sequentially.

[0022] In one specific example, the step of treating the supercritical carbon dioxide is carried out at a reactor temperature of 31 to 60°C. If the temperature in the reactor is lower than this temperature range, the decellularized substrate powder may not dissolve sufficiently, and conversely, if the temperature in the reactor is higher, the active ingredients such as physiologically active substances in the decellularized substrate may decompose or undergo other denaturation. For example, the step of processing supercritical carbon dioxide may be carried out under conditions where the temperature inside the reactor is 31 to 55°C, 31 to 50°C, 31 to 45°C, 31 to 40°C, 31 to 39°C, 31 to 38°C, 31 to 37°C, 32 to 40°C, 33 to 40°C, 34 to 40°C, 35 to 40°C, 32 to 39°C, 33 to 39°C, 34 to 39°C, 35 to 39°C, 32 to 38°C, 33 to 38°C, 34 to 38°C, 35 to 38°C, 32 to 37°C, 33 to 37°C, 34 to 37°C, or 35 to 37°C, but is not limited to these conditions.

[0023] In one specific example, the step of processing the supercritical carbon dioxide is carried out in a reactor, and carbon dioxide gas is injected into the reactor so that the pressure inside the reactor is 70 to 400 bar. If the pressure inside the reactor is lower than the aforementioned pressure range, the decellation substrate powder may not be sufficiently dissolved. For example, if the pressure inside the reactor is 80 to 400 bar, 90 to 400 bar, 100 to 400 bar, 110 to 400 bar, 120 to 400 bar, 130 to 400 bar, 140 to 400 bar, 150 to 400 bar, 160 to 400 bar, 170 to 400 bar, 180 to 400 bar, 190 to 400 bar, 150 to 390 bar, 150 to 380 bar, 150 to 370 bar, 150 to 360 bar, 150 to 350 bar, 1 Carbon dioxide gas is injected into the reactor to a pressure of 60 to 350 bar, 170 to 350 bar, 180 to 350 bar, 190 to 350 bar, 200 to 350 bar, 210 to 350 bar, 220 to 350 bar, 230 to 350 bar, 240 to 350 bar, 250 to 350 bar, 260 to 350 bar, 270 to 350 bar, 280 to 350 bar, 290 to 350 bar, or 300 to 350 bar, but is not limited to these.

[0024] In one specific example, the step of treating the supercritical carbon dioxide may be carried out for 2 to 24 hours, for example, by maintaining the pressure in the reactor for 2 to 24 hours. For example, 3 to 23 hours, 4 to 22 hours, 5 to 21 hours, 6 to 20 hours, 6 to 19 hours, 6 to 18 hours, 6 to 17 hours, 6 to 16 hours, 6 to 15 hours, 6 to 14 hours, 6 to 13 hours, or 6 to 12 hours, but not limited to these. For example, the step of treating the supercritical carbon dioxide may include the step of maintaining the pressure in the reactor for 6 to 12 hours.

[0025] In one specific example, the method for dissolving the decellularized substrate powder does not include the step of treating it with an acid or alkaline solution.

[0026] The method for dissolving the acellular matrix powder according to an embodiment includes a supercritical carbon dioxide treatment step performed at a specific temperature, which is different from the existing dissolution methods. As an effect starting from this, subsequent steps of treating with an acid or alkali solution and subsequent steps of treating with a digestive enzyme are not included, so the efficiency of the manufacturing process is high and a separate purification process is unnecessary. Thereby, the water-soluble acellular matrix composition produced by the method according to an embodiment maintains the content of the bioactive substance and its bioactive ability, maintains a low osmotic pressure, is easy to mix with other components or substances, and exhibits excellent biocompatibility.

[0027] The acellular matrix is produced by a decellularization method including a step of injecting supercritical carbon dioxide into a reactor containing a biological tissue and ethanol, such that the pressure in the reactor becomes 70 to 400 bar. The step of injecting supercritical carbon dioxide into the reactor containing the biological tissue and ethanol is performed at 31 to 60°C.

[0028] In one specific example, the biological tissue is also, but not limited to, the skin tissue, adipose tissue, heart tissue, corneal tissue, or cartilage tissue of a human or an animal such as a pig or a cow.

