Method for preparing high-function artificial organs using aptamers

Abstract

The present invention relates to a method for producing a highly functional artificial organ using aptamers, comprising blood vessels capable of transporting perfusate. When used as a coating agent for a decellularized scaffold, the aptamer according to the present invention enhances vascular adhesion, activity, and angiogenic potential, thereby enabling more effective vascular reconstruction than existing antibodies. Consequently, a vascularized artificial liver prepared using this aptamer reduces thrombosis in vivo and enhances liver function, and is therefore expected to be applicable to methods for treating diseases using artificial organs prepared using the aptamers according to the present invention.

Description

Technical Field

[0001] The present invention relates to a method for preparing a high-function artificial organ by using an aptamer or the like.

[0002] This application claims priority to and the benefit of Korean Patent Application Nos. 10-2020-0098314 and 10-2020-0150913, filed on August 6, 2020, and November 12, 2020, respectively, with the Korean Intellectual Property Office, and the entire contents of the specifications and drawings of these applications are incorporated into this application. Background Art

[0003] Artificial organ biotechnology is the technology for producing devices that replace human organs through various methods, including culturing stem cells and cell growth factors (such as cellular nutrients) on three-dimensional bio-artificial scaffolds and using this template to create artificial organs. Although the development of modern medicine has made almost all organs available for transplantation, many patients cannot benefit due to the lack of donors, and artificial organs have side effects due to their lack of biocompatibility.

[0004] Meanwhile, chronic liver diseases such as cirrhosis cause approximately two million deaths annually. In recent decades, breakthroughs in immunology and organ transplantation have enabled liver transplantation to be performed in many cases of end-stage liver disease. Liver transplantation is now recognized as the only treatment for congenital or acquired liver disease, but donors are scarce. In this regard, the production of artificial livers through tissue engineering as a donor organ alternative has been attracting considerable attention. Because the mortality rate for liver-related diseases in South Korea is very high compared to other diseases, the number of people waiting for transplants due to organ shortages is considerable, and South Korea is one of the countries with the highest number of liver transplants, there is a need to develop a technology that can improve these situations.

[0005] While various approaches have been investigated for producing liver scaffolds, including tissue engineering, the application of 3D printing technology, and organ reconstruction using stem cells (organoids), technical limitations such as the inability to simulate the delicate structure of organs and size restrictions have yet to be overcome. Decellularized scaffolds, in which all cells have been removed from animal organs, are being evaluated as useful substrates for safe and functional human organ-mimicking scaffolds while overcoming these issues. In these scaffolds, while cellular components that can induce an immune response are removed, an organ-specific microenvironment, such as microstructure and biochemical signals, is created, thus facilitating the three-dimensional organization of cells when human cells are injected into the scaffold. Therefore, decellularized scaffolds may serve as suitable substrates for the preparation of artificial organs, and research has been conducted on the use of decellularized scaffolds to reconstruct various organs.

[0006] Because the liver has a histologically complex vascular structure and is an organ through which 25% or more of the heart's blood output passes, organ vascularization may be crucial for the successful reconstruction of an artificial liver. After transplantation, artificial organs that have not yet developed a vascular structure connect to the recipient's bloodstream and then form intravascular thrombosis due to an acute immune response, with a high probability of subsequent transplant failure. Therefore, various coating agents for effectively reconstructing vascular structures have been studied.

[0007] Aptamers are single-stranded nucleic acids composed of short sequences. They are considered an alternative to antibodies due to their high affinity for specific proteins, low immunogenicity, and mass production. Aptamers are commonly used in medical diagnostics and have also attracted attention as therapeutic agents for cancer and viral diseases. However, the use of aptamers as coating agents for tissue engineering has not yet been elucidated.

[0008] In addition, human leukocyte antigen (HLA) is one of the human tissue compatibility antigens, and when homologous cells or artificial organs are transplanted into the patient's body, immune rejection may occur if the human leukocyte antigen type does not match. Recently, many studies have been conducted on universal cells established by gene editing to remove such human leukocyte antigens-A, B and C types. However, there is a limitation: cells from which human leukocyte antigens A, B and C have been removed still show sensitivity to NK cells during transplantation. Therefore, cells that can ultimately escape the immune response can be produced by overexpressing "don't eat me" signals that inhibit NK cell responses. In particular, cells differentiated into various lineages from ready-made universal stem cells (USCs) with such characteristics can be used as cell compositions to minimize the occurrence of immune rejection during artificial organ production.

[0009] Therefore, this technology uses aptamers as coating agents to maximize the efficiency of vascular reconstruction and establishes for the first time a technology that minimizes thrombosis after artificial organ transplantation. Furthermore, this technology uses liver-constituting cells derived from universal stem cells (USCs) to create a vascularized artificial liver with enhanced functionality that can be transplanted into the body.

[0010] [Related prior art documents]

[0011] KHHussein,KMPark,KSKang,HMWoo,Heparin-gelatin mixture improves vascular reconstruction efficiency and hepatic function inbioengineered livers,Acta Biomater 38(2016)82-93 Summary of the Invention

[0012] [Technical Issues]

[0013] Thus, the present inventors have maximized the efficiency of vascular reconstruction using nucleic acid aptamers as coating agents and, for the first time, have proposed a technology that can minimize thrombosis after artificial organ transplantation. Furthermore, the present inventors have demonstrated for the first time that nucleic acid aptamers can be used to prepare artificial organs that are transplantable and have enhanced function, thus completing the present invention.

[0014] Therefore, one object of the present invention is to provide a composition for producing an artificial organ, which comprises an aptamer.

[0015] Another object of the present invention is to provide a composition for coating a blood vessel, which comprises an aptamer.

[0016] Another object of the present invention is to provide a method for preparing an artificial organ using an aptamer.

[0017] Yet another object of the present invention is to provide an artificial organ produced by this method.

[0018] However, the technical problems to be solved by the present invention are not limited to the above problems, and a person skilled in the art will fully understand other problems not described herein from the following description.

[0019] [Technical solution]

[0020] To achieve the object of the present invention, the present invention provides a composition for preparing an artificial organ, wherein the composition comprises an aptamer.

[0021] Furthermore, the present invention provides a use of a composition comprising an aptamer for producing an artificial organ.

[0022] Furthermore, the present invention provides a method for producing an artificial organ, the method comprising the step of treating with an aptamer.

[0023] Furthermore, the present invention provides a composition for coating a blood vessel, the composition comprising an aptamer.

[0024] Furthermore, the present invention provides use of a composition comprising an aptamer for coating a blood vessel.

[0025] Furthermore, the present invention provides a method for coating a blood vessel, comprising the step of treating with a composition containing an aptamer.

[0026] Furthermore, the present invention also provides an artificial organ produced by the method.

[0027] In an exemplary embodiment of the present invention, the artificial organ may be biocompatible.

[0028] In another exemplary embodiment of the present invention, the artificial organ is not limited as long as it is an organ including a blood vessel, and specifically may be a liver, but is not limited thereto.

[0029] In yet another exemplary embodiment of the present invention, the aptamer may be an anti-CD31 aptamer, but is not limited thereto.

[0030] In yet another exemplary embodiment of the present invention, the aptamer may include the base sequence represented by SEQ ID NO: 1, but is not limited thereto.

[0031] In yet another exemplary embodiment of the present invention, the aptamer may be characterized by being specific to vascular endothelial cells, but is not limited thereto.

[0032] In yet another exemplary embodiment of the present invention, the aptamer may increase the expression of one or more selected from the group consisting of integrin β3 and phosphorylated Akt, but is not limited thereto.

[0033] In yet another exemplary embodiment of the present invention, the aptamer can reduce the expression of cleaved caspase-3, but is not limited thereto.

[0034] In yet another exemplary embodiment of the present invention, the composition may further include one or more cells selected from parenchymal cells and non-parenchymal cells, but is not limited thereto.

[0035] In yet another exemplary embodiment of the present invention, the cells may be stem cell-derived cells, but are not limited thereto.

[0036] In yet another exemplary embodiment of the present invention, the cells may be characterized as including differentiated cells of stem cell origin, but are not limited thereto.

[0037] In yet another exemplary embodiment of the present invention, the composition may further include stem cell-derived parenchymal cells, but is not limited thereto.

[0038] In yet another exemplary embodiment of the present invention, the composition may further include stem cell-derived non-parenchymal cells, but is not limited thereto.

[0039] In another exemplary embodiment of the present invention, the stem cells may be one or more selected from induced pluripotent stem cells (iPSCs), embryonic stem cells, mesenchymal stromal cells (MSCs), readily available universal stem cells (USCs), bone marrow-derived stem cells, adipose tissue-derived stem cells, and placenta-derived stem cells, but are not limited thereto.

