Method for producing highly functional artificial organs using aptamers

Abstract

To provide a composition for producing artificial organs comprising aptamers, to provide a composition for blood vessel coating comprising aptamers, to provide a method for producing artificial organs using aptamers, and to provide the produced artificial organs.SOLUTION: The present invention provides a composition for producing artificial organs comprising aptamers. When the aptamers are used as a coating agent of a decellularized scaffold, the composition enhances blood vessel adhesion ability, viability, and angiogenesis potential, thereby enabling more efficient reconstruction of vasculature than existing antibodies.SELECTED DRAWING: Figure 5a

Description

Technical Field

[0001] The present invention relates to a method for manufacturing a highly functional artificial organ using an aptamer, etc.

[0002] This application claims priority based on Korean Patent Application No. 10-2020-00983 filed on August 6, 2020, and Korean Patent Application No. 10-2020-01 50913 filed on November 12, 2020, and all of the content disclosed in the specifications and drawings of the said applications is incorporated herein by reference.

Background Art

[0003] Artificial organ biotechnology includes various methods of manufacturing substitute devices for body constituent organs, including culturing stem cells and cell growth factors (such as cell fluid nutrients) in a three-dimensional bioretainer and creating artificial organs using this mold. With the development of modern medicine, organ transplantation via donors is carried out for almost all organs. However, due to the shortage of donors, many patients cannot benefit, and currently, artificial organs have insufficient biocompatibility and side effects occur. On the other hand, chronic liver diseases such as cirrhosis are diseases that cause approximately two million patients to die every year. Recently, with the epoch-making development of immunology and organ transplantation over several decades, liver transplantation has been widely performed in end-stage liver diseases, and currently, liver transplantation is recognized as the only treatment method for congenital or acquired liver diseases, but there is a shortage of donors. From this perspective, the production of artificial livers through tissue engineering has attracted attention as a substitute for donor organs. In Korea, the mortality rate due to liver-related diseases is very high compared to other diseases, and due to the shortage of transplant organs, there are a considerable number of transplant candidates. .

[0004] and is one of the countries with the highest number of liver transplants, and there is a need to develop technologies that can improve such a situation. It is necessary.

[0005] For the production of liver scaffolds, various methods such as tissue engineering, application of 3D printing technology, and organoids using stem cells are being studied, but the technical limitations such as the inability to mimic the fine structure of organs and size limitations have not been overcome. As a human organ-mimicking scaffold with safety and functionality that overcomes this, a decellularized scaffold obtained by removing all cells from an animal organ has been evaluated as a useful substrate. While removing cell components that can induce an immune response, an organ-specific microenvironment such as the fine structure and biochemical signals within the scaffold is created, and when human cells are injected, there is an advantage that three-dimensional organization of the cells within the scaffold is easy. Therefore, the decellularized scaffold can be used as an appropriate substrate for producing artificial organs, and research is being conducted to reconstruct various organs using the decellularized scaffold. Since the liver has a very complex vascular structure histologically and is an organ through which more than 25% of the cardiac output passes, organ vascularization can be said to be the core for successful reconstruction of the artificial liver. An artificial organ without a formed vascular structure, after being connected to the bloodstream of the donor after transplantation, is likely to form intravascular thrombi due to an acute immune response, leading to a high possibility of graft failure. Therefore, various coating agents for efficiently reconstructing the vascular structure are being studied. An aptamer is a single-stranded nucleic acid consisting of a short sequence, and has advantages such as high binding affinity to a specific protein, low immunogenicity, and the ability to be mass-produced, so it can be used as an alternative to antibodies. As an alternative to antibodies.

[0006] The liver has a very complex vascular structure histologically and is an organ through which more than 25% of the cardiac output passes. Therefore, for the successful reconstruction of the artificial liver, organ vascularization is crucial. An artificial organ without a formed vascular structure, after being connected to the bloodstream of the donor after transplantation, is likely to form intravascular thrombi due to an acute immune response, leading to a high possibility of graft failure. Therefore, various coating agents for efficiently reconstructing the vascular structure are being studied. An aptamer is a single-stranded nucleic acid consisting of a short sequence, and has advantages such as high binding affinity to a specific protein, low immunogenicity, and the ability to be mass-produced, so it can be used as an alternative to antibodies. As an alternative to antibodies. Therefore, various coating agents for efficiently reconstructing the vascular structure are being studied.

[0007] An aptamer is a single-stranded nucleic acid consisting of a short sequence, and has advantages such as high binding affinity to a specific protein, low immunogenicity, and the ability to be mass-produced, so it can be used as an alternative to antibodies. Since it has advantages such as high binding affinity to a specific protein, low immunogenicity, and the ability to be mass-produced, it can be used as an alternative to antibodies. It is being applied. In addition, aptamers are mainly utilized in medical diagnosis and have attracted attention as therapeutic agents targeting cancer and viral diseases. However, it has not been clearly shown that aptamers have utility as coating agents from the perspective of tissue engineering. Moreover, human leukocyte antigen (HLA) is one of the human histocompatibility antigens. When allogeneic cells or artificial organs are transplanted into a patient, if the HLA types do not match, immune rejection reactions may occur. Recently, many studies have been conducted on universal cells established by removing such HLA-A, B, C types using gene editing. However, cells with HLA-A, B, C removed still have the limitation of being sensitive to NK cells during transplantation. Therefore, by overexpressing the "Don't eat me" signal that suppresses the reaction of such NK cells, it is finally possible to produce cells that can avoid immune reactions. In particular, cells differentiated into each lineage in off-the-shelf universal stem cells (USC)

[0008] can be applied as cell compositions for minimizing the occurrence of immune rejection reactions during the production of artificial organs. Furthermore, human leukocyte antigen (HLA) is one of the human histocompatibility antigens. When allogeneic cells or artificial organs are transplanted into a patient, if the HLA types do not match, immune rejection reactions may occur. Recently, many studies have been conducted on universal cells established by removing such HLA-A, B, C types using gene editing. However, cells with HLA-A, B, C removed still have the limitation of being sensitive to NK cells during transplantation. Therefore, by overexpressing the "Don't eat me" signal that suppresses the reaction of such NK cells, it is finally possible to produce cells that can avoid immune reactions. In particular, cells differentiated into each lineage in off-the-shelf universal stem cells (USC) can be applied as cell compositions for minimizing the occurrence of immune rejection reactions during the production of artificial organs. Moreover, human leukocyte antigen (HLA) is one of the human histocompatibility antigens. When allogeneic cells or artificial organs are transplanted into a patient, if the HLA types do not match, immune rejection reactions may occur. Recently, many studies have been conducted on universal cells established by removing such HLA-A, B, C types using gene editing. However, cells with HLA-A, B, C removed still have the limitation of being sensitive to NK cells during transplantation. Therefore, by overexpressing the "Don't eat me" signal that suppresses the reaction of such NK cells, it is finally possible to produce cells that can avoid immune reactions. In particular, cells differentiated into each lineage in off-the-shelf universal stem cells (USC) can be applied as cell compositions for minimizing the occurrence of immune rejection reactions during the production of artificial organs. (off-the-shelf universal stem cells, USC) can be applied as cell compositions for minimizing the occurrence of immune rejection reactions during the production of artificial organs. Therefore, in this technology, a technique was first established that can maximize the reconstruction efficiency of vascular structures by using nucleic acid aptamers as coating agents and minimize thrombosis after transplantation of artificial organs. Also, through this technology, cells derived from universal stem cells (USC) such as liver constituent cells are used for in vivo transplantation

[0009] Therefore, in this technology, a technique was first established that can maximize the reconstruction efficiency of vascular structures by using nucleic acid aptamers as coating agents and minimize thrombosis after transplantation of artificial organs. Also, through this technology, cells derived from universal stem cells (USC) such as liver constituent cells are used for in vivo transplantation Therefore, in this technology, a technique was first established that can maximize the reconstruction efficiency of vascular structures by using nucleic acid aptamers as coating agents and minimize thrombosis after transplantation of artificial organs. Also, through this technology, cells derived from universal stem cells (USC) such as liver constituent cells are used for in vivo transplantation Therefore, in this technology, a technique was first established that can maximize the reconstruction efficiency of vascular structures by using nucleic acid aptamers as coating agents and minimize thrombosis after transplantation of artificial organs. Also, through this technology, cells derived from universal stem cells (USC) such as liver constituent cells are used for in vivo transplantation It is possible to fabricate a vascularized artificial liver with improved functionality. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] KHHussein, KMPark, KSKang, HMWoo, Heparin-gelatin mixture improves vascular reconstruction efficiency and hepatic function in bioengineered livers, Acta Biomater 38(2016)82-93 Summary of the Invention [Problem to be solved by the invention]

[0011] Thus, the present inventors have demonstrated that nucleic acid aptamers can be used as coating agents to remodel the vasculature. We were the first to establish technology that can maximize construction efficiency and minimize side effects after transplantation of artificial organs. In addition, the present inventors have demonstrated that nucleic acid aptamers can be transplanted in vivo and have enhanced functionality. This led to the completion of the present invention by first confirming that it is possible to fabricate an artificial organ with the above structure. I did.

[0012] Therefore, an object of the present invention is to provide a composition for producing an artificial organ, which contains an aptamer. be.

[0013] Another object of the present invention is to provide a composition for coating blood vessels, which contains an aptamer. .

[0014] It is still another object of the present invention to provide a method for producing an artificial organ using an aptamer. do.

[0015] Another object of the present invention is to provide an artificial organ produced by the above method.

[0016] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those of ordinary skill in the art from the following description.

Means for Solving the Problems

[0017] To achieve the object of the present invention, the present invention provides a composition for producing an artificial organ containing an aptamer.

[0018] The present invention also provides a use of a composition containing an aptamer for producing an artificial organ.

[0019] The present invention also provides a method for producing an artificial organ including a step of treating an aptamer.

[0020] The present invention also provides a composition for vascular coating containing an aptamer.

[0021] The present invention also provides a use of a composition containing an aptamer for vascular coating.

[0022] The present invention also provides a vascular coating method including a step of treating a composition containing an aptamer.

[0023] The present invention also provides an artificial organ produced by the above method.

[0024] In one embodiment of the present invention, the artificial organ may be biocompatible.