[0029] The step of injecting supercritical carbon dioxide into the reactor containing the biological tissue and ethanol is performed such that the step of injecting the biological tissue and ethanol into the reactor and the step of injecting supercritical carbon dioxide are performed simultaneously or sequentially, which serves to remove components having immunogenicity, such as cell components in the biological tissue, through high pressure.

[0030] In one specific example, the step of injecting supercritical carbon dioxide into a reactor containing the biological tissue and ethanol is performed at 31 to 60°C. If the temperature inside the reactor is lower than the aforementioned temperature range, the decellularization process may not be carried out sufficiently, and there is a risk that the immunogenicity of the decellularized substrate may not be sufficiently removed. If the temperature inside the reactor is higher, there is a risk of denaturation, such as the decomposition of physiologically active substances and other active ingredients in the extracellular matrix during the decellularization process. For example, the step of processing supercritical carbon dioxide may be carried out under conditions where the temperature inside the reactor is 31 to 55°C, 31 to 50°C, 31 to 45°C, 31 to 40°C, 31 to 39°C, 31 to 38°C, 31 to 37°C, 32 to 40°C, 33 to 40°C, 34 to 40°C, 35 to 40°C, 32 to 39°C, 33 to 39°C, 34 to 39°C, 35 to 39°C, 32 to 38°C, 33 to 38°C, 34 to 38°C, 35 to 38°C, 32 to 37°C, 33 to 37°C, 34 to 37°C, or 35 to 37°C, but is not limited to these conditions.

[0031] In one specific example, the step of injecting supercritical carbon dioxide into a reactor containing the biological tissue and ethanol involves injecting carbon dioxide into the reactor so that the pressure inside the reactor is between 70 and 400 bar. If the pressure inside the reactor is lower than the aforementioned pressure range, the decellularization process may not be carried out sufficiently, and there is a risk that the immunogenicity of the decellularized substrate may not be sufficiently removed. For example, the pressure inside the reactor may be between 80 and 400 bar, 90 and 400 bar, 100 and 400 bar, 110 and 400 bar, 120 and 400 bar, 130 and 400 bar, 140 and 400 bar, 150 and 400 bar, 150 and 390 bar, 150 and 380 bar, 150 and 370 bar, 150 and 360 bar, 150 and 350 bar, 160 and 350 bar, or 170 and 350 bar. Supercritical carbon dioxide is injected to a pressure of 180 to 350 bar, 190 to 350 bar, 200 to 350 bar, 210 to 350 bar, 220 to 350 bar, 230 to 350 bar, 240 to 350 bar, 250 to 350 bar, 260 to 350 bar, 270 to 350 bar, 280 to 350 bar, 290 to 350 bar, or 300 to 350 bar, but is not limited to these.

[0032] In one specific example, the step of injecting supercritical carbon dioxide into a reactor containing the biological tissue and ethanol may be performed for 1 to 24 hours, for example, maintaining the pressure inside the reactor for 1 to 24 hours. For example, 1 to 23 hours, 1 to 22 hours, 1 to 21 hours, 1 to 20 hours, 1 to 19 hours, 1 to 18 hours, 1 to 17 hours, 1 to 16 hours, 1 to 15 hours, 1 to 14 hours, 1 to 13 hours, or 1 to 12 hours, 2 to 12 hours, 2 to 11 hours, 2 to 10 hours, 2 to 9 hours, 2 to 8 hours, 2 to 7 hours, or 2 to 6 hours, but is not limited to these. For example, the step of processing the supercritical carbon dioxide may include maintaining the pressure inside the reactor for 2 to 6 hours.

[0033] The decellularization method may further include a pretreatment step prior to the step of contacting the biological tissue with ethanol in a reactor. Through this pretreatment process, contaminants, fats, etc., can be removed from the biological tissue.

[0034] The decellularization method further includes the step of treating a mixture obtained by injecting supercritical carbon dioxide into a reactor containing biological tissue and ethanol with a DNA-degrading enzyme, which serves to degrade any remaining DNA in the mixture and remove the immunogenicity of the mixture. In one specific example, the DNA-degrading enzyme is contained in distilled water at a concentration of 0.01 to 1 ug / mL, and is, for example, 0.01 to 0.9 ug / mL, 0.01 to 0.8 ug / mL, 0.01 to 0.7 ug / mL, 0.01 to 0.6 ug / mL, 0.01 to 0.5 ug / mL, 0.01 to 0.4 ug / mL, 0.01 to 0.4 ug / mL, 0.01 to 0.3 ug / mL, 0.01 to 0.2 ug / mL, or 0.01 to 0.1 ug / mL, but is not limited to these.