[0040] In another embodiment of the present invention, the readily available universal stem cells may have one or more of the following characteristics, but are not limited thereto:

[0041] The HLA genes have been removed; and

[0042] The "don't eat me" signal is overexpressed.

[0043] In another embodiment of the present invention, the preparation method may include the following steps, but is not limited thereto:

[0044] a) Providing organs from tissue sources;

[0045] b) decellularizing the donated organ; and

[0046] c) Treatment of decellularized organs with aptamers.

[0047] In another embodiment of the present invention, the preparation method may further include one or more steps selected from the following steps, but not limited thereto:

[0048] d-1) recellularizing the decellularized organ into vascular endothelial cells;

[0049] d-2) recellularizing the decellularized organ into parenchymal cells; and

[0050] d-3) Recellularization of the decellularized organ into non-parenchymal cells.

[0051] [Beneficial Effects]

[0052] When used as a coating agent for decellularized scaffolds, the aptamers of the present invention can more effectively reconstruct the vasculature than existing antibodies by enhancing vascular adhesion, activity, and angiogenic potential. Consequently, vascularized artificial livers prepared using these aptamers can reduce thrombosis in vivo and enhance liver function, leading to their potential application in methods for treating diseases using artificial organs prepared using the aptamers of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figures 1A to 1G The results of characterization of anti-CD31 aptamers are shown in Figure 2: Figure 1A The functional structure of the anti-CD31 aptamer forming the CD31-aptamer complex is shown in FIG. Figures 1B to 1D Shows the results of validating the dose-dependent binding of cy5-labeled anti-CD31 aptamers to HUVEC, HepG2 cells, and MSCs; Figure 1B FACS profiles showing the dose-dependent binding efficiency of anti-CD31 aptamers to HUVECs, HepG2 cells, and MSCs; Figure 1C The quantification results of the binding rate are shown in ; Figure 1D The results of quantification of mean fluorescence intensity (MFI) of anti-CD31 aptamers binding to HUVECs, HepG2 cells, and MSCs are shown in the figure; Figure 1EThe middle shows immunocytochemistry images of HUVECs stained with cy5-labeled anti-CD31 aptamer (top, red) and anti-CD31 antibody (bottom, red); Figure 1F and 1G The middle shows immunocytochemistry images of HepG2 cells and MSCs stained with anti-CD31 aptamer (top, red) and anti-CD31 antibody (bottom, red).

[0054] Figures 2A to 2J Anti-CD31 aptamers were shown to enable integrin-mediated endothelial cell (EC) adhesion to the extracellular matrix (ECM) under shear stress: Figure 2A The ECM components are shown in; Figure 2B Representative phase contrast images of HUVECs subjected to shear stress are shown in the middle; Figure 2C The attachment rate is shown in the middle; Figure 2D The middle shows the results of qRT-PCR analysis of devices coated with PBS (blank, uncoated), anti-CD31 aptamer (APT-coated), and anti-CD31 antibody (Ab-coated); Figures 2E to 2G Figure 5 shows the results of Western blot analysis of integrin β3, total Akt, and phosphorylated Akt in static HUVECs and HUVECs exposed to shear stress in microfluidic devices coated with the respective coating agents; Figures 2H to 2J Figure 5 shows the results of Western blot analysis of the expression of cleaved caspase-3 in static HUVECs and exposed HUVECs after shear stress exposure in the groups coated with PBS (blank, uncoated), anti-CD31 aptamer (APT-coated), and anti-CD31 antibody (AB-coated), respectively; Figure 2J Shown in FIG are qRT-PCR results of nitric oxide synthase 3 (NOS3) expression in each group of shear-stressed HUVECs normalized to static HUVECs.

[0055] Figures 3A to 3L The results show that HUVECs can effectively reendothelialize decellularized liver scaffolds: Figure 3A The method of reendothelialization of decellularized rat liver scaffolds using HUVECs is shown in ; Figure 3B Representative immunofluorescence images of vascular endothelium with CFDA-labeled HUVECs on scaffolds coated or not with anti-CD31 aptamer (APT-coated) or anti-CD31 antibody (Ab-coated) are shown in Figure 5 . Figure 3C The endothelial coverage of the reendothelialized vessels for each stent is shown in the middle; Figure 3D The average number of reendothelialized vessels per stent was quantified; Figure 3E Shown in the figure are the results of quantifying intravascular dextran discharged from the inferior vena cava in each structure after dextran perfusion via the portal vein; Figure 3FMiddle shows a magnified confocal image of an APT-coated structure stained with zonula occludens-1 (ZO-1); Figure 3G Figure 5 shows the qRT-PCR results of VE-cadherin and Claudin-5 (CLDN5) in static HUVEC, uncoated, APT-coated, and Ab-coated reendothelialized structures; Figure 3H The results of detecting the activity of each construct using PrestoBlue reagent by performing resazurin reduction perfusion experiments on days 3, 5, and 7 are shown in FIG; Figure 3I Representative confocal images of each group of reendothelialized structures stained with cleaved caspase-3 (red) and DAPI (blue) are shown in Figure 5 ; Figure 3J The results of quantification of cells expressing cleaved caspase-3 in each group are shown in ; Figure 3K The amount of NO produced by each group, quantified by ELISA, in the reendothelialized structures is shown in FIG; and Figure 3L Figure 5 shows the ELISA quantification results of human VEGF secreted by the reendothelialized structures in each group on day 7.

[0056] Figures 4A to 4G The results show that reendothelialized structures have low thrombogenicity after perfusion with human blood: Figure 4A Methods for transferring human blood to reendothelialized structures to assess thrombus formation are shown in ; Figure 4B The macroscopic images of each stent after human blood perfusion are shown in the middle, where non-endothelialized DLM serves as a negative control; Figure 4C Representative immunohistochemical images of structures collected from each group stained with integrin αIIb (green) are shown in the middle; Figure 4D The fluorescence intensity of quantified integrin αIIb expression is shown in the middle; Figure 4E The middle shows the results of platelet quantification using a hematology analyzer after collecting blood perfusate at specific time points in the DLM group; Figure 4F The results show the platelet quantification using a hematology analyzer after collecting blood perfusate at specific time points in each group of reendothelialized structures; and Figure 4G RT-PCR analysis results of CD63, PLSCR1, TBXAS1, and THBS1, which are genes involved in platelet aggregation in each group of blood perfusion structures, are shown in FIG.

[0057] Figures 5A to 5M The results show the maturity of the enhanced functionality of the VBHL structure: Figure 5A Schematic diagram of the recellularization process of decellularized rat liver scaffolds using HepG2 cells, LX2 cells, HUVECs, and MSCs; Figure 5BShown in the middle is a set of representative immunofluorescence images of uncoated VBHL constructs (CTL-VBHL) or VBHL constructs coated with anti-CD31 aptamer (APT-VBHL) and anti-CD31 antibody (Ab-VBHL) on day 21; Figure 5C The endothelial coverage of the vessels in each group is shown in the middle; Figure 5D Results quantifying the average number of endothelialized vessels per field are shown in ; Figure 5E Shown in the middle is a set of representative immunohistochemical images of CTL-VBHL, APT-VBHL, and Ab-VBHL structures stained with α-SMA (red); Figure 5F The results of dextran perfusion analysis are shown in the middle; Figures 5G to 5H The ELISA quantitative results of VEGF and NO secretion in each group of VBHL structures at the indicated time points are shown in the middle; Figure 5I and 5J The ELISA quantification results of the amount of secreted albumin and urea in each group of VBHL structures at the indicated time points are shown in the figure; Figure 5K The results in Figure 2 show the increased activity of the APT-VBHL construct at day 17 and day 20. Figure 5L Representative confocal images of TUNEL experiments are shown in ; and Figure 5M The quantification results of TdT-positive cells in random fields are shown in Figure 2 .

[0058] Figures 6A to 6E Results illustrating successful in vivo reperfusion of anti-CD31 aptamer-coated VBHL constructs: Figure 6A A photograph of the assisted transplantation of a VBHL construct is shown, in which the renal vein (yellow arrow) is connected to the inferior vena cava of the VBHL construct, the renal artery (white arrow) is connected to the portal vein of the construct, and the VBHL construct is reperfused by the in vivo renal circulatory system after the vascular clamp (blue arrow) is removed; Figure 6B Representative images of structures collected after transplantation are shown in ; Figure 6C A representative set of confocal images of each collected structure stained with integrin αIIb (green) is shown in ; Figure 6D The fluorescence intensity quantification results of integrin αIIb expression are shown in FIG; and Figure 6E The results of RT-PCR analysis of Cd63, Plscr1, and Thbs1 in each VBHL construct after transplantation are shown in FIG.