[0025] In another embodiment of the present invention, the artificial organ is, for example, an organ including blood vessels, if so limited. Specifically, the target organ may be the liver, but is not limited thereto.

[0026] In yet another embodiment of the present invention, the aptamer is an anti-CD31 aptamer. may be used, but is not limited to this.

[0027] In yet another embodiment of the present invention, the aptamer has a base sequence represented by SEQ ID NO:1. This may include, but is not limited to.

[0028] In yet another embodiment of the present invention, the aptamer is specific to vascular endothelial cells. It is characterized by, but is not limited to,

[0029] In yet another embodiment of the present invention, the aptamer is an aptamer that binds integrin beta 3 and ribosomes. The expression of one or more selected from the group consisting of phosphorylated Akt and However, the present invention is not limited to the above.

[0030] In yet another embodiment of the present invention, the aptamer inhibits the expression of cleaved caspase-3. The present invention can reduce, but is not limited to,

[0031] In yet another embodiment of the present invention, the composition comprises parenchymal cells and non-parenchymal cells. The cell may further comprise one or more cells selected from the group including, but not limited to, .

[0032] In yet another embodiment of the present invention, the cells may be stem cell-derived cells. However, the present invention is not limited to the above.

[0033] In yet another embodiment of the present invention, the cells include stem cell-derived differentiated cells. but is not limited thereto.

[0034] In yet another embodiment of the present invention, the composition may further include stem cell-derived parenchymal cells but is not limited thereto.

[0035] In yet another embodiment of the present invention, the composition may further include stem cell-derived non-parenchymal cells but is not limited thereto.

[0036] In yet another embodiment of the present invention, the stem cells are induced pluripotent stem cells (iPSC), embryonic stem cells, mesenchymal stem cells (MSC), off-the-shelf universal stem cells (USC), marrow-derived stem cells, adipose tissue-derived stem cells, and placenta-derived stem cells Embr yonic Stem Cell), mesenchymal stro mal cells, MSC), off-the-shelf unive rsal stem cells, USC), marrow-derived stem cells, Marrow-deri ved Stem Cell), adipose tissue-derived stem cells, Adipose tissue-de rived Stem Cells) and placenta-derived stem cells, Placenta-der rived Stem Cells), and may be one or more selected from the group consisting of, but is not limited thereto.

[0037] In yet another embodiment of the present invention, the off-the-shelf universal stem cells may have one or more of the following characteristics but is not limited thereto. The HLA gene is removed; and The immunosuppressive signal is overexpressed.

[0038] In yet another embodiment of the present invention, the manufacturing method may include the following steps, but is not limited thereto. a) providing an organ from an individual; b) decellularizing the provided organ; and c) treating the decellularized organ with an aptamer.

[0039] In still another embodiment of the present invention, the manufacturing method may further include one or more steps selected from the group consisting of, but not limited to, 1) d-1) recellularizing the decellularized organ with vascular endothelial cells; d-2) recellularizing the decellularized organ with parenchymal cells; and d-3) recellularizing the decellularized organ with non-parenchymal cells.

Advantages of the Invention

[0040] When the aptamer of the present invention is used as a coating agent for a decellularized carrier, it enhances the ability to adhere to blood vessels, survival ability, angiogenesis ability, etc., enabling more efficient reconstruction of vascular structures than conventional antibodies. Therefore, the vascularized artificial liver produced using an aptamer not only reduces thrombosis in vivo but also shows the effect of enhancing liver functionality, and thus is expected to be applied as a method for treating diseases using an artificial organ produced using the aptamer of the present invention.

Brief Description of the Drawings

[0041]

Figure 1a-1g

Figure 2a-2j

Figure 3a-3l

Figure 4a-4g

Figure 5a-5m

Figure 6a-6e

Figure 7a-7f

Best Mode for Carrying Out the Invention

[0042] The present invention will be described in detail below.

[0043] The present invention provides a composition for manufacturing an artificial organ containing an aptamer.

[0044] The present invention also relates to a composition for vascular coating containing an aptamer.

[0045] Tissue engineering is a method of retaining cells in any structure (mold) using a combination of cells and various substances. Such a structure (mold) is called a scaffold, and the purpose of making it is to create a structure that can replace damaged tissues and organs, that is, to create an organ. There are various types, but a structure (mold) obtained by decellularizing a biological structure (i.e., an organ) to remove cells and leaving only the fine structure and the outline of the organ is called a bioscaffold. and leaving only the fine structure and the outline of the organ is called a bioscaffold. and leaving only the fine structure and the outline of the organ is called a bioscaffold. Although there are various types, a structure (mold) obtained by decellularizing a biological structure (i.e., an organ) to remove cells and leaving only the fine structure and the outline of the organ is called a bioscaffold. (i.e., an organ) to remove cells and leaving only the fine structure and the outline of the organ is called a bioscaffold. After removing the cells, the structure (mold) that retains only the fine structure and the outline of the organ is called a bioscaffold. After removing the cells, the structure (mold) that retains only the fine structure and the outline of the organ is called a bioscaffold.

[0046] The artificial organ of the present invention is characterized in that an aptamer is processed on a bioscaffold and the structure of the target organ, for example, the vascular structure, is reconstructed, but is not limited thereto. Specifically, the aptamer of the present invention can reconstruct or generate a vascular structure by being coated on the vascular wall of a decellularized carrier and specifically binding to vascular endothelial cells. The artificial organ of the present invention is characterized in that an aptamer is processed on a bioscaffold and the structure of the target organ, for example, the vascular structure, is reconstructed, but is not limited thereto. Specifically, the aptamer of the present invention can reconstruct or generate a vascular structure by being coated on the vascular wall of a decellularized carrier and specifically binding to vascular endothelial cells. The artificial organ of the present invention is characterized in that an aptamer is processed on a bioscaffold and the structure of the target organ, for example, the vascular structure, is reconstructed, but is not limited thereto. Specifically, the aptamer of the present invention can reconstruct or generate a vascular structure by being coated on the vascular wall of a decellularized carrier and specifically binding to vascular endothelial cells. The artificial organ of the present invention is characterized in that an aptamer is processed on a bioscaffold and the structure of the target organ, for example, the vascular structure, is reconstructed, but is not limited thereto. Specifically, the aptamer of the present invention can reconstruct or generate a vascular structure by being coated on the vascular wall of a decellularized carrier and specifically binding to vascular endothelial cells. The artificial organ of the present invention is characterized in that an aptamer is processed on a bioscaffold and the structure of the target organ, for example, the vascular structure, is reconstructed, but is not limited thereto. Specifically, the aptamer of the present invention can reconstruct or generate a vascular structure by being coated on the vascular wall of a decellularized carrier and specifically binding to vascular endothelial cells.

[0047] The inventors of the present invention confirmed that when the vascular wall of a decellularized carrier is coated with an anti-CD31 aptamer so that vascular endothelial cells can adhere to the wall of the decellularized carrier, a highly functional vascular structure that maintains the function of the vascular barrier is reconstructed (see the examples of the present invention). The inventors of the present invention confirmed that when the vascular wall of a decellularized carrier is coated with an anti-CD31 aptamer so that vascular endothelial cells can adhere to the wall of the decellularized carrier, a highly functional vascular structure that maintains the function of the vascular barrier is reconstructed (see the examples of the present invention). The inventors of the present invention confirmed that when the vascular wall of a decellularized carrier is coated with an anti-CD31 aptamer so that vascular endothelial cells can adhere to the wall of the decellularized carrier, a highly functional vascular structure that maintains the function of the vascular barrier is reconstructed (see the examples of the present invention).

[0048] In the present invention, the blood vessel may be a blood vessel of a decellularized carrier, but is not limited thereto. In the present invention, the blood vessel may be a blood vessel of a decellularized carrier, but is not limited thereto.

[0049] In the present invention, the blood vessel may be a hepatic blood vessel, for example, the portal vein (portal It may be a vein, hepatic sinusoid, hepatic vein, or hepatic artery, but is not limited thereto. No.

[0050] In the present invention, the artificial organ may be biocompatible. The term "biocompatibility" of the present invention is used interchangeably with "transplant compatibility" and means the property of not causing an immune rejection reaction during cell transplantation. By reducing the expression of genes that cause immune rejection reactions, cells, tissues, or organs can be made transplant-compatible. As a non-limiting example thereof, by reducing the expression level of the HLA gene, cells, tissues, or organs can be made transplant-compatible. Transplant-compatible cells include those in which the immune compatibility antigen is homozygous or null cells, but are not limited thereto.

[0051] In the present invention, the artificial organ can be, for example, a liver, kidney, heart, valve, ureter bladder, lung, pancreas, etc., but is not limited thereto.

[0052] In the present invention, the artificial organ may be an organ containing blood vessels, but is not limited thereto. No.

[0053] In the present invention, an aptamer is a substance that can specifically bind to a target substance and itself means a single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) having a stable tertiary structure, and can specifically bind to a target protein (substance). The production of aptamers is generally by the method for producing aptamers. After binding and synthesizing the sequence of an oligonucleotide having selective and high binding affinity for the target substance to be confirmed, the 5 The -SH, -COOH, -OH or NH 2 can be modified to be able to bind to the functional groups of the aptamer chip, but is not limited to this.

[0054] In the present invention, the aptamer may be an anti-CD31 aptamer, but is not limited thereto.

[0055] In the present invention, the anti-CD31 aptamer may contain the nucleotide sequence represented by SEQ ID NO: 1 or may consist of the nucleotide sequence represented by SEQ ID NO: 1, but is not limited thereto.

[0056] In the present invention, the aptamer is characterized by being specific for vascular endothelial cells, but is not limited thereto. Moreover, the aptamer of the present invention is not limited to targeting vascular endothelial cells to reconstruct blood vessels. When reconstructing organs, its applications can be expanded regardless of the type of organ, such as in the reconstruction of liver parenchyma and bile ducts. That is, when reconstructing the liver parenchyma, an aptamer that binds to a protein specifically expressed by liver parenchymal cells can be used, and when reconstructing the bile ducts, an aptamer that binds to a protein specifically expressed by bile duct cells can be used. When reconstructing the liver parenchyma, an aptamer that binds to a protein specifically expressed by liver parenchymal cells can be used. When reconstructing the bile ducts, an aptamer that binds to a protein specifically expressed by bile duct cells can be used.

