Recellularization of xenogeneic tissue

Abstract

The present invention relates to recellularization of xenogeneic tissue. Specifically, the present invention relates to recellularization of xenogeneic tissue, which enables transplantation of non-immunogenic biological tissue with improved durability and regenerative capacity by generating xenogeneic tissue having regenerative capacity through human cell recellularization after removal of galactose-alpha-1,3-galactose (α-Gal) xenoantigens and non-galactose (non-Gal) xenoantigens.

Description

Recellularization of xenogeneic tissue

[0001] This invention was carried out with funding from the Lee Kun-hee Pediatric Cancer and Rare Disease Overcoming Project, selected and supported by the Pediatric Cancer and Rare Disease Support Group of Seoul National University Hospital (Project No.: 25C-022-0100).

[0002] The present invention relates to the recellularization of heterologous tissues.

[0003] Specifically, the present invention relates to the recellularization of xenogeneic tissues, which enables the transplantation of non-immunogenic biological tissues with improved durability and regenerative capacity by utilizing human cell recellularization after removing galactose-alpha-1,3-galactose (α-Gal) xenoantigens and non-galactose xenoantigens.

[0004] Tissue engineering is an innovative technology that involves artificially creating tissues or organs to enable them to function when transplanted into the body. This technology aims for the ultimate regeneration of tissues or organs through the fusion of cells, biomaterials, and the biological environment, and holds significant potential for medical and clinical applications.

[0005] To develop ideal tissue-engineered heart valves, it is crucial to select appropriate tissues that serve as scaffolds for the optimal valve structure. Xenogeneic tissues have focused on appropriately removing cells capable of triggering an immune response while minimizing damage to the extracellular matrix (ECM). Decellularization protocols have been developed to treat the ECM into an immunologically resistant state while preserving its function. Generally, decellularization has been performed using chemical methods [alkali / acid, detergent, alcohol], physical methods [electroporation, pressurization, freeze / thaw], and biological methods (enzymes, etc.). Since nucleic acid residues attach to the ECM and attract circulating calcium salts, leading to calcification and subsequent tissue degeneration, the removal of nucleic acid residues is critical in all decellularization processes. Therefore, during the tissue decellularization period, proteolytic enzymes are used to break down proteins and effectively separate cellular components from connective tissue.

[0006] When heart valves made from xenogeneic tissue are transplanted into the human body, immune rejection reactions caused by galactose-alpha-1,3-galactose (α-Gal) and N-glycolylneuramic acid (Neu5Gc) lead to calcification and dysfunction of the valve. Although α-Gal can be removed by treatment with alpha-galactosidase, non-galactose xenoantigens, including Neu5Gc, are not removed, so there is a problem of reduced durability due to immune rejection.

[0007] In particular, when using porcine or bovine pericardial tissue, it is necessary to effectively remove α-Gal epitopes (alpha-gal epitopes; xenoepitopes) that induce xenogeneic immune rejection. The inventors have developed a decellularization protocol using α-galactosidase, which is used to manufacture artificial pericardial pulmonary artery valves. These valves have been successfully applied in clinical practice, and the results to date have been satisfactory. However, it has been found that the presence of non-Gal antigens (Neu5Gc) within the GalT-KO region does not induce hyperacute xenogeneic rejection, but can mediate acute graft damage to vascularized organs as well as cell transplants in xenografts. Furthermore, anti-non-Gal antibodies can be induced during xenografts and are expected to affect xenograft or tissue survival and durability. In xenografts, lectins are carbohydrate-binding proteins closely associated with immune rejection, and increased lectin levels are closely linked to immune rejection. By analyzing lectin expression, the effect of enzymatic treatment with PNGase-F on carbohydrate removal was investigated. In previous studies, the inventors have established that a combination of decellularization and PNGase-F treatment effectively removes non-Gal antigens without affecting the mechanical stability of the tissue. The removal of non-Gal antigens such as Neu5Gc requires deglycosylation treatment to effectively remove carbohydrates from N- and O-glycans.

[0008] As a result of the inventors' diligent research, all lectin binding signals decreased as the concentration of peptide-N-glycosidase F (PNGE-F) increased, and a synergistic effect of reduced lectin binding was observed when combined with low concentrations of α-galactosidase without changes in mechanical properties. The combination of PNGE-F and α-galactosidase effectively removes xenogenic carbohydrates from porcine pericardium, demonstrating its potential to reduce immunogenicity. The removal of lectin-bound xenogenic carbohydrates is more effective when decellularization is performed. The absence of cells within the decellularized xenograft tissue was confirmed using H&E staining and DAPI staining. Since it is essential to completely remove antigens that can induce a graft-versus-host reaction, the inventors propose a novel chemistry-based decellularization method that effectively removes residual cells while preserving the structural integrity of the tissue.