[0059] Figures 7A to 7F The results of transplantation of VBHL constructs in thioacetamide (TAA)-induced liver fibrosis are shown in: Figure 7A Schematic diagram of the method for transplanting VBHL constructs into TAA-induced cirrhotic rats; Figure 7BRepresentative H&E staining images of host liver tissue sections and representative Sirius red staining results of liver sections collected 4 weeks after transplantation of each construct are shown in the middle; Figure 7C The results of quantifying the degree of fibrosis in the host livers of each group are shown in the figure; Figure 7D The results of qRT-PCR analysis of fibrosis-related genes αSma, Vimentin, Tgf-beta1, and Timp1 in the livers of hosts in each group are shown in the figure; Figure 7E and 7F The ELISA measurement results of ALT and AST levels in rat serum are shown in Figure , where the red line indicates the normal range. DETAILED DESCRIPTION

[0060] Hereinafter, the present invention will be described in detail.

[0061] The present invention provides a composition for producing an artificial organ, which comprises an aptamer.

[0062] Furthermore, the present invention relates to a composition for coating a blood vessel, the composition comprising an aptamer.

[0063] Tissue engineering is a method that uses a combination of cells and various materials to anchor cells to a structure (template) called a scaffold. The goal is to create structures that can replace damaged tissues or organs, that is, organs. Although there are many types, bioscaffolds are biostructures (i.e., organs) that are decellularized and removed of cells, leaving only the structure (template) with the microstructure and outline of the organ.

[0064] The artificial organ described herein may be characterized by treating a biological scaffold with an aptamer to reconstruct the target organ structure, such as a vascular structure, but is not limited thereto. Specifically, the aptamer described herein can coat the vascular wall of a decellularized scaffold to specifically bind to vascular endothelial cells, thereby reconstructing or generating a vascular structure.

[0065] The present inventors demonstrated that when the vascular wall of a decellularized scaffold is coated with an anti-CD31 aptamer, vascular endothelial cells can adhere to the wall of the decellularized scaffold, thereby reconstructing a highly functional vasculature that maintains the function of the vascular barrier (see embodiments of the present invention).

[0066] In the present invention, the blood vessel may be a blood vessel of a decellularized scaffold, but is not limited thereto.

[0067] In the present invention, the blood vessel may be a hepatic blood vessel, such as the hepatic portal vein, hepatic sinusoid, hepatic vein or hepatic artery, but is not limited thereto.

[0068] In the present invention, artificial organs can be biocompatible. The term "biocompatibility" in the present invention is used interchangeably with "transplant compatibility" and refers to the property of cells that do not cause immune rejection when transplanted. Cells, tissues, or organs can be made transplant compatible by reducing the expression of genes responsible for immune rejection. As a non-limiting embodiment, reducing the expression level of HLA genes can make cells, tissues, or organs transplant compatible. Transplant-compatible cells include, but are not limited to, cells that are homozygous or null for immune compatibility antigens.

[0069] In the present invention, the artificial organ may be, for example, a liver, kidney, heart, valve, ureter, bladder, lung, pancreas, etc., but is not limited thereto.

[0070] In the present invention, the artificial organ may be an organ including a blood vessel, but is not limited thereto.

[0071] In the present invention, an aptamer refers to a single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) with a stable tertiary structure that is capable of specifically binding to a target substance, and the aptamer is capable of binding to a target protein (material). Aptamers can be prepared by determining and synthesizing an oligonucleotide sequence that has selectivity and binding ability for the target substance to be identified according to general aptamer preparation methods, and then modifying the 5' or 3' end of the oligonucleotide with -SH, -COOH, -OH, or NH2 to allow the end to bind to a functional group on the aptamer chip, but are not limited to this.

[0072] In the present invention, the aptamer may be an anti-CD31 aptamer, but is not limited thereto.

[0073] In the present invention, the anti-CD31 aptamer may include the base sequence represented by SEQ ID NO: 1, or may consist of the base sequence represented by SEQ ID NO: 1, but is not limited thereto.

[0074] In the present invention, aptamers may be characterized by, but are not limited to, specificity for vascular endothelial cells. Furthermore, in organ reconstruction procedures targeting only vascular endothelial cells, the aptamers of the present invention can be used not only for vascular reconstruction but also for liver parenchymal reconstruction, bile duct reconstruction, and other procedures, regardless of organ type. Specifically, aptamers that bind to proteins specifically expressed by hepatocytes can be used during liver parenchymal reconstruction, while aptamers that bind to proteins specifically expressed by bile duct cells can be used during bile duct reconstruction.

[0075] Therefore, the aptamer of the present invention may be specific to a protein specifically expressed by a target cell, but is not limited thereto.

[0076] In the present invention, the aptamer can increase the expression of one or more selected from the group consisting of integrin β3 and phosphorylated Akt, but is not limited thereto.

[0077] In the present invention, the aptamer can reduce the expression of cleaved caspase-3, but is not limited thereto.

[0078] In the present invention, the composition can generally be delivered to the tissue or organ matrix under physiological conditions (e.g., 37° C.) via a cell-miscible solution (e.g., a physiological composition). The composition may include, but is not limited to, a buffer, nutrients (e.g., sugars and carbohydrates), enzymes, proliferation and / or differentiation medium, cytokines, antibodies, inhibitory factors, growth factors, saline solutions, or serum-derived proteins.

[0079] In the present invention, the composition may further include parenchymal cells, but is not limited thereto. As used herein, the term "parenchymal cells" refers to cells that form the main part of the cell-intrinsic function. In the present invention, the parenchymal cells may specifically be hepatocytes, renal cells, corneal cells, vascular endothelial cells, etc., preferably hepatocytes, i.e., liver cells, but is not limited thereto. In the present invention, in a non-pathological state, hepatic parenchymal (parenchymal hepatocytes) cells account for 80% of the liver.

[0080] In the present invention, the parenchymal cells may be from the same species as the recipient, but are not limited thereto.

[0081] In the present invention, the composition may further include, but is not limited to, stem cell-derived parenchymal cells. The parenchymal cells may be, but are not limited to, parenchymal cells differentiated from readily available human universal stem cells, from which human leukocyte antigens have been removed, and / or which overexpress "don't eat me" signals.

[0082] In the present invention, the composition may further include non-parenchymal cells, but is not limited thereto. The non-parenchymal cells may be one or more selected from the group consisting of, but not limited to, hepatic endothelial cells, Kupffer cells, hepatic stellate cells, Ito cells, adipocytes, fat-storing cells, bile duct cells, crypt cells, vascular epithelial cells, and fibroblasts.

[0083] In the present invention, the composition may further include, but is not limited to, stem cell-derived non-parenchymal cells. The stem cell-derived non-parenchymal cells may be non-parenchymal cells differentiated from readily available universal human stem cells, from which human leukocyte antigens have been removed, and / or which overexpress "don't eat me" signals, but are not limited thereto.

[0084] In the present invention, the cells may be characterized as including differentiated cells derived from stem cells, but are not limited thereto.

[0085] The term "stem cell" as used herein refers to a cell that has the property of continuously producing cells identical to itself for a certain period of time in a differentiated state and the property of differentiating into a specific cell under appropriate conditions.

[0086] In the present invention, the stem cells may be one or more selected from the following: induced pluripotent stem cells (iPSCs), embryonic stem cells, mesenchymal stromal cells (MSCs), ready-made universal stem cells (USCs), bone marrow-derived stem cells, adipose tissue-derived stem cells and placenta-derived stem cells, but are not limited thereto.

[0087] In the present invention, the readily available universal stem cells may be characterized by, but are not limited to, HLA-depleted or overexpressed "don't eat me" signals. The present invention is expected to be applied clinically by constructing universal artificial organs comprising stem cell-derived constituent cells with human leukocyte antigens removed and / or overexpressing "don't eat me" signals to minimize immune rejection during transplantation.

[0088] In the present invention, the ready-made universal stem cells may be HLA type I knockout iPSC cells or HLA type I knockout MSC cells, but are not limited thereto.

[0089] In the present invention, the "don't eat me" signal may be CD47 or CD24, but is not limited thereto.

[0090] Furthermore, the present invention provides a method for producing an artificial organ, the method comprising treating with an aptamer.

[0091] In the present invention, the method for manufacturing the artificial organ may include the following steps, but is not limited thereto:

[0092] a) Providing tissue source organs;

[0093] b) decellularizing the donated organ; and

[0094] c) Treatment of decellularized organs with aptamers.

[0095] In the present invention, the method may further comprise one or more steps selected from:

[0096] d-1) recellularizing the decellularized organ into vascular endothelial cells;

[0097] d-2) recellularizing the decellularized organ into parenchymal cells; and

[0098] d-3) Recellularizing the decellularized organ into non-parenchymal cells, but not limited thereto.