[0057] Therefore, the aptamer of the present invention can be specific for a protein specifically expressed by target cells, but is not limited thereto.

[0058] In the present invention, the aptamer can increase the expression of one or more selected from the group consisting of integrin beta 3 and phosphorylated Akt, but is not limited thereto. ​​​​​​​​​

[0059] In the present invention, the aptamer can reduce the expression of cleaved caspase-3, but is not limited thereto.

[0060] In the present invention, the composition can usually be delivered to a tissue or organ matrix in a solution (e.g., a physiological composition) that can be mixed with cells under physiological conditions (e.g., 37°C). The composition may include, but is not limited to, buffers, nutrients (e.g., sugars, carbohydrates), enzymes, growth and / or differentiation media, cytokines, antibodies, inhibitory factors, growth factors, salt solutions, or serum-derived proteins.

[0061] In the present invention, the composition may further include parenchymal cells, but is not limited thereto. The term "parenchymal cells" used in the present invention means cells that constitute the main part performing cell-specific functions. In the present invention, the parenchymal cells specifically may be, for example, liver parenchymal cells, kidney parenchymal cells, corneal parenchymal cells, vascular endothelial cells, etc., preferably liver parenchymal cells, i.e., hepatocytes, but is not limited thereto. In the present invention, liver parenchymal (parenchymal hepatocyt e) cells account for 80% of the whole liver under non-diseased conditions.

[0062] In the present invention, the parenchymal cells may be derived from the same species as the donor, but are not limited thereto.

[0063] In the present invention, the composition may further include stem cell-derived parenchymal cells, but is not limited thereto. The parenchymal cells are those from which human leukocyte antigen has been removed and / or attacked Parenchymal cells differentiated from human universal stem cells that overexpress a signal for inactivation of attack may be used, but are not limited thereto. In the present invention, the composition may further contain non-parenchymal cells, but is not limited thereto.

[0064] The non-parenchymal cells may be one or more selected from the group consisting of liver endothelial cells, Kupffer cells, hepatic stellate cells (hepatic stellate cell, Ito cell, lipocytes, fat-storing cell), bile duct cells, pit cells, vascular epithelial cells, and fibroblasts, but are not limited thereto. In the present invention, the composition may further contain stem cell-derived non-parenchymal cells, but is not limited thereto. The stem cell-derived non-parenchymal cells may be non-parenchymal cells differentiated from human universal stem cells from which human leukocyte antigen has been removed and / or that overexpress a signal for inactivation of attack, but are not limited thereto. patic stellate cell, Ito cell, lipocytes, f at-storing cell), bile duct cells, pit cells, vascular epithelial cells, and fibroblasts fibroblast), but are not limited thereto. In the present invention, the cells are characterized by containing stem cell-derived differentiated cells, but are not limited thereto.

[0065] In the present invention, the term "stem cell" used herein means a cell having the property of continuously producing the same cells as itself over a certain period in an undifferentiated state and the property of differentiating into specific cells under appropriate conditions. In the present invention, the stem cells are induced pluripotent stem cells (Induced Pluripot ency stem cells, iPS cells), embryonic stem cells (Embryonic Stem Cells, ES cells), or tissue stem cells (somatic stem cells), but are not limited thereto.

[0066] In the present invention, the cells are characterized by including stem cell-derived differentiated cells, but are not limited thereto. In the present invention, the term "stem cell" means a cell having the property of continuously producing the same cells as itself over a certain period in an undifferentiated state and the property of differentiating into specific cells under appropriate conditions.

[0067] In the present invention, the stem cells may be induced pluripotent stem cells (Induced Pluripotency stem cells, iPS cells), embryonic stem cells (Embryonic Stem Cells, ES cells), or tissue stem cells (somatic stem cells), but are not limited thereto. In the present invention, the stem cells may be induced pluripotent stem cells (Induced Pluripotency stem cells, iPS cells), embryonic stem cells (Embryonic Stem Cells,

[0068] In the present invention, the stem cells are induced pluripotent stem cells (Induced Pluripot induced pluripotent stem cells (iPSC), embryonic stem cells stem cell), mesenchymal stromal cells , MSC), off-the-shelf universal stem cells (USC), marrow-derived stem cells cell), adipose tissue-derived stem cells m cells) and placenta-derived stem cells cells), and may be one or more selected from the group consisting of, but not limited to this. not.

[0069] In the present invention, the off-the-shelf universal stem cells are characterized by having HLA removed or overexpressing an attack-inactivating signal, but are not limited thereto. The present invention constructs a universal artificial organ containing constituent cells derived from stem cells in which human leukocyte antigen is removed and / or an "attack-inactivating" signal is overexpressed, thereby minimizing the immune rejection reaction during transplantation and being expected to be applicable to clinical practice. removed and / or "attack-inactivating" signal overexpressing stem cell-derived constituent cells By constructing a universal artificial organ, the immune rejection reaction during transplantation is minimized and it is expected to be applicable to clinical practice. possible.

[0070] In the present invention, the off-the-shelf universal stem cells may be HLA class I knockout iPSC cells or HLA class I knockout MSC cells, but are not limited thereto. not.

[0071] In the present invention, the attack-inactivating signal may be CD47 or CD24, but is not limited thereto. not.

[0072] Furthermore, the present invention provides a method for manufacturing an artificial organ including a step of treating an aptamer. In the present invention, the method for manufacturing the artificial organ may include the following steps, but is not limited thereto: : a) providing an organ from an individual; b) decellularizing the provided organ; and c) treating the decellularized organ with an aptamer.

[0073] In the present invention, the method may further include one or more steps selected from the group consisting of d-1) recellularizing the decellularized organ with vascular endothelial cells; ; d-2) recellularizing the decellularized organ with parenchymal cells; and d-3) recellularizing the decellularized organ with non-parenchymal cells, but is not limited thereto.

[0074] In the present invention, the individual can be selected from various mammalian groups such as humans, monkeys, mice, rats, pigs, cows, rabbits, etc.

[0075] In the present invention, the decellularization can be performed by methods known in the art. In one embodiment of the present invention, after washing the hepatic portal vein of the liver obtained from an individual with deionized water and PBS, it was washed with deoxyribonuclease and then sterilized with peracetic acid. For example, the following references describe the perfusion-based decellularization of the lung, liver, kidney, brain, and limbs: Van Put te et al., 2002, Ann. Thorac. Surg., 74(3): 89 3 - 8; den Butter et al., 1995, Transpl. Int., 8: 466 - 71; Firth et al., 1989, Clin. Sci. (Lon d.), 77(6): 657 - 61; Mazzetti et al., 2004, B ​​​rain Res.,999(1):81-90;Wagner et al.,200 3,J.Artif.Organs,6(3):183-91. Alternative to decellularization of perfusion substrate As an alternative, biological tissues and organs can be decellularized by immersion in a decellularization solution that removes cells. See, for example, U.S. Patent Nos. 6,376,244 and 6,75 3,181. See also

[0076] In the present invention, a physiological buffer compatible with perfusion is a nutrient supply that can be used for storage and / or organ perfusion including transplantation, for example, a buffer containing glucose, EGM-2, EGM-2 MV, DMEM, Promocell endothelial cell medium, Medium 200, DMEMF / 12, but is not limited thereto. The buffer also includes a culture medium solution compatible with the culture of endothelial cells or phosphate buffered saline (PBS), but is not limited thereto. but is not limited thereto. but is not limited thereto. but is not limited thereto.

[0077] In the present invention, alternating the direction of perfusion (e.g., forward and reverse) during decellularization can help effectively remove cells from the whole organ or tissue. Decellularization as described herein essentially removes cells from the inside of the organ with little damage to the ECM, but is not limited thereto. Organs or tissues can be decellularized at an appropriate temperature between 4 and 40°C. Depending on the size and weight of the organ or tissue and the concentration of a particular detergent and detergent in the cell lysis medium, the organ or tissue is generally perfused with the cell lysis medium for about 2 to about 12 hours per gram of solid organ or tissue. Organs containing the washing solution can be perfused for about 1 to about 12 hours per gram of tissue. but is not limited thereto. but is not limited thereto. but is not limited thereto. but is not limited thereto. but is not limited thereto. Perfusion is generally regulated by physiological conditions including pulsatile blood flow, flow rate, and pressure and

[0078] In the present invention, recellularization for generating an organ or tissue may involve the number of regenerative cells introduced into and onto a decellularized organ, which may vary depending on the organ (e.g., what kind of organ, and its size and weight), or tissue, as well as the type and developmental stage of the regenerative cells. Different types of cells can have different tendencies regarding the population density they reach. Similarly, different organs or tissues can be recellularized at different densities. For example, a decellularized organ or tissue may be "seeded" with at least 1,000, 10,000, 100,000, 1,000,000, 10,000,000, or 100,000,000 regenerative cells; or may have, after recellularization, from about 1,000 cells / tissue mg (wet weight, i.e., the weight before decellularization) to about 100,000,000 cells / tissue mg (wet weight), but is not limited thereto. and all of the tissue and the type and developmental stage of the regenerative cells. Different types of cells can have different tendencies regarding the population density (p opulation density) they reach. Similarly to this, different organs or tissues can be recellularized at different densities. For example, a decellularized organ or tissue may be "seeded" with at least 1,000, 10,000, 100,000, 1, 000,000, 10,000,000, or 100,000,000 regenerative cells ; or may have, after recellularization, from about 1,000 cells / tissue mg (wet weight i.e., the weight before decellularization) to about 100,000,000 cells / tissue mg (wet weight) but is not limited thereto.

[0079] In the present invention, the method may further include culturing the aptamer-treated organ in a bioreactor for 1 to 100 days, 1 to 90 days, 1 to 80 days, 1 to 70 days, 1 to 60 days, 1 to 50 days, 1 to 40 days, 1 to 1 month, 1 to 3 weeks, 1 to 15 days, 1 to 2 weeks, 5 to 15 days, 5 to 2 weeks, 1 week to 3 weeks, or about 2 weeks, but is not limited thereto. and

[0080] In addition, the present invention provides an artificial organ produced by the above method.