[0009] The present invention aims to achieve the realization of an immunogenic biological tissue transplant with improved durability and regenerative capacity by providing a method for the recellularization of xenogeneic tissue.

[0010] One embodiment of the present invention aims to provide a method for manufacturing an immunogenic xenograft tissue with improved durability and regenerative capacity.

[0011] In addition, one embodiment of the present invention aims to provide an immunogenic xenograft tissue with improved durability and regenerative ability manufactured according to the above manufacturing method.

[0012] The present invention comprises the steps of: (a) decellularizing porcine pericardia tissue; (b) treating the decellularized porcine pericardia tissue with α-galactosidase to remove galactose-alpha-1,3-galactose (α-Gal) xenoantigen; (c) a step of treating porcine pericardial tissue from which galactose-alpha-1,3-galactose (α-Gal) xenoantigens have been removed with peptide-N-glycosidase F to remove non-Gal xenoantigens, including N-glycolylneuramic acid (Neu5Gc); (d) a step of modifying decellularized porcine pericardial tissue treated with α-galactosidase and peptide-N-glycosidase F by coating the decellularized porcine pericardial tissue treated with α-galactosidase and peptide-N-glycosidase F with fibrin, heparin and vascular endothelial growth factor (VEGF); and (e) a recellularization step of co-culturing human adipose-derived stem cells (ADSC) and human umbilical vein endothelial cells (HUVEC) in the decellularized porcine pericardial tissue modified in step (d), thereby providing a method for producing an immunogenic xenograft tissue with improved durability and regenerative capacity.

[0013] In the method for preparing an immunogenic xenograft tissue with improved durability and regenerative capacity according to the present invention, the decellularization of step (a) is performed using sodium dodecyl sulfate (SDS) and Triton X-100. The decellularization of step (a) may be performed using one or more methods selected from the group consisting of a method of decellularizing by repeatedly freezing and thawing, a method of removing cells from the surface of the extracellular matrix (ECM) by stirring, a method of inducing cell lysis by creating micropores in the cell membrane through electroporation, a method of lysing cells by pressurization, and a method using a supercritical fluid. Additionally, the decellularization of step (a) is performed by treatment with one or more enzymes selected from the group consisting of nucleases, dispases, and phospholipases.

[0014] In the method for manufacturing an immunogenic xenograft tissue with improved durability and regenerative capacity according to the present invention, it is preferable that step (b) and step (c) be performed simultaneously.

[0015] In the method for producing an immunogenic xenograft tissue with improved durability and regenerative ability according to the present invention, step (e) may involve culturing human adipose-derived stem cells (ADSC) and subsequently culturing human umbilical vein endothelial cells (HUVEC); or culturing human adipose-derived stem cells (ADSC) and human umbilical vein endothelial cells (HUVEC) simultaneously.

[0016] In the method for producing a non-immunogenic xenograft tissue with improved durability and regenerative ability according to the present invention, prior to step (d), the method may further include the step of fixing porcine pericardial tissue from which galactose-alpha-1,3-galactose (α-Gal) xenoantigen and non-Gal xenoantigen have been removed by treating it with glutaraldehyde; and the step of detoxifying the fixed porcine pericardial tissue by treating it with glycine.

[0017] In addition, the present invention provides an immunogenic xenograft tissue with improved durability and regenerative ability, manufactured according to the above manufacturing method, wherein non-Gal xenoantigens, including galactose-alpha-1,3-galactose (α-Gal) xenoantigen and N-glycolylneuramic acid (Neu5Gc), are removed and recellularized.

[0018] The immunogenic xenograft tissue of the present invention, with improved durability and regenerative capacity, has increased expression of vimentin, a marker of mesenchymal cells.

[0019] The non-immunogenic xenograft tissue of the present invention, with improved durability and regenerative capacity, exhibits increased expression of calponin, a marker for differentiation into vascular smooth muscle cells.

[0020] The immunogenic xenograft tissue with improved durability and regenerative capacity according to the present invention has increased expression of fibronectin, which promotes cell adhesion and regeneration as a major component of the extracellular matrix (ECM).

[0021] The non-immunogenic xenograft tissue of the present invention, with improved durability and regenerative capacity, has increased expression of CD31, which plays an important role in angiogenesis, intercellular adhesion, and inflammatory response as a major component of endothelial cells.