[0099] In the present invention, the tissue source can be selected from various mammals such as humans, monkeys, mice, rats, pigs, cows and rabbits.

[0100] In the present invention, the decellularization can be performed by methods known in the art, but in an exemplary embodiment of the present invention, the hepatic portal vein obtained from the tissue source is washed with deionized water and PBS, then washed with deoxyribonuclease, and then sterilized with peracetic acid. For example, the following references describe perfusion-based decellularization of lung, liver, kidney, brain, and limbs: Van Putte et al., 2002, Ann. Thorac. Surg., 74(3):893-8; den Butter et al., 1995, Transpl. Int., 8:466-71; Firth et al., 1989, Clin. Sci. (Lond.), 77(6):657-61; Mazzetti et al., 2004, Brain Res., 999(1):81-90; Wagner et al., 2003, J. Artif. Organs, 6(3):183-91. As an alternative to perfusion-based decellularization, biological tissues and organs can be decellularized by immersion in a decellularization solution. References: eg, US Patent Nos. 6,376,244 and 6,753,181.

[0101] In the present invention, physiological buffers suitable for perfusion include nutrient supply sources that can be used for storage and / or organ perfusion (including transplantation). Examples include, but are not limited to, glucose-containing buffers, EGM-2, EGM-2MV, DMEM, Promocell Endothelial Cell Culture Medium, Medium 200, and DMEM F / 12. Furthermore, buffers include, but are not limited to, culture medium solutions or phosphate-buffered saline (PBS) suitable for culturing endothelial cells.

[0102] In the present invention, during the decellularization process, alternating perfusion directions (e.g., antegrade and retrograde) can help to effectively remove cells from the entire organ or tissue. The decellularization described in this specification can be performed by substantially removing cells with little damage to the ECM within the organ without damaging the ECM, but is not limited thereto. The organ or tissue can be decellularized at a suitable temperature between 4 and 40°C. Depending on the size and weight of the organ or tissue, the specific detergent in the cell disintegration medium, and the concentration of the detergent, the organ or tissue is typically perfused with the cell disintegration medium for about 2 to about 12 hours per gram of solid organ or tissue. The organ, including the perfusion fluid, can be perfused for about 1 to about 12 hours per gram of tissue. Perfusion is typically regulated by physiological conditions, including blood flow pulsation, flow rate, and pressure.

[0103] In the present invention, the number of regenerative cells introduced into and onto a decellularized organ for recellularization to produce an organ or tissue may vary depending on the type and developmental stage of the organ (e.g., what organ it is and its weight and size), or the tissue and the regenerative cells. Different cell types may have different tendencies in the population density achieved by these cells. Similarly, different organs or tissues can be recellularized at different densities. For example, a decellularized organ or tissue can be "seeded" with at least 1,000, 10,000, 100,000, 1,000,000, 10,000,000, or 100,000,000 regenerative cells; or, after recellularization, can have from about 1,000 cells / mg of tissue (wet weight, i.e., weight before decellularization) to about 100,000,000 cells / mg of tissue (wet weight), but is not limited thereto.

[0104] In the present invention, the method may further include culturing the organ treated with the aptamer 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 to 3 weeks or about 2 weeks, but is not limited thereto.

[0105] Furthermore, the present invention also provides an artificial organ produced by the method.

[0106] The artificial organ as an organ for transplantation may not cause immune rejection in a recipient after transplantation, but is not limited thereto.

[0107] The artificial organ can be used for screening therapeutic agents, preferably for screening therapeutic agents for liver diseases, but is not limited thereto.

[0108] Throughout the specification of the present application, when a part includes a constituent element, unless otherwise specifically stated, this does not mean that another constituent element is excluded, but rather that another constituent element may also be included. Throughout the specification of the present application, terms of degree, such as "approximately" or "substantially", are used in corresponding numerical values, or when natural manufacturing and material tolerance errors are presented in a descriptive sense, as a meaning close to the numerical value, and are used to prevent unlawful infringement of the disclosure, including numerical values ​​described as accurate or absolute in order to help understand the present invention. Throughout the specification of the present application, terms such as step ... or step of ... do not mean steps for ...

[0109] Throughout the specification of the present application, the terms and combinations thereof included in the Markush expression refer to a mixture or combination of at least one selected from the group consisting of constituent elements described in the Markush expression, and mean including at least one selected from the group consisting of constituent elements.

[0110] Throughout the specification of this application, the description A and / or B means A or B, or A and B.

[0111] In the case of certain embodiments that can be implemented differently, specific steps may not be performed in the order described. For example, two steps described in succession may be performed substantially simultaneously, or may be performed in an order opposite to the order described.

[0112] The terms or words used in this specification and claims should not be construed as being limited to typical or dictionary meanings, but should be construed with meanings and concepts consistent with the technical spirit of the present invention, based on the principle that the inventor can appropriately define the concepts of the terms in order to best describe his / her own invention.

[0113] Hereinafter, preferred embodiments will be provided to help understand the present invention. However, the following embodiments are provided only to make the present invention easier to understand, and the content of the present invention is not limited to the following embodiments.

[0114] Implementation methods and materials

[0115] 1. Preparation and Analysis of Aptamers

[0116] Aptamer Sciences Inc. (Korea) synthesized and characterized an anti-CD31 single-stranded DNA aptamer (76mer, 21966-18-01) with a core sequence of SEQ ID NO: 1. The anti-CD31 aptamer was dissolved in sterile Ultrapure DEPC-treated water (Invitrogen, USA), and the resulting solution was stored at -20°C. Before the experiment, the aptamer was heated at 95°C for 10 minutes and then cooled at room temperature for proper folding. To confirm that the anti-CD31 aptamer specifically targets CD31+EC, immunofluorescence staining and flow cytometry were performed using an aptamer labeled with cy5 at the 5' end (5'-cy5-aptamer-3').

[0117] <Anti-CD31 Aptamer>

[0118] 5-TCA GCC GCC AGC CAG TTC (primer)-G6A GAG GAG G6A CG6 AA6 G6C 6GG G6A6AC CCC GA6 AA6 6 (SEQ ID NO: 1; core sequence)-GAC CAG AGC ACC ACA GAG (primer)-3

[0119] 6 = NapdU [5-(N-naphthylcarboxyamide)-2'-deoxyuridine]

[0120]

[0121] 2 Cell culture

[0122] Human umbilical vein endothelial cells (HUVECs) and human umbilical cord blood-derived mesenchymal stem cells (MSCs) purchased from ATCC (USA) were maintained in endothelial cell growth medium-2 (EGM-2, Lonza, Switzerland). MSCs were established as described in Donor-dependent variation of human umbilical cord blood mesenchymal stem cells in response to hypoxic preconditioning and amelioration of limb ischemia, Exp Mol Med 50(4)(2018)35, with approval from the Institutional Review Board of Seoul National University (IRB No. 1608 / 001-021). Hepatocellular carcinoma cells (HepG2 cells) purchased from ATCC and LX-2 human stellate cells purchased from Millipore (USA) were cultured in DMEM (Dulbecco's Modified Eagle Medium, Hyclone, USA) containing high glucose. All culture media were supplemented with 10% fetal bovine serum (FBS, Gibco), antibiotics (100 U / ml penicillin and 100 μg / ml streptomycin (1% P / S, Gibco)), and 20 μg / ml Primocin (InvivoGen, USA). The culture medium was changed every 48 hours. Cells were cultured in a humidified incubator at 37°C with 5% CO2.

[0123] 3. Microfluidic Device Design and Cell Isolation Analysis

[0124] A 3D microfluidics system was used to evaluate the cell separation efficiency based on the coating agent. A device with three microchannels was fabricated using polydimethylsiloxane (PDMS, Sylgard 184; Dow Corning, USA) by soft lithography and replica molding. A PDMS prepolymer consisting of a mixture of PDMS matrix and curing agent (10:1) was poured onto a silicon wafer and cured. The PDMS block was then completely solidified and separated from the wafer.

[0125] After the inlet and outlet ports were punched using a biopsy punch, the PDMS block was attached to a vacuum-adhesive polycarbonate membrane. Before performing additional experiments, the device was placed in a drying oven overnight to restore hydrophobicity and sterilized by UV irradiation. The microfluidic device was coated with a mixture of 100 μg / ml type 1 collagen (rat tail, #354236, Corning, USA), vitronectin (#A31804, Gibco), and fibronectin (#356008, Corning).

[0126] Thereafter, the device was coated with each of the following coating agents: PBS (negative control), 600 nM anti-CD31 aptamer, and 50 μg / mL anti-CD31 antibody. 5 HUVECs were seeded into each channel and incubated for 2 hours. Subsequently, fluid shear stress was applied to the HUVEC monolayer using an infusion syringe pump (PHD 2000 Infusion, Harvard device, USA). Shear stress was calculated according to the modified Poiseuille equation (see Equation 1).