[0081] The artificial organ is an organ for transplantation and does not induce the immune rejection reaction of the donor after transplantation. It may be so, but it is not limited thereto.

[0082] The artificial organ may be used for therapeutic agent screening applications, preferably for liver disease treatment agent screening applications, but it is not limited thereto.

[0083] Throughout this specification, when any part states that a certain component "includes", this means that, unless otherwise stated to the contrary, it does not exclude other components, but may further include other components. The terms "about", "substantially", etc. used throughout this specification are used to present manufacturing and material tolerances inherent in the stated meaning, and are used as a meaning equal to or close to that numerical value, in order to prevent unscrupulous infringers from improperly using the disclosed content where an exact or absolute numerical value is mentioned to assist in the understanding of the present invention. The terms "~ (at the stage of)" or "~ stage" used throughout this specification do not mean "stage for ~".

[0084] Throughout this specification, the term "these combinations" included in the Markush format expression means one or more mixtures or combinations selected from the group consisting of the components described in the Markush format expression, and means including one or more selected from the group consisting of the above-mentioned components.

[0085] Throughout this specification, the description "A and / or B" means "A or B, or A and B".

[0086] If any embodiment can be implemented separately, a particular step may be performed in an order different from the order described. For example, two steps described in sequence may be performed substantially simultaneously or in an order opposite to the described order.

[0087] The terms and words used in this specification and claims should not be construed as being limited to their ordinary or dictionary meanings. Instead, the inventor should construe them in accordance with the technical idea of the present invention based on the principle that the inventor can appropriately define the concept of the terms in order to best explain his or her invention. That is, they should be construed in meanings and concepts that conform to the technical idea of the present invention.

Embodiments for Carrying Out the Invention

[0088] Hereinafter, preferred embodiments are presented to assist in understanding the present invention. However, the following embodiments are provided only to more easily understand the present invention, and the content of the present invention is not limited by the following embodiments.

[0089] Experimental Methods and Materials 1. Aptamer Preparation and Analysis An anti-CD31 single-stranded DNA aptamer having the core sequence of SEQ ID NO: 1 ( 76mer, 21966-18-01) was synthesized and characterized by Aptamer Sciences Inc. (Korea). After dissolving the anti-CD31 aptamer in sterile Ultrapure DEPC-treated water (Invitrogen, USA), it was stored at -20°C. Before conducting the experiment, the aptamer was heated at 95°C for 10 minutes and cooled to room temperature to induce proper folding. To confirm that the anti-CD31 aptamer specifically targets CD31+ ECs, an aptamer labeled with cy3 at the 5'-end (5'-cy3-apt ​ Immunofluorescence staining and flow cytometry were performed with amer-3’).

[0090] <Anti-CD31 aptamer> 5’-TCA GCC GCC AGC CAG TTC(primer)-G6A G AG GAG G6A CG6 AA6 G6C 6GG G6A 6AC CCC G A6 AA6 6(SEQ ID NO: 1; core sequence)-GAC CAG AG C ACC ACA GAG(primer)-3’ 6=NapdU[5-(N-Napthylcarboxyamide)-2’-deo xyuridine]

[0091]

Chemical formula

[0092] 2. Cell Culture Human umbilical vein endothelial cells (HUVEC, Human umb ilical vein endothelial cells) and human umbilical cord blood-derived mesenchymal stem cells (MSC, mesenchymal stem cells) were maintained in endothelial growth medium-2 (EGM-2, Lonza, Switzerland). Under the approval of the Institutional Review Board of Seoul National University (IRB No. 1608 / 001-021), MSC is, Donor-depen dent variation of human umbilical cord b lood mesenchymal stem cells in response to hypoxic preconditioning and ameliorat ion of limb ischemia, Exp Mol Med 50(4)(2 to hypoxic preconditioning and amelioration ion of limb ischemia, Exp Mol Med 50(4)(2 018) Established as described in 35. Liver cancer cells (Hep G2 cells) purchased from ATCC and LX-2 human stellate cells (ste llate cells) purchased from Millipore (USA) were cultured in Dulbecco's Modified Eagle Medium (DMEM, Hyclone, USA) containing high glucose. All media were supplemented with 10% fetal bovine serum (FBS , Gibco) and antibiotics (100 U / ml penicillin and 100 μg / ml stre ptomycin (1% P / S, Gibco)) and 20 μg / ml primocin (Prim ocin, InvivoGen, USA). The culture medium was changed every 48 hours. The cells were maintained at 37 °C in a 5% CO 2 humidified incubator.

[0093] 3. Microfluidic Device Design and Cell Separation Analysis The cell separation rate by the coating agent was evaluated using a 3D microfluidic system. Polydi methylsiloxane (PDMS, Sylga rd 184; Dow Corning, USA) was used to fabricate a device with three microchannels by soft lithography and replica molding. A PDMS prepolymer composed of a mixture of PDMS base and a curing agent (10:1) was poured onto a silicon wafer and cured. After the PDMS block was completely solidified, it was separated from the wafer. .

[0094] After punching the inlet and outlet using a biopsy punch, the PDMS block was attached to a pressure-sensitive adhesive polycarbonate film. Before performing additional experiments, the device was maintained in a drying oven overnight to recover its hydrophobicity and sterilized by UV irradiation. The microfluidic device was of type 1. Collagen (rat tail, #354236, Corning, USA), vitronectin (#A31804, Gibco) and fibronectin (#356008, Corn ing) were coated at a mixture concentration of 100 μg / ml.

[0095] Subsequently, the device was coated with each of the following coating agents; PBS (negative control group), 600 nM anti-CD31 aptamer and 50 μg / mL anti-CD31 antibody, 5×10 5 HUVECs were seeded into each channel and incubated for 2 hours. Then, fluid shear stress was applied to the monolayer of HUVECs using an infusion syringe pump (PHD 2000 Infusion, Harvard device , USA). Shear stress was calculated based on the modified Poiseuille equation (see Equation 1).

[0096] [Equation 1] Tw = 6μQ / wH 2

[0097] (Tw: shear stress (dynes / cm 2 ), μ: viscosity at 37°C (Poise), Q : flow rate (mL / s), w: channel width (cm) and H: channel height (cm))

[0098] EGM-2 supplemented with 1% FBS was passed through the channels at the indicated flow rate for 10 minutes. For each flow, after the flow, images of the cells attached to the surface were captured using an optical microscope (IX7 0, Olympus, Japan) and counted using Image J software ware. To analyze the changes in HUVECs due to the response to shear stress, after exposure to a shear stress of 10 dynes / cm for 30 minutes, RNA and proteins were extracted from the cells. 2 ​ Protein was extracted.

[0099] 4. Fabrication of Decellularized Rat Liver Scaffold After systemic heparinization, native livers were obtained from 8-week-old female Sprague-Dawley rats (250 - 300 g). The portal vein was cannulated with a 24 G catheter and perfused with 0.1% SDS (Sigma Aldrich, USA) in deionized water for 6 hours, followed by washing with PBS for 10 hours. Then, the scaffold was perfused with 0.1 mg / ml deoxyribonuclease 1 (DNase 1; Sigma Aldrich) for 1 hour and washed with PBS for 2 hours. To remove the bioburden from the scaffold, it was sterilized with 0.1% peracetic acid (Sigma Aldrich) and stored at 4 °C in PBS containing antibiotics. All rat experiments were approved by the Animal Care Committee of Seoul National University (SNU-170113-2).

[0100] 5. Re-Endothelialization of Rat Liver Scaffold To stabilize the sterilized decellularized liver matrix scaffolds, they were perfused with EGM-2 for 1 hour, and then they were coated at 4 °C for 1 hour by injecting the following coating agents through the portal vein: PBS, 600 nM anti-CD31 aptamer and 50 μg / mL anti-CD31 antibody (14-0319-80, eBioscience, USA). A total of 1 × 10 HUVECs were labeled with carboxyfluorescein diacetate succinimidyl ester (CFDA) 7 using the Vybrant CFDA SE Cell Tracer Kit (Invitrogen).

[0101] ​​CFDA-labeled HUVEC suspended in EGM-2 supplemented with 10% FBS and antibiotics were delivered at a rate of 0.5 ml / min via the portal vein. After maintaining a 2-hour static culture, the re-endothelialized construct was perfused at a rate of 1.5 ml / min together with the culture medium in a bioreactor using a peristaltic pump. The culture medium was alternated every 48 hours, and samples were harvested on day 7 for additional analysis. Endothelial coverage and the number of re-endothelialized blood vessels were quantified by Image J software.

[0102] 6. Delivery of Hepatocytes and Non-Parenchymal Cells to Decellularized Rat Liver When producing the decellularized liver scaffold, the bile duct was cannulated with a 26G catheter and the portal vein was cannulated with a 24G catheter. After decellularization, a total of 4×10 He 7 pG2 cells were delivered in half via the bile duct into the parenchymal space of the scaffold and maintained in a bioreactor for 2 days. After 2 days, another 4×10 HepG2 cells were delivered further into the parenchyma, and the construct 7 was cultured for up to 14 days.

[0103] On day 14, 6×10 6 CFDA-labeled HUVEC and 3×10 6 MSC were delivered into the vascular lumen via the portal vein, and simultaneously 1×10 7 LX 2 cells were injected via the bile duct. All cells were delivered at a rate of 0.5 ml / min. After maintaining a static culture for 2 hours, the construct was perfused at a rate of 1.5 ml / min using a peristaltic pump. Up to day 14, the VBHL construct was maintained in DMEM high glucose medium with 10% FBS and antibiotics. From day 14 to day 21, the medium was E ​​It alternated with GM-2. Additional analysis was performed on the VBHL structures obtained on day 21.

[0104] 7. Statistical Analysis Statistical analysis was performed using GraphPad Prism version 5.0. All values were reported using the mean ± standard deviation. Values with p < 0.05 were considered significant . Statistical analysis between two groups was performed using an unpaired, two-tailed Student 's t-test. For multiple group comparisons, two-way ANOVA was performed with a Bonferroni post hoc test. The data presented represent the results of at least three independent experiments.