[0035] The decellularization method may further include a washing step of treating the mixture obtained by the step of treating the DNA-degrading enzyme with distilled water to remove residual contaminants.

[0036] Another embodiment provides a method for producing a water-soluble decellularizing substrate composition, comprising the step of treating a solution containing a decellularizing substrate powder with supercritical carbon dioxide, wherein the step of treating with supercritical carbon dioxide is performed at 31 to 60°C.

[0037] Another embodiment provides a water-soluble decellularized substrate composition produced by the method for producing the water-soluble decellularized substrate composition described above.

[0038] The terms or elements used in the description of the method for producing the water-soluble decellularizing substrate composition and the water-soluble decellularizing substrate composition produced thereby are the same as those already described above.

[0039] The aforementioned water-soluble decellularizing substrate composition means a composition comprising an aqueous solution containing a decellularizing substrate, and also a composition comprising a decellularizing substrate dissolved by the method for dissolving decellularizing substrate powder according to one specific example. [Effects of the Invention]

[0040] According to one embodiment of the method, the content and activity of physiologically active substances within the decellularized substrate can be maintained during the dissolution process of the decellularized substrate through supercritical carbon dioxide treatment, and a low osmotic pressure can be maintained.

[0041] Therefore, a water-soluble decellularized substrate composition produced by one embodiment and a decellularized substrate material containing the same have low biotoxicity, excellent tissue regeneration capabilities, and can be efficiently compounded with other substances, making them effective for use as cosmetic or medical materials. [Brief explanation of the drawing]

[0042] [Figure 1] This is the result of visually inspecting the freeze-dried powder of a decellularized matrix derived from porcine skin tissue according to one example. [Figure 2] This is the result of visually inspecting the freeze-dried powder of a decellularized substrate derived from porcine heart tissue according to one example. [Figure 3] This is a confirmation of the results obtained by dissolving a freeze-dried powder of decellularized porcine skin tissue-derived substrate by supercritical carbon dioxide injection, as described in one example. [Figure 4] This is a confirmation of the results obtained by dissolving a freeze-dried powder of decellularized porcine skin tissue-derived substrate by supercritical carbon dioxide injection, as described in one example. [Figure 5] This is a confirmation of the results obtained by dissolving a freeze-dried powder of a decellularized substrate derived from porcine heart tissue, as described in one example, through supercritical carbon dioxide injection. [Figure 6] This is a comparison of the pH of compositions obtained by dissolving the lyophilized substrate powder according to one example. [Figure 7] This is a comparison of the osmotic pressure of compositions obtained by dissolving a lyophilized substrate powder according to one embodiment. [Figure 8] This is a comparison of the molecular weight of collagen in the decellularized substrate using a dissolution method for lyophilized substrate powder according to one embodiment. [Figure 9] This is a comparison of the Poly Dispersity Index (PDI) of collagen in decellularized substrates using a dissolution method for freeze-dried decellularized substrate powder according to one example. [Modes for carrying out the invention]

[0043] The invention will be explained in more detail below through the examples provided. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

[0044] Manufacturing Example 1. Production of Decellularized Substrate 1-1. Production of decellularized matrix derived from skin tissue Pig skin tissue was prepared, and the keratin was removed with a knife. This was followed by a pretreatment process to remove fat by stirring with 70% ethanol at room temperature for 4 hours. Subsequently, 20 g of the skin tissue and 100 ml of 100% ethanol were placed in a supercritical fluid reactor (ILSHIN AUTOCLAVE, South Korea), and the temperature was set to 35°C. Carbon dioxide was then injected to adjust the pressure to 300 bar, and the reaction was carried out at the above temperature and pressure conditions for 6 hours. The reacted tissue was treated with a DNA-degrading enzyme solution (0.01 ug / mL), stirred for 24 hours, and then washed with distilled water. After washing, the tissue was freeze-dried and powdered using a freeze-miller. The results of visual inspection of the decellularized matrix powder derived from pig skin tissue produced in this way are shown in Figure 1.