[0127] [Equation 1]

[0128] Tw=6μQ / wH 2

[0129] (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).

[0130] EGM-2 supplemented with 1% FBS was passed through the channel at a specified flow rate for 10 minutes. For each flow, after the flow, images of cells adhered to the surface were captured using an optical microscope (IX70, Olympus, Japan) and counted using Image J software. To analyze changes in HUVECs in response to shear stress, the cells were placed under a pressure of 10 dynes / cm 2 After 30 min of shear stress, RNA and protein were extracted from the cells.

[0131] 4. Production of Decellularized Rat Liver Scaffolds

[0132] Native livers were obtained from 8-week-old female Sprague-Dawley rats (250 to 300 g) after systemic heparinization. The hepatic portal vein was cannulated with a 24G catheter and perfused with 0.1% SDS in deionized water (Sigma Aldrich, USA) for 6 hours, followed by washing with PBS for 10 hours. Subsequently, 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 on the scaffold, the scaffold 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).

[0133] 5. Reendothelialization of Rat Liver Scaffolds

[0134] After being perfused with EGM-2 for 1 hour to stabilize the sterile decellularized liver matrix scaffold, the scaffold was 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 cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFDA) using the Vybrant CFDA SE Cell Tracker Kit (Invitrogen). 7 HUVECs.

[0135] CFDA-labeled HUVECs suspended in EGM-2 supplemented with 10% FBS and antibiotics were delivered via the portal vein at a rate of 0.5 ml / min. After 2 hours of static culture, medium was perfused into the reendothelialized constructs using a peristaltic infusion pump in the bioreactor at a rate of 1.5 ml / min. Medium was replaced every 48 hours, and samples were collected on day 7 for further analysis. Endothelial coverage and the number of reendothelialized vessels were quantified using Image J software.

[0136] 6. Delivery of Hepatocytes and Non-parenchymal Cells into Decellularized Rat Liver

[0137] When making acellular liver scaffolds, a 26G catheter was used for bile duct cannulation and a 24G catheter was used for portal vein cannulation. After decellularization, a total of 4×10 7 HepG2 cells were delivered into the parenchyma of the scaffold and cultured in the bioreactor for 2 days. 7 HepG2 cells were cultured in this construct for up to 14 days.

[0138] On the 14th day, 6x106 CFDA-labeled HUVEC and 3x10 6 The mixture of MSCs was delivered to the vascular lumen via the portal vein, and 1x10 7 LX2 cells. All cells were delivered at a rate of 0.5 ml / min. After 2 hours of static culture, the constructs were perfused at a rate of 1.5 ml / min using a peristaltic infusion pump. The VBHL constructs were maintained in DMEM high glucose medium containing 10% FBS and antibiotics for up to 14 days. From day 14 to day 21, the medium was replaced with EGM-2 containing 10% FBS and antibiotics. Additional analysis was performed on the VBHL constructs collected on day 21.

[0139] 7. Statistical Analysis

[0140] Statistical analysis was performed using GraphPad Prism version 5.0. All values ​​are reported using mean ± standard deviation. Values ​​of p < 0.05 were considered significant. Statistical analysis between the two groups was performed using an unpaired, two-tailed Student's t-test. For multiple group comparisons, a two-way ANOVA analysis was performed using the Bonferroni post hoc test. The data provided represent the results of at least three independent experiments.

[0141] 8. Immunofluorescence Staining

[0142] Cells or tissue sections were fixed with 4% formaldehyde for 10 minutes. Subsequently, samples were permeabilized with 0.2% Triton X-100 (Sigma Aldrich) for 10 minutes and blocked with 5% chlorothiazide serum (Vector Laboratories, Switzerland) for 1 hour. Subsequently, the samples were probed with the following primary antibodies overnight at 4°C: anti-human CD31 (14-0319-80, eBioscience, USA), anti-albumin (GTX102419, GeneTex, USA), anti-vimentin (ab45939, Abcam, UK), anti-β-galactosidase α (GTX101178, GeneTex), anti-ZO-1 (#40-2200, Invitrogen), anti-cleaved caspase-3 (#9664, Cell Signaling Technology, USA), anti-phospho-Akt (#9271, Cell Signaling Technology), anti-integrin β3 (#13166, Cell Signaling Technology), anti-integrin αⅡb (sc-365938, Santa Cruz biotechnology, USA), and anti-α-smooth muscle actin (ab7814, Abcam). The cells were treated with fluorescent dye-coupled secondary antibodies (Alexa Fluor 488 and 549; Invitrogen) for 1 hour. The nuclei were stained with DAPI (sc3598, Santa Cruz Biotechnology) for 10 minutes and the cells were fixed in fluorescent mounting medium (S302380, DAKO, Denmark). The staining signals were observed using an Eclipse TE 2000 confocal laser scanning microscope (Nikon, Japan). Similarly, paraffin-embedded tissue sections were deparaffinized, hydrated, fixed with 4% formaldehyde, and then stained as described above.

[0143] 9. Flow Cytometry

[0144] To evaluate the binding kinetics of anti-CD31 aptamers in a concentration-dependent manner, HUVECs were incubated with various concentrations of Cy5-labeled anti-CD31 aptamers at 4°C for 20 minutes in a reaction buffer consisting of 1 mM MgCl2 and 2 mg / ml bovine serum albumin. HepG2 cells and MSCs were compared to test the specificity of the anti-CD31 aptamers. The cells were washed with 1 ml of reaction buffer, suspended in PBS, and analyzed by FACS Calibur (BD Bioscience, USA). A PE-conjugated anti-CD31 antibody (BD555446, BD Biosciences) was used as a positive control. Data were analyzed using FlowJo software (USA).

[0145] 10. Cell Adhesion Assay

[0146] The rat abdominal aorta was collected and immersed in stirred 0.05% SDS for decellularization. The decellularized artery scaffold was cut longitudinally to reveal the internal structure and disinfected with 0.1% peracetic acid. Subsequently, the scaffold was immersed in the coating agent for 2 hours: PBS (negative control), 600nM anti-CD31 aptamer and 50μg / mL anti-human CD31 antibody (14-0319-80, eBioscience, positive control). Subsequently, the coated scaffold was transferred to a 24-well plate and 1x10 6 HUVECs were seeded onto the inner surface of the arterial stent. After maintaining static culture at the specified time points (2 hours and 4 hours), the cell-distributed stents were washed with PBS and transferred to a new well plate. The stents were subjected to a tetrazolium-based MTT assay to quantify the number of viable cells. For the MTT assay, the samples were stored at 37°C for 4 hours in a culture medium containing 10% MTT stock solution (Sigma Aldrich). Subsequently, the supernatant was removed and dimethyl sulfoxide was added to each well to dissolve the purple formazan. The absorbance at a wavelength of 540 nm was detected using a microplate reader (Infinite M200 pro, Tecan, Switzerland).

[0147] 11.qRT-PCR

[0148] Total RNA was extracted from cells or constructed constructs using NucleoZOL (Macherey-Nagel, Germany). Complementary DNA was then synthesized from the extracted RNA using the Superscript III First-Strand Synthesis System (Invitrogen). qRT-PCR was performed using the ABI 7300 Real-Time PCR System (Applied Biosystems) with SYBR Green PCR MasterMix (Applied Biosystems, USA). Relative quantification of target mRNA expression levels was performed using 2 (ΔΔ threshold cycle )(2- ΔΔCT ) method. The expression level of each gene was normalized to the expression of the housekeeping gene. The primer sequences used are shown in Table 1.

[0149]

Table 1

[0150]

[0151] 12. Immunoblotting

[0152] Protein was extracted using PRO-PREP (iNtRon Biotechnology, South Korea), and whole-cell lysates were sonicated. To extract protein from tissue samples, the tissue was homogenized with steel beads and PRO-PREP, followed by sonication. After centrifugation, the supernatant was transferred to a fresh tube and analyzed. Protein concentration was quantified using a DC assay kit (Bio-Rad, USA). Equal amounts of protein (10 μg) were loaded onto 8% to 15% acrylamide Tris-glycine gels. The following primary antibodies were used to detect target proteins: anti-GAPDH (AB2302, Millipore), anti-integrin beta 3 (#13166, Cell Signaling Technology), anti-Akt (#4691, Cell Signaling Technology), anti-phospho-Akt (#9271, Cell Signaling Technology), anti-cleaved caspase-3 (#9664, Cell Signaling Technology), anti-galactosidase α (GTX101178, GeneTex), anti-fibronectin (ab2413, Abcam), anti-collagen IV (ab19808, Abcam), anti-laminin (ab11575, Abcam), anti-CK18 (MAB3234, Millipore), anti-CYP2E1 (CSB-PA006425EA01HU, CUSABIO, China), and anti-AFP (A0008, DAKO). After incubation with primary antibodies overnight at 4°C, HRP-conjugated secondary antibodies were used. After binding using the Amersham enhanced chemiluminescence detection kit (GE Healthcare, USA), specific protein bands were detected by FluorChem HD2 (Alpha Innotech., USA).