[0105] 8. Immunofluorescence Staining Cells or tissue sections were fixed with 4% formaldehyde for 10 minutes. Then, the samples were permeabilized with 0.2% Triton X-100 (Sigma Aldrich) for 10 minutes and blocked with 5% goat serum (Vector Laboratories, Switzerland) for 1 hour. Then, the samples were probed with the following primary antibodies at 4°C overnight: a nti-human CD31 (14-0319-80, eBioscience, US A), anti-Albumin (GTX102419, GeneTex, USA), a nti-Vimentin (ab45939, Abcam, UK), anti-Gala ctosidase alpha (GTX101178, GeneTex), anti- ZO-1 (#40-2200, Invitrogen), anti-cleaved C aspase-3 (#9664, Cell Signaling Technology , USA), anti-phospho-Akt (#9271, Cell Signal ing Technology), anti-Integrin beta 3 (#13 166, Cell Signaling Technology), anti-Inte grinαIIb (sc-365938, Santa Cruz biotechnolo gy, USA) and anti-α-Smooth muscle actin (ab7 814, Abcam). Fluorescent-dye conjugated secondary antibodies (Alexa Fluor 48 8-labeled and 549-labeled; Invitrogen) were applied for 1 hour. The nuclei were stained with DAPI (s c3598, Santa Cruz Biotechnology) for 10 minutes, and the cells were mounted in fluorescent mounting medium (S302380, DAKO, Denmark). The stained signals were visualized by an Eclipse TE 2000 confocal laser scanning microscope (Ni kon, Japan). Similarly, after removing paraffin, hydrating, and fixing with 4% formaldehyde from paraffin-embedded tissue sections, they were stained as described above.

[0106] 9. Flow Cytometry To estimate the binding kinetics of the anti-CD31 aptamer in a concentration-dependent manner, HUVECs were incubated with different concentrations of cy5-labeled anti-CD31 aptamer in a reaction buffer composed of 1 mM MgCl 2 and 2 mg / ml bovine serum albumin at 4°C for 20 minutes. To examine the specificity of the anti-CD31 apt amer, HepG2 cells and MSCs were compared. The cells were washed with 1 m l of reaction buffer, suspended in PBS, and analyzed by FACS Calibur (BD bio l and MSC were compared. The cells were washed with 1 m ​analyzed in (science, USA). The PE-conjugated anti-CD31 antibody (BD 555446, BD Biosciences) was used as the positive control group. FlowJo software ware (USA) was used to analyze the data.

[0107] 10. Analysis of Cell Adhesion Ability The abdominal aorta of rats was harvested and immersed in 0.05% SDS stirred for decellularization . The decellularized arterial scaffold was cut longitudinally to expose the internal structure and sterilized with 0.1% peracetic acid. Then, the scaffold was immersed in the coating agent for 2 hours ; PBS (negative control group), 600 nM anti-CD31 aptamer, and 50 μg / mL anti-human CD31 antibody (14-0319-80, eBioscience, positive control group). Then , the coated scaffolds were transferred to 24-well plates, and 1×10 6 cells of HU VEC were seeded on the inner surface of the arterial scaffold. After maintaining static culture at the indicated time points (2 hours and 4 hours), the scaffolds seeded with cells were washed with PBS and transferred to a new plate. The scaffolds were subjected to tetrazolium-based MTT analysis to quantify the number of viable cells. For MTT analysis, the samples were maintained at 37 °C for 4 hours in medium containing 10% MTT stock solution (Sigma Aldrich). Then, the supernatant was removed, and dimethyl sulfoxide was added to each well for solubilization of purple formazan . The absorbance at a wavelength of 540 nm was measured using a microplate reader (Infinite M200 pro, Tecan, Switzerland) . (Infinite M200 pro, Tecan, Switzerland) was used .

[0108] 11. qRT-PCR Total RNA was extracted from cells or engineered products using NucleoZOL (Macherey-Nagel, Germany). Subsequently, complementary DNA was synthesized from the extracted RNA using Superscrip t III First-Strand Synthesis System (Invit rogen). qRT-PCR was performed using SYBR Green PCR Master Mix (Ap plied Biosystems, USA) on an ABI 7300 Real time PCR system (Applied Bios ystems). Relative quantification of the target mRNA expression level was determined by the 2(ΔΔ threshold cycle )(2- ΔΔCT ) method. The expression level of each gene was normalized by the housekeeping gene expression. The primer sequences used are shown in Table 1. Protein was extracted using PRO-PREP (iNtRon biotechnology, Korea), and the whole cell lysate was sonicated. To extract protein from tissue samples, the tissue was homogenized with steel beads and PRO-PREP and then sonicated. After centrifugation, the supernatant was transferred to a new tube and analyzed. Protein concentration was quantified using a DC assay kit (Bio-Ra

[0109]

Table 1

[0110] 12. Western Blot d, USA). The same amount of protein (10 μg) was loaded onto an 8–15% acrylamide Tris–glycine gel. The target protein For tissue samples, the tissue was homogenized with steel beads and PRO-PREP and then sonicated. After centrifugation, the supernatant was transferred to a new tube and analyzed. Protein concentration was quantified using a DC assay kit (Bio-Ra d, USA). The same amount of protein (10 μg) was loaded onto an 8–15% acrylamide Tris–glycine gel. The target protein It was probed with the following primary antibodies: anti-GAPDH (AB2302, Millipo re), anti-Integrin beta 3 (#13166, Cell Sig naling Technology), anti-Akt (#4691, Cell S ignaling Technology), anti-phospho-Akt (#9 271, Cell Signaling Technology), anti-clea ved Caspase-3 (#9664, Cell Signaling Techn ology), anti-Galactosidase alpha (GTX10117 8, GeneTex), anti-Fibronectin (ab2413, Abcam ), anti-CollagenIV (ab19808, Abcam), anti-Lam inin (ab11575, Abcam), anti-CK18 (MAB3234, Mi llipore), anti-CYP2E1 (CSB-PA006425EA01HU, CUSABIO, China) and anti-AFP (A0008, DAKO). After incubating overnight with the primary antibodies at 4°C, an HRP-conjugated secondary antibody was applied . After probing with the Amersham Enhanced chemiluminescence d etection kit (GE Healthcare, USA), specific protein bands were derived using FluorChem HD2 (Alpha Innotech., USA). The samples were washed three times with PBS and fixed in 4% paraformaldehyde overnight at 4°C.

[0111] 13. Histological Examination ​​​The tissue was paraffin-embedded and serially sectioned at a thickness of 10 μm. After deparaffinization of the tissue sections, a series of ethanol in descending order in the range of 100% to 70% was used for hydration . Subsequently, the tissue slices were stained with H&E and picrosirius red solution (0.1% Direct Red 80 and 0.1% Fast Green FCF). All reagents were purchased from Sigma Aldrich. PAS staining was performed using a PAS staining kit (ab150680, Abcam). After staining, the sections were washed with tap water, dehydrated using a series of ethanol in ascending concentration, and mounted. Visualization was performed with Nikon NIS-Elements software and an optical microscope (Nikon), and quantification of fibrosis was performed with Image J software .

[0112] 14. Analysis of Resazurin Reduction Perfusion PrestoBlue Cell Viability Reagent (A13261, Invitrogen), a non-toxic resazurin-based solution, was used according to the manufacturer's guide . To draw a standard curve for HUVEC, 0.02, 0.05, 0.1, 0.2, 0.5, 1, and 2 million cells were seeded in 6-well plates. After culturing for 6 hours, the cells were washed and the medium was replaced with 2 ml of PrestoBlue reagent mixed with complete EGM-2 (1:20 ratio). After culturing at 37 °C for 1 hour, the medium was collected and then the absorbance at a wavelength of 570 nm was measured using a microplate reader. Corresponding to the standard curve, 30 ml of PrestoBlue-medium mixture was perfused into the engineered construct at 37 °C for 1 hour. The harvested medium was analyzed as described above

[0113] ​​​​​​​​​​​​​15. Analysis of FITC-Conjugated Dextran Perfusion To evaluate the permeability of the re-endothelialized blood vessels in the operated tissue, perfusion of 500 kDa FITC-dextran (FD500S, Sigma Aldrich) was used . 25 mL of FITC-dextran (0.2 mg / mL) was administered at 20 mmHg. The dextran in the fluid drained from the inferior vena cava was regarded as intravascular dextran, while the peripheral blood was regarded as extravascular dextran. The total amount of dextran was measured by multiplying the amount of the drained fluid by the fluorescence intensity (absorbance at 490 nm).

[0114] 16. TUNEL Assay To detect the dead cells from the operated structure, ApopTag Red In situ Apoptosis Detection Kit (Millipore) was used. The tissue sections were deparaffinized and digested with proteinase K. After applying the equilibration buffer to the sections, they were probed with TdT enzyme in a 37 °C incubator for 1 hour. After washing with the stop / wash buffer, anti-digoxigenin (rhodamine- conjugated) was applied to the sections. Then, these sections were washed with PBS and the nuclei were stained with DAPI. TUNEL-positive cells were detected by confocal microscopy.

[0115] 17. Quantification of Secreted NO and VEGF The concentration of NO secreted from the biotechnological structure was measured using the NO Plus detection kit (iNtRON ). The conditioned medium was collected at the indicated time points and cell debris was removed by centrifugation . Then, NO was measured from the supernatant of the conditioned medium according to the manufacturer's instructions. The absorbance at a wavelength of 540 nm was measured using a microplate reader . measured using. The constructs were cultured in EBM-2 (endothelial basal medium-2) without growth factors and exposed to a human VEGF Quantikine ELISA kit (DVE00, R&D Systems, USA). After 12 hours, supernatants were obtained for VEGF quantification of the conditioned medium from each engineered construct. Absorbance at a wavelength of 450 nm was measured using a microplate reader. asal medium-2) and exposed to a human VEGF Quantikine ELISA kit (DVE00, R&D Systems, USA). After 12 hours, supernatants were obtained for VEGF quantification of the conditioned medium from each engineered construct. Absorbance at a wavelength of 450 nm was measured using a microplate reader. ISA kit (DVE00, R&D Systems, USA). After 12 hours, supernatants were obtained for VEGF quantification of the conditioned medium from each engineered construct. Absorbance at a wavelength of 450 nm was measured using a microplate reader. were obtained for VEGF quantification of the conditioned medium from each engineered construct. Absorbance at a wavelength of 450 nm was measured using a microplate reader. Absorbance at a wavelength of 450 nm was measured using a microplate reader.