[0045] 1-2. Production of decellularized substrate derived from cardiac tissue Pig heart tissue was prepared and pretreated by stirring with a 100 U / ml heparin solution for 4 hours to remove blood, and then stirring with 70% ethanol at room temperature for 4 hours to remove fat. Subsequently, 20 Gg of the heart tissue and 100 ml of 100% ethanol were placed in a supercritical fluid reactor (ILSHIN AUTOCLAVE, South Korea), and the temperature was set to 35°C. Carbon dioxide was then injected to adjust the pressure to 300 bar, and the reaction was carried out at the above temperature and pressure conditions for 6 hours. The reacted tissue was treated with a DNA-degrading enzyme solution (0.01 ug / mL), stirred for 24 hours, and then washed with distilled water. After washing, the tissue was freeze-dried and powdered using a freeze-miller. The results of visual inspection of the porcine heart tissue-derived decellularized substrate powder produced in this way are shown in Figure 2.

[0046] Example 1. Dissolution of decellularized substrate powder via supercritical carbon dioxide process 1-1. Dissolution of decellularized matrix powder derived from skin tissue The dried powder of the decellularized substrate, produced as described in Production Example 1-1, was mixed with distilled water at a concentration of 1.0% by weight. This mixture was then placed in a supercritical fluid-equipped reactor (ILSHIN AUTOCLAVE, South Korea), and the temperature was set to 37°C. Subsequently, carbon dioxide was injected to maintain a reactor pressure of 350 bar, and the reaction was carried out under these conditions for 12 hours. As a result, as shown in Figure 3, it was confirmed that the dried powder of the decellularized substrate was sufficiently dissolved in the distilled water.

[0047] The dried powder of the decellularized substrate, prepared as described in Production Example 1-1, was mixed with distilled water at concentrations of 1.0, 3.0, and 5.0% by weight, respectively. This mixture was then placed in a supercritical fluid-equipped reactor (ILSHIN AUTOCLAVE, South Korea), and the temperature was set to 37°C. Subsequently, carbon dioxide was injected to maintain a reactor pressure of 190 bar, and the reaction was carried out under these conditions for 12 hours. As a result, as shown in Figure 4, it was confirmed that the dried powder of the decellularized substrate was sufficiently dissolved in the distilled water.

[0048] 1-2. Preparation of aqueous solution of decellularized substrate powder derived from cardiac tissue The dried powder of the decellularized substrate, prepared as described in Production Example 1-2 above, was mixed with distilled water at a concentration of 3.0% by weight. This mixture was then placed in a supercritical fluid-equipped reactor (ILSHIN AUTOCLAVE, South Korea), and the temperature was set to 37°C. Subsequently, carbon dioxide was injected to maintain a reactor pressure of 190 bar, and the reaction was carried out under these conditions for 12 hours. The hydrogel obtained from this reaction was then diluted 10-fold with physiological saline to produce a 0.3% aqueous solution of the decellularized substrate. As a result, as shown in Figure 5, it was confirmed that the dried powder of the decellularized substrate was sufficiently dissolved in the distilled water.

[0049] The method for dissolving the decellularized substrate dry powder through the supercritical carbon dioxide process in this embodiment used distilled water as the solvent and was carried out at a specific temperature below 60°C. Furthermore, it did not involve any additional dissolution methods using acidic or alkaline solvents. In other words, it was confirmed that the decellularized substrate powder could be dissolved through the supercritical carbon dioxide process described above without the need for acidic or alkaline solvents or high temperatures. These results indicate that the method for dissolving the decellularized substrate powder according to this embodiment maintains the content of effective physiologically active substances in the decellularized substrate, prevents denaturation such as thermal decomposition, increases its physiological activity, has low or no toxicity to biological tissues, possesses excellent biocompatibility, and enables a low osmotic pressure of the composition, thus contributing to efficient compounding with other components.

[0050] Experimental Example 1. Comparison of pH of decellularized substrates by dissolution method (Figure 3) In this experimental example, the pH of the decellularized substrate composition produced by the dissolution method according to one embodiment was compared with the pH of a decellularized substrate composition produced by an existing dissolution method in order to evaluate its pH.

[0051] Comparative Example 1 was prepared by dispersing the decellularization substrate dry powder produced by Production Example 1-1 in 0.1 N HCl at a concentration of 1.0% by weight, and then dissolving it at 60°C for 1 hour. Its pH was compared with that of the decellularization substrate composition produced by Example 1-1.