[0153] 13. Organize inspections

[0154] The samples were washed three times with PBS and fixed in 4% paraformaldehyde at 4°C overnight. The tissue was embedded in paraffin and serially cut into 10 μm thickness. After dewaxing, the tissue sections were hydrated using a series of decreasing concentrations of ethanol from 100% to 70%. Subsequently, the tissue sections were stained with H&E and Sirius 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 rinsed with tap water, dehydrated using a series of increasing concentrations of ethanol, and sealed. Visualization was performed using Nikon NIS-Elements software and an optical microscope (Nikon), and fibrosis was quantified using Image J software.

[0155] 14. Resazurin Reduction Perfusion Test

[0156] PrestoBlue Cell Viability Reagent (A13261, Invitrogen) is a non-toxic solution based on resazurin and was used according to the manufacturer's instructions. 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 a 6-well plate. After 6 hours of culture, the cells were washed and the culture medium was replaced with 2 ml of PrestoBlue reagent mixed with complete EGM-2 (ratio of 1:20). After incubation at 37°C for 1 hour, the culture medium was collected and the absorbance at a wavelength of 570 nm was measured using a microplate reader. According to the standard curve, 30 ml of the PrestoBlue intermediate mixture was perfused into the constructed structure at 37°C for 1 hour. The collected culture medium was analyzed as described above.

[0157] 15. FITC-conjugated dextran perfusion assay

[0158] In order to assess the permeability of reendothelialized blood vessels in the tissue constructed, 500kDa FITC-dextran (FD500S, Sigma Aldrich) was perfused through the portal vein. 25mL of FITC-dextran (0.2mg / mL) was used under 20mmHg. The dextran in the fluid discharged from the inferior vena cava is considered to be intravascular dextran, while the dextran in the peripheral blood is considered to be extravascular dextran. The total amount of dextran is determined by fluorescence intensity (absorbance at 490nm) multiplied by the amount of fluid discharged.

[0159] 16.TUNEL assay

[0160] To detect apoptotic cells from the constructs, the ApopTag Red In situ Apoptosis Detection Kit (Millipore) was used. Tissue sections were deparaffinized and digested with proteinase K. Sections were treated with equilibration buffer and incubated with TdT enzyme for 1 hour in a 37°C incubator. After washing with stop / wash buffer, anti-digoxigenin (rhodamine conjugate) was applied to the sections. Subsequently, the sections were washed with PBS, and the nuclei were stained with DAPI. TUNEL-positive cells were detected under a confocal microscope.

[0161] 17. Quantification of Secreted NO and VEGF

[0162] The NO concentration secreted by the bioengineered structure was measured using the NO Plus detection kit (iNtRON). Conditioned culture medium was collected at the specified time point and cell debris was removed by centrifugation. Afterwards, NO was measured from the supernatant of the conditioned culture medium according to the manufacturer's instructions. The absorbance at a wavelength of 540 nm was measured using a microplate reader. The structure was cultured in endothelial basal medium 2 (EBM-2) without growth factors and exposed to human VEGF Quantikine ELISA kit (DVE00, R&D Systems, USA). Twelve hours later, supernatant was obtained for quantification of VEGF in the conditioned culture medium from each structure. The absorbance at a wavelength of 450 nm was measured using a microplate reader.

[0163] 18. In vitro human blood perfusion test

[0164] Use 1x10 7 The HUVEC-refilled scaffolds were cultured in EGM-2 supplemented with 10% FBS and antibiotics for 7 days and then perfused with human blood. Heparinized human blood obtained from the Korean Red Cross Central Blood Center was mixed with culture medium (1:1) and perfused through the portal vein of each scaffold. At the specified time points, vascular effluent was collected and the number of platelets in the blood perfusate was counted using an Advia 2120i hematology system (Siemens Healthineers, Germany). 24 hours after perfusion, the samples were washed with PBS and subjected to gene expression analysis and immunostaining to assess the extent of intra-stent thrombosis. cDNA was synthesized from the extracted RNA and then amplified by PCR using primers targeting thrombosis genes. In addition, paraffin-embedded tissue blocks were sectioned and stained with integrin αIIb according to the procedures described in the immunofluorescence staining method.

[0165] 19. Albumin / urea ELISA analysis

[0166] The amount of albumin secreted from the constructs in the conditioned medium was determined using a human albumin ELISA kit (ab108788, Abcam). Conditioned medium from the VBHL constructs was collected at the designated time points. After centrifugation, the concentration of secreted albumin in the supernatant was quantified using an ELISA according to the manufacturer's recommendations. The absorbance at a wavelength of 450 nm was measured using a microplate reader. In addition, 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 Quanti ChromUrea Assay Kit (BioAssay Systems, USA).

[0167] 20. In Vivo Reperfusion of Vascularized Liver Constructs

[0168] First, the following decellularized liver constructs were prepared by catheterization: 22G catheter - portal vein, 26G catheter - bile duct, and 20G catheter - inferior vena cava. The VBHL constructs were generated as described in Experimental Methods Section 6. The VBHL constructs were cultured in a bioreactor for 21 days and then directly connected to the host renal circulatory system through catheters of various sizes. For the above procedures, the left kidney was exposed by anesthetizing healthy 1-year-old rats. After the renal artery and vein were fixed, each 24G catheter was inserted. The catheters placed in the portal vein and inferior vena cava of the construct were connected to the catheters placed in the renal artery and vein, respectively. Each connection was fixed with Vetbond glue (3M, USA). After removing the vascular clamps, blood was perfused in vivo for 2 hours. Thereafter, additional analyses were performed on the collected liver constructs.

[0169] 21. TAA-induced chronic liver injury rat model and VBHL construct transplantation

[0170] To evaluate the in vivo function of VBHL constructs, chronic liver injury was induced in 4-week-old female rats. After the rats were continuously administered drinking water containing 0.3 g / L TAA (Sigma Aldrich) for 12 weeks, the induced liver fibrosis was analyzed. ALT and AST levels in rat serum were measured using an ALT activity colorimetric assay kit (K752-100, Biovision, China) and an AST activity colorimetric assay kit (K753-100, Biovision). After 8 weeks of induction of cirrhosis in rats, the livers were exposed by performing laparotomy on the rats. Subsequently, the fibrous capsules were removed from the VBHL constructs in each group and then transplanted and fixed between the medial and right lobes of the host liver. The VBHL constructs were generated using decellularized rat livers as described in Experimental Methods Section 6. After 4 weeks, the livers of the hosts transplanted with the liver constructs were collected again and subjected to additional analysis. Serum samples were also obtained before and after surgery.

[0171] Embodiment 1: Characteristics of anti-CD31 aptamers

[0172] like Figure 1A As shown, an anti-CD31 aptamer that can specifically bind to CD31 protein was prepared, and then flow cytometry was used to verify the binding strength between CD31-expressing cells and the aptamer.

[0173] Results, such as Figure 1B and 1C As shown, aptamers at concentrations of 600 nM and 800 nM were able to bind to approximately 99% of vascular endothelial cells (HUVECs), and to approximately 2% of HepG2 and MSCs, which do not express CD31. Furthermore, when the binding strength to vascular endothelial cells was quantified based on aptamer concentration, the binding strength saturated at an aptamer concentration of 600 nM, thus optimizing the aptamer concentration condition.

[0174] In addition, if Figures 1E to 1G As shown, cell immunostaining confirmed that 600 nM aptamer bound to HUVECs, but not to HepG2 and MSCs. Based on the above results, it was confirmed that the anti-CD31 aptamer specifically bound to CD31.

[0175] Implementation 2: Evaluation of the Adhesion Ability of Vascular Endothelial Cells During Aptamer Treatment and Verification of the Effect on Vascular Endothelial Cells

[0176] like Figure 2A As shown, the interior of the microfluidic device was first coated with extracellular matrix (ECM) components, and then coated with anti-CD31 aptamer (APT-coated) and anti-CD31 antibody (Ab-coated, positive control), respectively. HUVECs were injected, and the cell adhesion ability was evaluated while the liquid was flowing at a constant speed.

[0177] Results, such as Figure 2B and 2C As shown, compared with the uncoated group, more than 80% of the cells in the APT-coated group showed a significant increase in the number of cells after application of 10 dynes / cm 2 Even when a strong shear stress (20 dynes / cm 2 ) also showed a high adhesion capacity of 61.5%. As a positive control, the Ab coating group was coated with 20 dynes / cm 2 The results showed that the adhesion capacity of vascular endothelial cells increased by 51% when treated with shear stress. This indicates that the anti-CD31 aptamer is more effective than the anti-CD31 antibody in enhancing the adhesion capacity of vascular endothelial cells.