[0116] 18. Analysis of Ex vivo Human Blood Perfusion 1×10 7 scaffolds further seeded with HUVECs were maintained in EGM-2 supplemented with 10% FBS and antibiotics for 7 days before perfusion with human blood. Heparinized human blood obtained from the Korean Red Cross Central Blood Center was mixed with the culture medium (1:1) and perfused through the portal vein of each scaffold. At the indicated time points, blood perfusates were collected and the number of platelets in the perfusates was counted using an Advia 2120i hematology system (Siemens Healthcareers, Germany). After 24 hours of perfusion, samples were washed with PBS and gene expression analysis and immunostaining were performed to evaluate the degree of thrombus formation within the scaffolds. After synthesizing cDNA from the extracted RNA, it was amplified by PCR using primers targeting thrombogenic genes. Also, paraffin-embedded tissue blocks were sectioned according to the procedure described for immunofluorescence staining and stained with integrin αIIb. scaffolds further seeded with HUVECs were maintained in EGM-2 supplemented with 10% FBS and antibiotics for 7 days before perfusion with human blood. Heparinized human blood obtained from the Korean Red Cross Central Blood Center was mixed with the culture medium (1:1) and perfused through the portal vein of each scaffold. At the indicated time points, blood perfusates were collected and the number of platelets in the perfusates was counted using an Advia 2120i hematology system (Siemens Healthcareers, Germany). After 24 hours of perfusion, samples were washed with PBS and gene expression analysis and immunostaining were performed to evaluate the degree of thrombus formation within the scaffolds. After synthesizing cDNA from the extracted RNA, it was amplified by PCR using primers targeting thrombogenic genes. Also, paraffin-embedded tissue blocks were sectioned according to the procedure described for immunofluorescence staining and stained with integrin αIIb. scaffolds further seeded with HUVECs were maintained in EGM-2 supplemented with 10% FBS and antibiotics for 7 days before perfusion with human blood. Heparinized human blood obtained from the Korean Red Cross Central Blood Center was mixed with the culture medium (1:1) and perfused through the portal vein of each scaffold. At the indicated time points, blood perfusates were collected and the number of platelets in the perfusates was counted using an Advia 2120i hematology system (Siemens Healthcareers, Germany). After 24 hours of perfusion, samples were washed with PBS and gene expression analysis and immunostaining were performed to evaluate the degree of thrombus formation within the scaffolds. After synthesizing cDNA from the extracted RNA, it was amplified by PCR using primers targeting thrombogenic genes. Also, paraffin-embedded tissue blocks were sectioned according to the procedure described for immunofluorescence staining and stained with integrin αIIb. scaffolds further seeded with HUVECs were maintained in EGM-2 supplemented with 10% FBS and antibiotics for 7 days before perfusion with human blood. Heparinized human blood obtained from the Korean Red Cross Central Blood Center was mixed with the culture medium (1:1) and perfused through the portal vein of each scaffold. At the indicated time points, blood perfusates were collected and the number of platelets in the perfusates was counted using an Advia 2120i hematology system (Siemens Healthcareers, Germany). After 24 hours of perfusion, samples were washed with PBS and gene expression analysis and immunostaining were performed to evaluate the degree of thrombus formation within the scaffolds. After synthesizing cDNA from the extracted RNA, it was amplified by PCR using primers targeting thrombogenic genes. Also, paraffin-embedded tissue blocks were sectioned according to the procedure described for immunofluorescence staining and stained with integrin αIIb. scaffolds further seeded with HUVECs were maintained in EGM-2 supplemented with 10% FBS and antibiotics for 7 days before perfusion with human blood. Heparinized human blood obtained from the Korean Red Cross Central Blood Center was mixed with the culture medium (1:1) and perfused through the portal vein of each scaffold. At the indicated time points, blood perfusates were collected and the number of platelets in the perfusates was counted using an Advia 2120i hematology system (Siemens Healthcareers, Germany). After 24 hours of perfusion, samples were washed with PBS and gene expression analysis and immunostaining were performed to evaluate the degree of thrombus formation within the scaffolds. After synthesizing cDNA from the extracted RNA, it was amplified by PCR using primers targeting thrombogenic genes. Also, paraffin-embedded tissue blocks were sectioned according to the procedure described for immunofluorescence staining and stained with integrin αIIb. scaffolds further seeded with HUVECs were maintained in EGM-2 supplemented with 10% FBS and antibiotics for 7 days before perfusion with human blood. Heparinized human blood obtained from the Korean Red Cross Central Blood Center was mixed with the culture medium (1:1) and perfused through the portal vein of each scaffold. At the indicated time points, blood perfusates were collected and the number of platelets in the perfusates was counted using an Advia 2120i hematology system (Siemens Healthcareers, Germany). After 24 hours of perfusion, samples were washed with PBS and gene expression analysis and immunostaining were performed to evaluate the degree of thrombus formation within the scaffolds. After synthesizing cDNA from the extracted RNA, it was amplified by PCR using primers targeting thrombogenic genes. Also, paraffin-embedded tissue blocks were sectioned according to the procedure described for immunofluorescence staining and stained with integrin αIIb. scaffolds further seeded with HUVECs were maintained in EGM-2 supplemented with 10% FBS and antibiotics for 7 days before perfusion with human blood. Heparinized human blood obtained from the Korean Red Cross Central Blood Center was mixed with the culture medium (1:1) and perfused through the portal vein of each scaffold. At the indicated time points, blood perfusates were collected and the number of platelets in the perfusates was counted using an Advia 2120i hematology system (Siemens Healthcareers, Germany). After 24 hours of perfusion, samples were washed with PBS and gene expression analysis and immunostaining were performed to evaluate the degree of thrombus formation within the scaffolds. After synthesizing cDNA from the extracted RNA, it was amplified by PCR using primers targeting thrombogenic genes. Also, paraffin-embedded tissue blocks were sectioned according to the procedure described for immunofluorescence staining and stained with integrin αIIb. scaffolds further seeded with HUVECs were maintained in EGM-2 supplemented with 10% FBS and antibiotics for 7 days before perfusion with human blood. Heparinized human blood obtained from the Korean Red Cross Central Blood Center was mixed with the culture medium (1:1) and perfused through the portal vein of each scaffold. At the indicated time points, blood perfusates were collected and the number of platelets in the perfusates was counted using an Advia 2120i hematology system (Siemens Healthcareers, Germany). After 24 hours of perfusion, samples were washed with PBS and gene expression analysis and immunostaining were performed to evaluate the degree of thrombus formation within the scaffolds. After synthesizing cDNA from the extracted RNA, it was amplified by PCR using primers targeting thrombogenic genes. Also, paraffin-embedded tissue blocks were sectioned according to the procedure described for immunofluorescence staining and stained with integrin αIIb. scaffolds further seeded with HUVECs were maintained in EGM-2 supplemented with 10% FBS and antibiotics for 7 days before perfusion with human blood. Heparinized human blood obtained from the Korean Red Cross Central Blood Center was mixed with the culture medium (1:1) and perfused through the portal vein of each scaffold. At the indicated time points, blood perfusates were collected and the number of platelets in the perfusates was counted using an Advia 2120i hematology system (Siemens Healthcareers, Germany). After 24 hours of perfusion, samples were washed with PBS and gene expression analysis and immunostaining were performed to evaluate the degree of thrombus formation within the scaffolds. After synthesizing cDNA from the extracted RNA, it was amplified by PCR using primers targeting thrombogenic genes. Also, paraffin-embedded tissue blocks were sectioned according to the procedure described for immunofluorescence staining and stained with integrin αIIb. scaffolds further seeded with HUVECs were maintained in EGM-2 supplemented with 10% FBS and antibiotics for 7 days before perfusion with human blood. Heparinized human blood obtained from the Korean Red Cross Central Blood Center was mixed with the culture medium (1:1) and perfused through the portal vein of each scaffold. At the indicated time points, blood perfusates were collected and the number of platelets in the perfusates was counted using an Advia 2120i hematology system (Siemens Healthcareers, Germany). After 24 hours of perfusion, samples were washed with PBS and gene expression analysis and immunostaining were performed to evaluate the degree of thrombus formation within the scaffolds. After synthesizing cDNA from the extracted RNA, it was amplified by PCR using primers targeting thrombogenic genes. Also, paraffin-embedded tissue blocks were sectioned according to the procedure described for immunofluorescence staining and stained with integrin αIIb. scaffolds further seeded with HUVECs were maintained in EGM-2 supplemented with 10% FBS and antibiotics for 7 days before perfusion with human blood. Heparinized human blood obtained from the Korean Red Cross Central Blood Center was mixed with the culture medium (1:1) and perfused through the portal vein of each scaffold. At the indicated time points, blood perfusates were collected and the number of platelets in the perfusates was counted using an Advia 2120i hematology system (Siemens Healthcareers, Germany). After 24 hours of perfusion, samples were washed with PBS and gene expression analysis and immunostaining were performed to evaluate the degree of thrombus formation within the scaffolds. After synthesizing cDNA from the extracted RNA, it was amplified by PCR using primers targeting thrombogenic genes. Also, paraffin-embedded tissue blocks were sectioned according to the procedure described for immunofluorescence staining and stained with integrin αIIb.

[0117] 19. Albumin / Urea ELISA Analysis The amount of albumin protein secreted from the constructs in the conditioned medium was determined using a human albumin ELISA kit. Determined using the LISA kit (ab108788, Abcam). The VBHL construct Conditioned medium was harvested from at the indicated time points. After centrifugation, the concentration of secreted albumin in the supernatant was quantified using ELISA according to the manufacturer's recommendations . Absorbance at a wavelength of 450 nm was measured using a microplate reader . Also, the amount of urea secreted from the supernatant collected in the conditioned medium was measured. The concentration of urea was quantified by measuring the absorbance at a wavelength of 520 nm using the QuantiChrom Urea Assay Kit (BioAssay Systems , USA) .