[0052] As a result, as shown in Figure 6, it was confirmed that the decellularized substrate composition prepared by the dissolution method of one embodiment also maintained a neutral pH. This result indicates that the dissolution method of one embodiment does not require a further step of correcting the pH with an acidic or alkaline substance.

[0053] Experimental Example 2. Comparison of osmotic pressure of decellularized substrates by dissolution method. To compare the osmotic pressure of decellularized substrate compositions based on the dissolution method of the decellularized substrate powder, the osmotic pressure of the compositions from Comparative Example 1 and Example 1-1 was measured using an osmotic pressure analyzer (Osmomat 3000, Gonatec).

[0054] As a result, as shown in Figure 7, it was confirmed that the osmotic pressure of the decellularized substrate composition prepared by the dissolution method of one embodiment was significantly lower than that of the comparative example. This result indicates that the water-soluble decellularized substrate composition prepared by the dissolution method of one embodiment is easily compounded with other substances and has the potential to become an effective material for composite cosmetic or medical materials.

[0055] Experimental Example 3. Measurement of the molecular weight and distribution map of collagen in decellularized substrate by dissolution method. To confirm the collagen content within the decellularized substrate composition based on the dissolution method of the decellularized substrate powder, the following experiment was conducted.

[0056] Comparative Example 2 was prepared by dispersing the decellularized substrate dry powder from Production Example 1-1 at 1% by weight in 0.1N HCl containing pepsin, and then dissolving it at 4°C for 48 hours. Comparative Example 3 was prepared by dispersing the decellularized substrate dry powder from Production Example 1-1 at 1.0% by weight in 0.1N HCl containing pepsin, and then dissolving it at 60°C for 1 hour. The collagen molecular weight and molecular weight distribution of Comparative Examples 2 and 3 were compared with the composition from Example 1-1. The number average molecular weight (Mn), weight average molecular weight (Mw), and Z-average molecular weight (Mz) of the collagen in the decellularized material prepared for each were measured by GPC (gel permeation chromatography) analysis, and the results are shown in Figure 8. Furthermore, the Poly Dispersity Index (PDI = Mw / Mn) was calculated from the above results, and the results are shown in Figure 9.

[0057] Through this, it was confirmed that the decellularized substrate composition prepared by the dissolution method of one example contained collagen not only while maintaining a high molecular weight, but also while maintaining a uniform molecular weight distribution, compared to the comparative examples (Comparative Examples 2 and 3). These results indicate that the physiological activity of the decellularized substrate prepared by the containment method of one example is better maintained.

[0058] The above description of the present invention is illustrative, and a person with ordinary skill in the art to which the invention pertains will understand that it can be readily modified into other specific forms without altering the technical idea or essential features of the invention. Therefore, the above-described embodiments should be understood to be illustrative and not limiting in all respects.

Claims

1. A method for dissolving a decellularizing substrate, comprising the step of treating a solution containing the decellularizing substrate powder with supercritical carbon dioxide, The method involves performing the step of processing the supercritical carbon dioxide at a temperature of 31 to 60°C.

2. The method according to claim 1, wherein the step of treating the supercritical carbon dioxide is carried out in a reactor, and carbon dioxide is injected so that the pressure in the reactor is 70 to 400 bar.

3. The method according to claim 1, wherein the step of treating the supercritical carbon dioxide is performed for 6 to 12 hours.

4. The method according to claim 1, wherein the solution contains 0.1 to 5% by weight of a decellularized substrate powder.

5. The method according to claim 1, wherein the matrix is ​​an extracellular matrix selected from the group consisting of skin tissue, cardiac tissue, adipose tissue, corneal tissue, or cartilage tissue.

6. The aforementioned decellularizing substrate is It is produced by a decellularization method that includes the step of injecting supercritical carbon dioxide into a reactor containing biological tissue and ethanol so that the pressure inside the reactor is between 70 and 400 bar. The method according to claim 1, wherein the step of injecting supercritical carbon dioxide into a reactor containing the biological tissue and ethanol is performed at 31 to 60°C.

7. A method for producing a water-soluble decellularizing substrate composition, comprising the step of treating a solution containing a decellularizing substrate powder with supercritical carbon dioxide, The method involves performing the step of processing the supercritical carbon dioxide at a temperature of 31 to 60°C.

8. A water-soluble decellularization substrate composition produced by the method described in claim 7.

Images