[0178] Furthermore, to investigate the mechanism by which the differences in HUVEC adhesion ability were mediated by each coating agent, mRNA expression patterns were analyzed by extracting RNA from cells in the microfluidic device.

[0179] Integrins are known to consist of an extracellular domain, a transmembrane domain, and an intracellular domain. When the extracellular domain binds to the extracellular matrix, signals are transmitted into the cell, activating various signaling systems. Therefore, we investigated whether shear stress could activate integrins.

[0180] Results, such as Figures 2E to 2G As shown in the figure, it is proved that in the actual microfluidic device coated with aptamers, the 2 In HUVECs subjected to shear stress, not only integrins but also Akt signaling (a lower signaling system) are activated. It has been reported that Akt signaling is related to cell survival and angiogenic potential of vascular endothelial cells. Therefore, Figure 2I As shown, it was also confirmed that the expression of cleaved caspase-3, a marker of cell apoptosis, was reduced in the APT-coated group.

[0181] Implementation 3: Validation of aptamer efficacy in the process of reconstructing vascular structure through recellularization of endothelial cells in decellularized scaffolds

[0182] like Figure 3A As shown, a decellularized scaffold was fabricated from rat liver, and the vascular lining was then coated with a coating agent. CFDA (green)-labeled HUVECs were recellularized and cultured for 7 days to confirm the reconstructed vascular structure.

[0183] Results, such as Figure 3B As shown, it was demonstrated that vascular endothelialization was better performed in the stent treated with the anti-CD31 aptamer as a coating agent.

[0184] In addition, if Figure 3C and 3D As shown, it was demonstrated that approximately 80% endothelialization was achieved per blood vessel in the APT-coated group, and approximately 80% endothelialization was achieved within the stent. Thus, it was demonstrated that the endothelialization efficiency of the aptamer-treated group was maximized compared to the uncoated group.

[0185] In addition, if Figure 3E As shown, when dextran was injected through the portal vein catheter to evaluate the barrier function of the reconstructed blood vessels, the amount of intravascular dextran that did not leak out of the vessels was significantly increased in the APT-coated group. Therefore, it was demonstrated that when the scaffolds were treated with anti-CD31 aptamers as a coating agent, a highly functional vascular structure was effectively reconstructed, in which the barrier function was well maintained in the decellularized scaffolds.

[0186] In addition, since the viability of cultured cells during in vitro culture can significantly affect the function of reconstructed artificial organs, the degree of apoptosis of cultured cells was analyzed by immunostaining method.

[0187] Results, such as Figure 3I and 3J As shown, the cell apoptosis in the APT-coated group was reduced compared with the uncoated group or the anti-CD31 antibody-coated group.

[0188] Thereafter, enzyme-linked immunosorbent assay (ELISA) was performed to verify the vessel-specific functions of the reconstructed vascularized organs.

[0189] Results, such as Figure 3K and 3L As shown, it was demonstrated that when the amounts of nitric oxide (NO) and VEGF secreted from the HUVEC recellularized scaffolds were analyzed, the amounts of NO and VEGF secreted were significantly increased in the APT-coated group compared to the other groups.

[0190] Overall, it has been demonstrated that when anti-CD31 aptamers were used as coating agents, not only the revascularization efficiency and barrier function were enhanced, but also the vasoactivity and angiogenic potential were enhanced.

[0191] Example 4: Evaluation of thrombosis in stents by in vitro human blood perfusion

[0192] like Figure 4A To further verify the extent of vascular remodeling, human blood was perfused into HUVEC-recellularized scaffolds and cultured for 7 days to assess the extent of thrombosis in vitro. This assay allows for the evaluation of thrombosis when artificial organs are implanted into real people.

[0193] Results, such as Figure 4B As shown in Figure 2, thrombus formation was visually reduced in the APT-coated group. Figure 4C and 4D As shown, it was demonstrated by immunostaining that the expression of integrin αIIb, a thrombus marker, was decreased in the APT-coated group.

[0194] In addition, if Figure 4E and 4F As shown, it was confirmed that when the number of platelets in the perfusate was quantified after blood perfusion, in the decellularized liver matrix (DLM) group without reendothelialization, the amount of platelets remaining in the perfusate decreased to less than 20% after 4 hours of perfusion due to platelet aggregation, while approximately 60% of the platelets remained in the APT-coated group even 24 hours after perfusion.

[0195] In addition, if Figure 4G As shown, the expression of platelet aggregation-related markers (CD63, PLSCR1, TBXAS1, and THBS1) was decreased in the APT coating group.

[0196] Based on the above results, it was demonstrated that aptamer coating enabled the formation of perfused blood vessels through more efficient vascular endothelialization and minimized thrombosis after blood perfusion.

[0197] Implementation 5: Preparation of vascularized artificial liver using aptamers and functional evaluation

[0198] To reconstruct vascularized bioengineered human liver (VBHL) using aptamers, hepatocytes (HepG2), mesenchymal cells (hepatic stellate cells, mesenchymal stem cells), and vascular endothelial cells (HUVECs) were cultured as shown in FIG5 .

[0199] Results, such as Figures 5B to 5D As shown, albumin immunostaining demonstrated that in the artificial liver tissue (APT-VBHL) coated with the aptamer and then endothelialized, reconstruction of the liver parenchyma and more than 90% endothelialization of the blood vessels were achieved.

[0200] In addition, if Figure 5E As shown in Figure 3, aSMA immunostaining demonstrated that mesenchymal stem cells engrafted well into the perivascular area during aptamer treatment, whereas no perivascular area was observed in the uncoated or antibody-treated groups.

[0201] In addition, if Figure 5F The results showed that when dextran flowed through the hepatic portal vein and intravascular dextran was quantified, dextran significantly increased in the APT-VBHL group, confirming that the vascular barrier function of liver tissue was maintained.

[0202] Therefore, not only vascular endothelialization but also the remodeling of the perivascular area constituting the vascular wall during aptamer treatment may lead to the acquisition of well-functioning blood vessels that well maintain their barrier function.

[0203] In addition, the extent of cell apoptosis in artificial liver tissues was quantified by TUNEL assay.

[0204] Results, such as Figure 5L As shown, the APT-VBHL group exhibited the least apoptosis, which was related to the function of the artificial liver.

[0205] Furthermore, to confirm the vascular-specific function, the NO production and VEGF secretion of the artificial liver were measured.

[0206] Results, such as Figure 5G and 5H As shown, the APT-VBHL group showed excellent function compared with the other groups.

[0207] In addition, ELISAs for quantifying albumin and urea components secreted from the artificial liver were performed to confirm liver-specific functions.

[0208] Results, such as Figure 5I and 5J As shown, the APT-VBHL group also showed the highest secretion of albumin and urea on culture day 21. In particular, the differences in liver function after revascularization indicate that high-function revascularization via aptamer coating plays a crucial role in maintaining the overall function of artificial liver tissue.

[0209] Example 6: Evaluation of thrombosis in vivo in vascularized artificial liver treated with aptamers

[0210] like Figure 6A As shown, the extent of thrombosis in a vascularized artificial liver was evaluated in vivo by connecting the portal vein and inferior vena cava of the artificial liver to the renal artery and renal vein of a rat, respectively, for in vivo blood perfusion. Specifically, the renal vein (yellow arrow) was connected to the inferior vena cava of the VBHL construct, and the renal artery (white arrow) was connected to the portal vein of the construct. After the vascular clamp (blue arrow) was removed, the VBHL construct was reperfused with the renal circulatory system in vivo.

[0211] Results, such as Figure 6B As shown, no thrombus formation was observed macroscopically in the aptamer-treated artificial liver.

[0212] In addition, if Figure 6C and 6D As shown, it was demonstrated that the expression of integrin αIIb, a thrombus marker, was also reduced.

[0213] In addition, if Figure 6E As shown, when the expression of coagulation-related mRNA markers (Cd63, Plscr1, and Thbs1) was detected, the expression of coagulation-related markers decreased the most in the APT-VBHL group.

[0214] Therefore, based on the above results, it was demonstrated that artificial liver culture capable of minimizing thrombosis in vivo was successfully achieved through aptamer treatment.

[0215] Therefore, it is expected that when the aptamer-coated agent of the present invention is used, the success rate of artificial liver transplantation can be improved by minimizing the most serious side effects that may occur after artificial liver transplantation.