[0022] According to the present invention, after removing α-Gal and non-Gal xenoantigens, human cell recellularization is utilized to generate a xenogeneic tissue with regenerative capacity, thereby enabling the transplantation of an immunogenic biological tissue with improved durability and regenerative capacity.

[0023] Furthermore, non-immunogenic xenograft tissues with improved durability and regenerative capacity can be utilized, for example, as artificial heart valves.

[0024] Figure 1 shows the histological characteristics and DNA content of native and decellularized porcine pericardiums. (A) shows native and decellularized porcine pericardiums stained with hematoxylin-eosine (H&E) and imaged under a microscope. (B) shows native and decellularized porcine pericardium tissues stained with 4',6-diamidino-2-phenylindole (DAPI) and fluorescence signals detected using a fluorescence microscope. (a) and (b) are images of native porcine pericardiums (images magnified 200 times), and (c) and (d) are images of decellularized porcine pericardiums (images magnified 400 times). (C) shows the difference in DNA content between native and decellularized porcine pericardium tissues. DNA content for the two groups was measured using a spectrophotometer. Decellularized porcine pericardial tissue showed a significantly reduced DNA content compared to natural porcine pericardial tissue. The graphs represent the quantification results. Data are presented as the mean ± standard deviation of three independently conducted experiments. Statistical significance was calculated using one-way ANOVA based on an unpaired t-test (* p < 0.05, ** p < 0.01, *** p , 0.001).

[0025] Figure 2 shows the percentage area of ​​collagen deposition in porcine pericardial tissue stained with Masson's Trichrome. (A) shows the Masson's Trichrome staining results of natural porcine pericardium and decellularized porcine pericardium. (a) and (b) are images of natural porcine pericardium (magnified 200 times), and (c) and (d) are images of decellularized porcine pericardium (magnified 400 times). (B) shows the difference in the percentage area of ​​collagen deposition between the two groups. Quantification results showed no significant difference in the percentage area of ​​collagen deposition between the natural porcine pericardium group and the decellularized porcine pericardium group.

[0026] Figure 3 shows the histological observation results of the untreated group, α-galactosidase treated group, and PNGase treated group of porcine pericardium. The porcine pericardium samples were H&E stained and then imaged using a microscope (200x magnification). (A) is the untreated group in its natural state, (B) is the group treated with PNGase 500 unit / mL, (C) is the group treated with PNGase 1000 unit / mL, (D) is the group treated with PNGase 1500 unit / mL, (E) is the group treated with PNGase 2000 unit / mL, (F) is the group treated with α-galactosidase 0.1 unit / mL, (G) is the group treated with α-galactosidase 0.2 unit / mL, and (H) is the group treated with α-galactosidase 0.1 unit / mL + PNGase 1000 unit / mL.

[0027] Figure 4 shows the results of toluidine blue staining and immunohistochemical staining of porcine pericardial tissue. (A) shows the toluidine blue staining results of natural porcine pericardium and decellularized porcine pericardium. (B) shows the difference in the percentage area of ​​glycosaminoglycans (GAGs) between each group. The graph indicates that decellularized porcine pericardial tissues treated with or without α-galactosidase have lower glycosaminoglycan (GAF) content compared to natural porcine pericardial tissue. (C) shows the results of α-Gal epitope immunohistochemical (IHC) staining of natural porcine pericardium and decellularized porcine pericardium after α-galactosidase treatment. (D) shows the difference in the relative change of α-Gal epitope expression between each group. The graph shows that α-galactosidase-treated natural porcine pericardium and α-galactosidase-treated or untreated decellularized porcine pericardium have lower α-Gal epitope expression compared to natural porcine pericardium. (a) and (b) are 200x magnified images of a porcine pericardium not treated with α-galactosidase, and (b) and (d) are 200x magnified images of a porcine pericardium treated with α-galactosidase.

[0028] Figure 5 shows the biomechanical properties of a group of porcine pericardial tissues in their natural state and a group of porcine pericardial tissues treated with α-galactosidase and / or PNGase-F. The tissue samples include tissue samples in their natural state and enzyme-treated tissue samples, i.e., samples treated with PNGase-F (greater than 0 unit / mL and less than or equal to 1000 unit / mL) for 24 hours in the absence or presence of α-galactosidase (0.1-0.2 unit / mL) or untreated samples. (A) to (C) show the uniaxial tensile properties of the porcine pericardium analyzed using a tensile testing machine [(A): tensile stress at break, (B): tensile stress at maximum force, (C): tensile displacement at break]. (D) shows the permeability of the porcine pericardium analyzed using a permeability testing machine. Data were expressed as mean ± standard deviation. Statistical significance was calculated using one-way ANOVA based on the Dunnet multiple contrast test (* p < 0.05, ** p < 0.01, *** p , 0.001).