[0118] 20. In Vivo Reperfusion of Vascularized Liver Structures First, decellularized liver constructs were produced by catheter insertion as follows: 22G catheter - portal vein, 26G catheter - bile duct, and 20G catheter - inferior vena cava. The VBHL construct was produced as described in Experimental Method 6. The VBHL construct was maintained in a bioreactor for 21 days and then directly connected to the host kidney circulation system by catheters of various sizes. For the above procedure, healthy 1-year-old rats were anesthetized and the left kidney was exposed. After fixing the renal artery and vein , catheters were inserted with each 24G catheter. The catheters placed in the portal vein and inferior vena cava of the construct were respectively connected to the catheters placed in the renal artery and vein. Each connection was fixed with Vetbond adhesive (3M, USA) . After removing the vascular clamp, the blood was perfused in vivo for 2 hours. Then, the harvested liver construct was subjected to additional analysis .

[0119] 21. TAA-Induced Chronic Liver Injury Rat Model and Transplantation of VBHL Structures ​​​​To evaluate the in vivo function of the VBHL construct, chronic liver injury was induced in 4-week-old female rats as follows. 0.3 g / L of TAA (Sigma Aldrich) containing drinking water was administered to the rats continuously for 12 weeks, and then the induced liver fibrosis was analyzed. ALT and AST levels in rat serum were measured using the ALT Activity Colorimetric assa y kit (K752-100, Biovision, China) and the AST Ac tivity Colorimetric assay kit (K753-100, B iovision). After inducing cirrhosis in the rats for 8 weeks, laparotomy was performed on the rats to expose the liver. Then, after removing the fibrous caps ules from the VBHL constructs of each group, they were transplanted between the central lobe and the right lateral lobe of the host liver and fixed. The VBHL construct was manufactured as described in Part 6 of the experimental method using decellularized rat liver. Four weeks later, the liver of the host transplanted with the liver construct was harvested for further analysis. On the other hand, serum samples were obtained before and after the surgery.

[0120] Example 1. Verification of the Characteristics of Anti-CD31 Aptamer After fabricating an anti-CD31 aptamer that can specifically bind to the CD31 protein as shown in Fig. 1a, the binding affinity between the cells expressing CD31 and the aptamer was verified using a flow cytometer.

[0121] As a result, as shown in Figs. 1b and 1c, the aptamers at 600 nM and 800 nM could bind to approximately 99% of vascular endothelial cells (HUVECs), and HepG2 and MSC that do not express CD31 bound to approximately 2% of the cells. Also, according to the aptamer concentration ​When quantifying the binding ability to vascular endothelial cells, the binding ability was saturated with an aptamer at 600 nM. Therefore, the aptamer concentration condition was optimized at 600 nM.

[0122] Also, as shown in FIGS. 1e to 1g, it was confirmed that the 600 nM aptamer binds to HUVEC but not to HepG2 and MSC using the cell immunostaining method. Based on the above results, it was verified that the anti-CD31 aptamer binds specifically to CD31.

[0123] Example 2. Evaluation of the Adhesion Ability of Vascular Endothelial Cells and Confirmation of the Effect on Vascular Endothelial Cells during Aptamer Treatment Example 3. Verification of the Efficacy of Aptamer during Vascular Structure Reconstruction through Re-Cellularization of Decellularized Retained In Vivo Vascular Endothelial Cells As shown in FIG. 2a, after coating the inside of a microfluidic device with an extracellular matrix (ECM) component, an anti-CD31 aptamer (APT-coated) and an anti-CD31 antibody (Ab-coated, positive control group) were each coated, and after injecting HUVEC, the cell adhesion ability was evaluated while flowing the liquid at a constant speed. As a result, as shown in FIGS. 2b and 2c, compared with the non-coated group, in the APT-coated group, when a shear stress of 10 dynes / cm was applied, more than 80% of the cells strongly adhered, and even when a strong shear stress of 20 dynes / cm was applied, a high adhesion ability of 61.5% was shown. In the case of the Ab-coated group, which is the positive control group, when treated with a shear stress of 20 dynes / cm, an adhesion ability of 51% was shown. This shows that the anti-CD31 aptamer enhances the adhesion ability of vascular endothelial cells with higher efficiency compared to the anti-CD31 antibody.

[0124] 2 2 2

[0125] ​​​​​​​​​​​​​ Also, to examine the mechanism mediating the difference in the adhesion ability of HUVECs by each coating agent, RNA was extracted from the cells in the microfluidic device and the mRNA expression pattern was analyzed.

[0126] Integrin proteins are composed of an extracellular domain, a transmembrane domain, and an intracellular domain. When the extracellular domain binds to the extracellular matrix, signals are transmitted intracellularly and are known to activate various signal transduction systems. Thus, the presence or absence of activation of integrin proteins by shear stress was confirmed. As a result, as shown in FIGS. 2e to 2g, in HUVECs subjected to a shear stress of 10 dynes / cm in the microfluidic device actually coated with the aptamer,

[0127] not only integrin but also Akt signaling, a downstream signal transduction system, was confirmed to be activated. Akt signaling has been reported to be related to cell survival and angiogenesis ability in vascular endothelial cells. Thus, as shown in FIG. 2i, it was also confirmed that the expression of cleaved caspase-3, a cell death marker, decreased in the APT-coated group. 2 In the APT-coated group, it was also confirmed that the expression of cleaved caspase-3, a cell death marker, decreased as shown in FIG. 2i. In the APT-coated group, it was also confirmed that the expression of cleaved caspase-3, a cell death marker, decreased as shown in FIG. 2i. Akt signaling is reported to be related to cell survival and angiogenesis ability in vascular endothelial cells. In the APT-coated group, it was also confirmed that the expression of cleaved caspase-3, a cell death marker, decreased as shown in FIG. 2i. In the APT-coated group, it was also confirmed that the expression of cleaved caspase-3, a cell death marker, decreased as shown in FIG. 2i. In the APT-coated group, it was also confirmed that the expression of cleaved caspase-3, a cell death marker, decreased as shown in FIG. 2i.

[0128] Example 4. Evaluation of In Vivo Thrombosis through Ex vivo Human Blood Perfusion Example 5. Fabrication and Functional Evaluation of Vascularized Artificial Liver Using Aptamer As shown in FIG. 3a, after fabricating a decellularized scaffold from rat liver, the inner wall of the blood vessel was coated with a coating agent. After recellularizing with HUVECs labeled with CFDA (green), it was cultured for 7 days and each reconstructed blood vessel structure was confirmed.

[0129] As a result, as shown in Fig. 3b, the anti-CD31 aptamer was treated with a coating agent In the holding body, it was confirmed that vascular endothelialization was sufficiently performed.

[0130] Also, as shown in Figs. 3c and 3d, in the APT-coated group, about 80% endothelialization was performed per blood vessel, and it was confirmed that about 80% vascular endothelialization was performed in the holding body. As a result, it was confirmed that the endothelialization efficiency was maximized in the group treated with the aptamer compared to the uncoated group

[0131] Also, as shown in Fig. 3e, in order to evaluate the barrier function of the reconstructed blood vessels, when dextran was injected via a portal catheter, the amount of intravascular dextran (intravascular dextran) that did not leak outside the blood vessels was significantly increased in the APT-coated group. Therefore, when the anti-CD31 aptamer is treated with a coating agent, it was verified that a highly functional vascular structure with good maintenance of the barrier function in the acellular holding body is efficiently

[0132] In addition, since the survival rate of the cells cultured during in vitro culture can greatly affect the functionality of the reconstructed artificial organ, the degree of cell death of the cultured cells was analyzed using immunostaining.

[0133] As a result, as shown in Figs. 3i and 3j, cell death was decreased in the APT-coated group compared to the group without coating or coated with an anti-CD31 antibody.

[0134] Subsequently, to verify the vascular-specific functionality of the reconstructed vascularized organ, an enzyme-linked immunosorbent assay (ELISA) was performed.

[0135] As a result, as shown in FIGS. 3k and 3l, when analyzing the amounts of NO (nitric oxide) and VEGF secreted from the carriers recellularized with HUVEC, the secretion amounts of NO and VEGF in the APT -coated group were significantly increased compared to those in other groups. This was confirmed.

[0136] Overall, when using the anti-CD31 aptamer as a coating agent, it was verified that not only the vascular reconstruction efficiency and barrier function increased, but also the viability of blood vessels and angiogenesis potential were enhanced.

[0137] Example 6. Evaluation of In Vivo Thrombosis of Vascularized Artificial Liver Treated with Aptamer As shown in FIG. 4a, to closely verify the degree of vascular reconstruction, human blood was perfused into the carriers recellularized with HUVEC and cultured for 7 days, and the degree of thrombosis was evaluated in vitro. By the above analysis method, when an artificial organ was actually transplanted into a human, the degree of thrombus formation could be indirectly evaluated. As a result, as shown in FIG. 4b, visual inspection showed a decrease in thrombosis in the APT-coated group. Moreover, as shown in FIGS. 4c and 4d, through immunostaining, it was confirmed that the expression of integrin αIIb, a thrombus marker, decreased in the APT-coated group. This was confirmed.

[0138] Also, as shown in FIGS. 4e and 4f, when quantifying the number of platelets in the perfusate, the re- Decellularized liver matrix without endothelialization (Decellularized liver In the matrix, DLM) group, due to the in vivo coagulation phenomenon, the number of platelets remaining in the perfusion fluid decreased to less than 20% after 4 hours of perfusion. However, in the APT-coated group, it was confirmed that about 60% of the platelets remained even after 24 hours of blood perfusion.

[0139] Moreover, as shown in Figure 4g, the expression of markers related to platelet aggregation (CD63, PLSCR1, TBXAS1, and THBS1) decreased in the APT-coated group.

[0140] Based on the above results, it was verified that during aptamer coating, the formation of perfusable blood vessels was made possible through more efficient vascular endothelialization, and thrombosis was minimized during blood perfusion.

[0141] Example 7. Evaluation of In Vivo Liver Function of Vascularized Artificial Liver Treated with Aptamer To reconstruct a vascularized bioengineered human liver (VBHL) with a reconstructed vascular structure using an aptamer, the following acellular in vivo liver parenchymal cells (HepG2), hepatic stellate cells (hepatic stell ate cell, mesenchymal stem cell), vascular endothelial cells (H UVEC), etc. were cultured in the method shown in Figure 5a.