[0216] Embodiment 7: In vivo liver function assessment of aptamer-treated vascularized artificial liver

[0217] like Figure 7AIn vivo liver-specific function was assessed by transplanting a vascularized artificial liver into a rat model of liver fibrosis induced by thioacetamide (TAA). Eight weeks after TAA induction, rats received xenografts of either acellular liver matrix (DLM transplant) or VBHL constructs (CTL-VBHL; CTL transplant, APT-VBHL; APT transplant, Ab-VBHL; Ab transplant).

[0218] The specific experimental groups are as follows:

[0219] 1) Sham

[0220] 2) DLM implantation; cell-free decellularized scaffold implantation alone

[0221] 3) CTL implantation; implantation of uncoated reconstructed artificial liver

[0222] 4) APT implantation: Implantation of artificial liver treated with anti-CD31 aptamer as coating agent

[0223] 5) Antibody implantation; Implantation of artificial liver treated with anti-CD31 antibody as coating agent

[0224] Four weeks later, the livers of the recipient rats were collected and subjected to H&E tissue staining and Sirius red tissue staining (red: collagen, green: cytoplasm).

[0225] Results, such as Figure 7B and 7C As shown, compared with the sham surgery group, the changes in eosinophils and the degree of fibrosis in the liver were reduced when the decellularized scaffolds and artificial livers were transplanted. Among them, the degree of liver fibrosis was reduced the most in the artificial liver treated with aptamers.

[0226] In addition, if Figure 7D As shown, when the mRNA expressed in the recipient liver was detected, it was demonstrated that the expression of fibrosis-related markers (α-smooth muscle actin (Sma), vimentin (Vimentin), Tgf-beta1 and Timp1) was significantly reduced in the APT implantation group.

[0227] In addition, if Figure 7E and 7F As shown, it was demonstrated that when ALT and AST levels, which are indicators of liver damage, were verified in rat serum levels, the serum ALT and AST levels of the APT implantation group were maintained at normal levels after surgery (between the red lines).

[0228] Therefore, the aptamer-treated artificial liver of the present invention was shown to maintain enhanced liver-specific functions in vivo, thereby helping the recipient's impaired liver function.

[0229] As described above, the present inventors demonstrated for the first time that aptamers can be applied to artificial organs.

[0230] Based on these results, aptamers used as coatings for decellularized scaffolds can more effectively reconstruct the vasculature than the anti-CD31 antibodies used in existing studies by enhancing vascular adhesion, activity, and angiogenic potential via the integrin-Akt signaling system. Therefore, the vascularized artificial liver produced using this invention not only reduces thrombosis in vivo but also enhances liver function. Furthermore, these results demonstrate the applicability of artificial livers as therapeutic agents for liver disease in tissue engineering.

[0231] The above description of the present invention is for illustrative purposes. It should be understood by those skilled in the art that the present invention can be easily modified into other specific forms without departing from the technical spirit or essential characteristics of the present invention. Therefore, the above exemplary embodiments should be interpreted as illustrative and not limiting in any respect.

[0232] Industrial Applicability

[0233] When used as a coating agent for decellularized scaffolds, the aptamers of the present invention can more effectively reconstruct the vasculature than existing antibodies by enhancing vascular adhesion, activity, and angiogenic potential. Consequently, vascularized artificial livers prepared using these aptamers have been shown to reduce thrombosis in vivo and enhance liver function. Therefore, they are expected to be applicable to methods for treating diseases using artificial organs prepared using these aptamers, thus possessing industrial applicability. <110> Kang Stem Cell Biotechnology Co., Ltd. <120> Method for preparing high-function artificial organs using aptamers <130> MPCT21-066 <150> KR 10-2020-0098314 <151> 2020-08-06 <150> KR 10-2020-0150913 <151> 2020-11-12 <160> 41 <170> KoPatentIn 3.0 <210> 1 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Anti-CD31 aptamer, where n is NapdU [5-(N-naphthylcarboxamide)-2'-deoxyuridine] <400> 1 gnagaggagg nacgnaangn cngggnanac cccganaann 40 <210> 2 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> People GAPDH_F <400> 2 tgatgacatc aagaaggtgg tg 22 <210> 3 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> People GAPDH_R <400> 3 accctgttgc tgtagccaaa t 21 <210> 4 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Human ITGB1_F <400> 4 cgtagcaaag gaacagcaga g 21 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Human ITGB1_R <400> 5 ggtagtagag gtcaatggga tagtc 25 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Human ITGB3_F <400> 6 cctcatcacc atccacgacc 20 <210> 7 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Human ITGB3_R <400> 7 gttgttggct gtgtcccatt t 21 <210> 8 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Human VE-cadherin_F <400> 8 cttcacccag accaagtaca ca 22 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Human VE-cadherin_R <400> 9 aatggtgaaa gcgtcctggt 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Human CLDN5_F <400> 10 aagtgtacga ctcggtgctg 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Human CLDN5_R <400> 11 aaacaggtag agcacgcctc 20 <210> 12 <211> twenty three <212> DNA <213> Artificial Sequence <220> <223> Person NOS3_F <400> 12 aaagacaagg cagcagtgga aat 23 <210> 13 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> NOS3_R <400> 13 tccacgatgg tgactttggc ta 22 <210> 14 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> human CD63_F <400> 14 cagctagaga gccccgga 18 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> human CD63_R <400> 15 ttcattcctc cttccaccgc 20 <210> 16 <211> twenty four <212> DNA <213> Artificial Sequence <220> <223> human PLSCR1_F <400> 16 ggcagccaga gaactgtttt aatc 24 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> human PLSCR1_R <400> 17 ggcaagtttg tttccgggtg 20 <210> 18 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Human TBXAS1_F <400> 18 tgcagagcac ggttccc 17 <210> 19 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Human TBXAS1_R <400> 19 gtggagtacc atttcaggag gg 22 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Human THBS1_F <400> 20 agtcgtctct gcaacaaccc 20 <210> twenty one <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> Human THBS1_R <400> twenty one acaggcatcc atcaattgga ca 22 <210> twenty two <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> PeopleALB_F <400> twenty two cgctattagt tcgttacacc a 21 <210> twenty three <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ALB_R <400> twenty three tttacaacat ttgctgccca 20 <210> twenty four <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Human CYP1A2_F <400> twenty four cggacagcac ttccctgaga 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Human CYP1A2_R <400> 25 aggcaggtag cgaaggatgg 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Rat Gapdh_F <400> 26 accacagtcc atgccatcac 20 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Rat Gapdh_R <400> 27 tccaccaccc tgttgctgta 20 <210> 28 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Rat Cd63_F <400> 28 catcaagaag cgtcgggga 19 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Rat Cd63_R <400> 29 acctgaactg ctacgccaat 20 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Rat Plscr1_F <400> 30 ctgcgaggct ttagggagag 20 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Rat Plscr1_R <400> 31 ctctgaggtc tgcaaggtgg 20 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Rat Thbs1_F <400> 32 tagctggaaa tgtggtgcgt 20 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Rat Thbs1_R <400> 33 agcaagcatc aggcacttct 20 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Rat alpha-Sma_F <400> 34 catcaccaac tgggacgaca 20 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Rat alpha-Sma_R <400> 35 tccgttagca aggtcggatg 20 <210> 36 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Rat Vimentin_F <400> 36 cactcacctg cgaagtggat 20 <210> 37 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Rat Vimentin_R <400> 37 tggtattcac gaaggtggcg 20 <210> 38 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Rat Tgf-beta1_F <400> 38 ctgctgaccc ccactgatac 20 <210> 39 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Rat Tgf-beta1_R <400> 39 agccctgtat tccgtctcct 20 <210> 40 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Rat Timp1_F <400> 40 tgctcaaagg attcgacgct 20 <210> 41 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> rat Timp1_R <400> 41 agcagggctc agattatgcc 20

Claims

1. A use of a composition comprising an anti-CD31 aptamer, vascular endothelial cells, and mesenchymal stromal cells (MSCs) for producing an artificial organ, wherein the anti-CD31 aptamer is coated with blood vessels of a decellularized liver comprising blood vessels and has the base sequence shown in SEQ ID NO:

1.

2. The use according to claim 1, wherein the composition further comprises one or more cells selected from hepatocytes and non-parenchymal cells.

3. The use according to claim 2, wherein the cells comprise differentiated cells derived from stem cells.

4. A composition for coating hepatic blood vessels of a decellularized liver comprising blood vessels, comprising an anti-CD31 aptamer, vascular endothelial cells, and mesenchymal stromal cells (MSCs), wherein the anti-CD31 aptamer has the base sequence shown in SEQ ID NO:

1.

5. The composition according to claim 4, wherein The aptamer increases the expression of one or more selected from the group consisting of integrin β3 and phosphorylated Akt. The composition of claim 4 , wherein the aptamer reduces the expression of cleaved caspase-3.

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