[0029] Figure 6 shows the lectin histochemistry results of groups of porcine pericardial tissue in their natural state and groups of porcine pericardial tissue treated with α-galactosidase and / or PNGase-F. The porcine pericardial tissue samples of (a) to (e) include tissue samples in their natural state and enzyme-treated tissue samples, i.e., samples treated with PNGase-F (greater than 0 unit / mL and less than or equal to 1000 unit / mL) for 24 hours in the absence or presence of α-galactosidase (0.1-0.2 unit / mL) or samples that were not treated. Negative control sample (a) derived from natural porcine pericardial tissue, natural porcine pericardial tissue sample (b), porcine pericardial tissue sample treated with α-galactosidase 0.1 unit / mL (d), and porcine pericardial tissue sample treated with α-galactosidase 0.1 unit / mL + PNGase-F 1000 unit / mL (e) were longitudinally sliced ​​and stained with Jacalin (A), MAL-I (B), WGA (C), RCA-I (D), GSL (E), ECA (F), PNA (G), SBA (H), WFA (I), and DSL (J). Each sample of carbohydrate-binding lectin was stained red and imaged at 200x magnification.

[0030] Figure 7 shows the quantitative evaluation of lectin binding to soluble proteins in porcine pericardium. Porcine pericardium tissue samples included natural tissue samples and enzyme-treated tissue samples, specifically samples treated with PNGase-F (greater than 0 unit / mL and less than 1000 unit / mL) for 24 hours in the absence or presence of α-galactosidase (0.1–0.2 unit / mL) or untreated samples. Each tissue sample was homogenized with lysis buffer, and detection of lectin binding to soluble extracellular matrix (ECM) proteins on the tissue surface was performed to measure the expression of WGA (A), MAL-I (B), ECA (C), RCA-I (D), GSL (E), WFA (F), and DSL (G). The graph represents the quantification graph. Data are presented as the mean ± standard deviation of five independently performed experiments. Statistical significance was calculated using one-way ANOVA based on the Dunnett multiple contrast test (* p < 0.05, ** p < 0.01, *** p , 0.001.).

[0031] Figure 8 shows the expression of lectins on enzymatically treated or non-enzymatically treated porcine pericardium. (a) to (b) show porcine pericardium tissue samples in their natural state, and (c) to (f) show porcine pericardium samples decellularized with Triton X-100 + sodium dodecyl sulfate (SDS). The porcine pericardium tissue samples include tissue samples in their natural state and enzymatically treated tissue samples, i.e., samples treated or untreated with PNGase-F (greater than 0 unit / mL and less than or equal to 1000 unit / mL) for 24 hours in the absence or presence of α-galactosidase (0.1-0.2 unit / mL). Negative control sample derived from natural porcine pericardial tissue (a), natural porcine pericardial tissue sample (b), decellularized porcine pericardial tissue sample (c), decellularized porcine pericardial tissue sample treated with α-galactosidase 0.1 unit / mL (d), decellularized porcine pericardial tissue sample treated with α-galactosidase 0.2 unit / mL (e), and porcine pericardial tissue sample treated with α-galactosidase 0.1 unit / mL + PNGase-F 2000 unit / mL (f) were longitudinally sectioned and stained with Jacalin (A), MAL-I (B), WGA (C), RCA-I (D), GSL (E), ECA (F), PNA (G), SBA (H), WFA (I), and DSL (J). Each sample of carbohydrate-binding lectin was stained red and imaged at 200x magnification.

[0032] Figure 9 shows the histological observation results of ADSCs and HUVECs inoculated onto modified porcine pericardial tissue. Decellularized porcine pericardial tissue samples included samples treated with PNGase-F (greater than 0 unit / mL and less than or equal to 1000 unit / mL) or untreated for 24 hours in the absence or presence of α-galactosidase (0.1 unit / mL). Natural porcine pericardial tissue samples (a), decellularized porcine pericardial tissue samples (b), and decellularized porcine pericardial tissue samples treated with α-galactosidase / PNGase-F were coated with fibrin mesh + heparin (Fb + H), and the coated porcine pericardial tissue samples were incubated with vascular endothelial growth factor (VEGF) (Fb + H + VEGF). Subsequently, human adipose tissue-derived stem cells (ADSCs) were inoculated onto all modified porcine pericardial tissue samples, followed by human umbilical vein endothelial cells (HUVECs), and these samples were stained with H&E. H&E staining was imaged using a microscope (magnified 200 times).