[0142] As a result, as shown in Figures 5b to 5d, in the artificial liver tissue (APT-VBHL) with vascular endothelialization after aptamer coating, more than 90% of vascular endothelialization was achieved, and at the same time the hepatic parenchymal part was also well reconstructed, which was confirmed by albumin immunostaining.

[0143] ​​​​​​In addition, as shown in Fig. 5e, it was confirmed that mesenchymal stem cells were favorably engrafted in the perivascular region during aptamer treatment using aSMA immunostaining. On the other hand, it was observed that the perivascular region was not developed in the group without coating or treated with antibodies. enchymal stem cells were favorably engrafted in the perivascular region during aptamer treatment using aSMA immunostaining. On the other hand, it was observed that the perivascular region was not developed in the group without coating or treated with antibodies. erivascular region was not developed in the group without coating or treated with antibodies. erivascular region was not developed in the group without coating or treated with antibodies.

[0144] In addition, as shown in Fig. 5f, when dextran was flowed through the portal vein and intravascular dextran was quantified, it was confirmed that there was a significant increase in the APT-VBHL group. In addition, as shown in Fig. 5f, when dextran was flowed through the portal vein and intravascular dextran was quantified, it was confirmed that there was a significant increase in the APT-VBHL group. This confirmed that the vascular barrier function of the liver tissue was maintained.

[0145] Therefore, during aptamer treatment, by reconstructing not only endothelialization but also the perivascular region that constitutes the blood vessel wall, highly functional blood vessels that can maintain the barrier function well were obtained. Therefore, during aptamer treatment, by reconstructing not only endothelialization but also the perivascular region that constitutes the blood vessel wall, highly functional blood vessels that can maintain the barrier function well were obtained. Therefore, during aptamer treatment, by reconstructing not only endothelialization but also the perivascular region that constitutes the blood vessel wall, highly functional blood vessels that can maintain the barrier function well were obtained.

[0146] In addition, the degree of cell death in the artificial liver tissue was quantified using the TUNEL assay.

[0147] As a result, as shown in Fig. 5l, the APT-VBHL group showed the least cell death, which is associated with the functionality of the artificial liver. As a result, as shown in Fig. 5l, the APT-VBHL group showed the least cell death, which is associated with the functionality of the artificial liver.

[0148] In addition, to confirm the vascular-specific functionality, the amount of NO production and the amount of VEGF secreted from the artificial liver were measured. In addition, to confirm the vascular-specific functionality, the amount of NO production and the amount of VEGF secreted from the artificial liver were measured.

[0149] As a result, as shown in Figs. 5g and 5h, the APT-VBHL group showed superior functionality compared to the other groups. As a result, as shown in Figs. 5g and 5h, the APT-VBHL group showed superior functionality compared to the other groups.

[0150] In addition, to confirm liver-specific functionality, Albumin and Ure secreted from the artificial liver The ELISA for quantifying the a component was performed.

[0151] As a result, as shown in FIGS. 5i and 5j, it was confirmed that the amounts of albumin and urea secreted on the 21st day of culture were also the highest in the APT-VBHL group. In particular, the liver function showed a difference after vascular reconstruction, so it can be seen that highly functional vascular reconstruction via aptamer coating plays an important role in maintaining the overall functionality of the artificial liver tissue.

[0152] ​ As shown in FIG. 6a, the portal vein and inferior vena cava of the artificial liver were respectively connected to the renal artery and renal vein of the rat and the degree of in vivo thrombosis of the vascularized artificial liver was evaluated by perfusing blood in vivo. Specifically, the renal vein (yellow arrow) was connected to the inferior vena cava of the VBHL structure, and the renal artery (white arrow) was connected to the portal vein of the structure. After removing the vascular clamp (blue arrow), the VBHL structure was reperfused into the in vivo renal circulation system.

[0153] As a result, as shown in FIG. 6b, no thrombosis was visually observed in the aptamer-treated artificial liver.

[0154] In addition, as shown in FIGS. 6c and 6d, it was confirmed that the expression of integrin α IIb, a thrombus marker, was also decreased.

[0155] Not only that, as shown in FIG. 6e, when analyzing the expression of mRNA markers related to blood coagulation (Cd 63, Plscr1, Thbs1), the expression of blood coagulation-related markers was most decreased in the APT-VBHL group. ​​

[0156] Therefore, based on the above results, it is expected that thrombosis in the living body can be minimized through aptamer treatment for those individuals It was verified that successful artificial liver culture had been achieved.

[0157] Therefore, when using the aptamer coating agent of the present invention, it is expected that the most significant side effects that may occur after transplantation of the artificial liver can be minimized, thereby increasing the success rate of artificial liver transplantation. be awaited.

[0158] ​ As shown in Fig. 7a, a rat model with liver fibrosis induced using thioacetamide (TAA) was transplanted with a vascularized artificial liver to evaluate liver-specific functionality in vivo. Eight weeks after TAA induction, the rats received heterologous (transplant) transplantation of a decellularized liver matrix (DLM transplant) or a VBHL structure (CTL-VBHL; CTL transplant, APT-VBHL; APT transplant, Ab-VBHL; Ab transplant).

[0159] The specific experimental groups were as follows: 1) Sham 2) DLM implanted; transplantation with a decellularized carrier alone without cells 3) CTL implanted; transplantation of a reconstructed artificial liver without coating 4) APT implanted; transplantation of an artificial liver treated with an anti-CD31 aptamer as a coating agent 5) Ab implanted; transplantation of an artificial liver treated with an anti-CD31 antibody as a coating agent

[0160] Four weeks later, the livers of the donor rats were sampled for H&E tissue staining and picrosirius (Pic​​​​​​​ rosirius) Red tissue staining (red: collagen, green: cytoplasm of cells) was performed. .

[0161] As a result, as shown in FIGS. 7b and 7c, when the acellular carrier and the artificial liver were transplanted, the eosinophilic change and the degree of fibrosis of the liver were reduced compared to the sham group. Among them, when the artificial liver treated with the aptamer was transplanted, it was confirmed that the degree of liver fibrosis was most significantly reduced.

[0162] Also, as shown in FIG. 7d, when analyzing the mRNA expressed in the liver of the donor, the markers (α-smooth muscle actin (Sma), Vimentin, Tgf-beta1, Timp1) related to fibrosis were clearly reduced in the APT implanted group.

[0163] Also, as shown in FIGS. 7e and 7f, when confirming the levels of ALT and AST, which are liver injury indicators, in the serum of rats, in the APT implanted group, it was confirmed that the serum ALT and AST levels were maintained at normal levels (between the red lines) after the surgery.

[0164] Therefore, the artificial liver treated with the aptamer of the present invention maintains improved liver-specific functionality in vivo, indicating that it can assist the damaged liver function of the donor.

[0165] Thus, the present inventors first confirmed that aptamers can be applied to artificial organs.

[0166] From the above results, when the aptamer is used as a coating agent for the acellular carrier, inte Via the Grin-Akt signaling system, the ability to adhere to blood vessels, viability, angiogenesis ability, etc. are enhanced, enabling more efficient reconstruction of the vascular structure than the anti-CD31 antibody used in conventional research. Therefore, the vascularized artificial liver produced using the present invention not only reduces thrombus formation in vivo but also shows the effect of enhancing liver functionality. Moreover, this presents the applicability of the artificial liver as a therapeutic agent for liver diseases from the perspective of tissue engineering. This is the result of presenting the applicability as a therapeutic agent for liver diseases of the artificial liver.

[0167] The above description of the present invention is for illustrative purposes, and those with ordinary knowledge in the technical field to which the present invention pertains can understand that it can be easily transformed into other specific forms without changing the technical idea and essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all aspects and not limiting.

Industrial Applicability

[0168] When the aptamer of the present invention is used as a coating agent for the decellularized carrier, it enhances the ability to adhere to blood vessels, viability, angiogenesis ability, etc., enabling more efficient reconstruction of the vascular structure than conventional antibodies. Therefore, the vascularized artificial liver produced using the aptamer not only reduces thrombus formation in vivo but also shows the effect of enhancing liver functionality. Since the application as a disease treatment method using the artificial organ produced using the aptamer of the present invention is expected, it has industrial applicability. it not only reduces thrombus formation in vivo but also shows the effect of enhancing liver functionality. Since the application as a disease treatment method using the artificial organ produced using the aptamer of the present invention is expected, it has industrial applicability. Therefore, there is industrial applicability.

Claims

1. A composition for producing an artificial organ comprising an anti-CD31 aptamer and vascular endothelial cells, wherein the anti-CD31 aptamer binds to the vascular endothelial cells to coat a decellularized scaffold, and the anti-CD31 aptamer comprises the base sequence represented by sequence number 1.

2. The composition of claim 1, further comprising one or more cells selected from the group consisting of parenchymal cells and non-parenchymal cells.

3. The composition of claim 2 , wherein the cells comprise differentiated cells derived from stem cells.

4. 4. The composition of claim 3, wherein the stem cells are at least one selected from the group consisting of induced pluripotent stem cells (iPSCs), embryonic stem cells (embryonic stem cells), mesenchymal stromal cells (MSCs), off-the-shelf universal stem cells (USCs), bone marrow-derived stem cells, adipose tissue-derived stem cells, and placenta-derived stem cells.

5. 5. The composition of claim 4, wherein the universal stem cells have one or more of the following characteristics: The HLA (Human leukocyte antigen) gene is removed; and Attack neutralization signals are overexpressed.

6. The composition described in claim 1, characterized in that the composition further contains hepatocytes, hepatic stellate cells and mesenchymal stem cells.

7. The composition described in claim 1, characterized in that the artificial organ is a liver.

8. A method for manufacturing the artificial organ described in claim 1, comprising the steps of: a) providing a tissue-derived organ; b) decellularizing the donated organ; c) treating the decellularized organ with an anti-CD31 aptamer, wherein the anti-CD31 aptamer comprises the base sequence represented by SEQ ID NO: 1; and d-1) Recellularizing the decellularized organ with vascular endothelial cells.

9. 9. The method of claim 8, further comprising one or more steps selected from the group consisting of: d-2) recellularizing the decellularized organ with parenchymal cells; and d-3) Recellularizing the decellularized organ with non-parenchymal cells.

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