[0033] Figure 10 shows the results of immunofluorescence staining of vimentin in the co-culture medium of ADSCs and HUVECs on modified porcine pericardial tissue. Decellularized porcine pericardial tissue samples included samples treated with PNGase-F 2000 unit / mL or untreated for 24 hours in the absence or presence of α-galactosidase (0.1 unit / mL). All porcine pericardial tissue samples were coated with fibrin, heparin, and VEGF (Fb + H + VEGF). Subsequently, ADSCs and HUVECs were inoculated into all modified porcine pericardial tissue samples. Natural porcine pericardium samples (a, a'), decellularized porcine pericardium samples (b, b'), and decellularized porcine pericardium samples treated with α-galactosidase / PNGase-F (c, c') were incubated on day 7 (A) and day 28 (B), and these samples were stained with vimentin. The fluorescence signal of vimentin was measured using a fluorescence microscope. Cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) and imaged at 200x magnification.

[0034] Figures 11 and 10 show the results of immunohistochemical staining of calponin in the co-culture medium of ADSCs and HUVECs on modified porcine pericardial tissue. Decellularized porcine pericardial tissue samples included samples treated with PNGase-F 2000 unit / mL for 24 hours in the absence or presence of α-galactosidase (0.1 unit / mL) or untreated samples. All porcine pericardial tissue samples were coated with fibrin, heparin, and VEGF (Fb + H + VEGF). Subsequently, ADSCs and HUVECs were inoculated into all modified porcine pericardial tissue samples. Natural porcine pericardium samples (a, a'), decellularized porcine pericardium samples (b, b'), and decellularized porcine pericardium samples treated with α-galactosidase / PNGase-F (c, c') were incubated on day 7 (A) and day 28 (B), and these samples were stained with calponin. The fluorescence signal of calponin was measured using a fluorescence microscope. Cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) and imaged at 200x magnification.

[0035] Figure 12 shows the results of immunocytochemical staining of fibronectin in ADSC and HUVEC co-culture medium on modified porcine pericardial tissue. Decellularized porcine pericardial tissue samples included samples treated with PNGase-F 2000 unit / mL or untreated for 24 hours in the absence or presence of α-galactosidase (0.1 unit / mL). All porcine pericardial tissue samples were coated with fibrin, heparin, and VEGF (Fb + H + VEGF). Subsequently, ADSC and HUVEC were inoculated into all modified porcine pericardial tissue samples. Natural porcine pericardium samples (a, a'), decellularized porcine pericardium samples (b, b'), and decellularized porcine pericardium samples treated with α-galactosidase / PNGase-F (c, c') were incubated on day 7 (A) and day 28 (B), and these samples were stained with fibronectin. The fluorescence signal of fibronectin was measured using a fluorescence microscope. Cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) and imaged at 200x magnification.

[0036] Figure 13 shows the results of immunocytochemical staining of CD31 in co-culture medium of ADSC and HUVEC on modified porcine pericardial tissue. Decellularized porcine pericardial tissue samples included samples treated with PNGase-F 2000 unit / mL for 24 hours in the absence or presence of α-galactosidase (0.1 unit / mL) or untreated samples. All porcine pericardial tissue samples were coated with fibrin, heparin, and VEGF (Fb + H + VEGF). Subsequently, ADSC and HUVEC were inoculated into all modified porcine pericardial tissue samples. Natural porcine pericardial samples (a, a'), decellularized porcine pericardial samples (b, b'), and decellularized porcine pericardial samples treated with α-galactosidase / PNGase-F (c, c') were incubated at a temperature on day 7 (A) and day 28 (B), and these samples were stained with CD31. The fluorescence signal of CD31 was measured using a fluorescence microscope. Cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) and imaged at 200x magnification.

[0037] Figure 14 shows the results of immunohistochemical staining of von Willebrand factor (vWF) in co-culture medium of human adipose tissue-derived stem cells (ADSCs) and human umbilical vein endothelial cells (HUVECs) on modified porcine pericardium. Decellularized porcine pericardium samples treated with or untreated with 2000 unit / mL of PNGase-F for 24 hours in the absence or presence of α-galactosidase (0.1 unit / mL) were modified with fibrin, heparin, and VEGF (Fb+H+VEGF). Subsequently, ADSCs and HEVECs were inoculated into all modified porcine pericardium samples. Natural porcine pericardium samples (a, a'), decellularized porcine pericardium samples (b, b'), and decellularized porcine pericardium samples treated with α-galactosidase / PNGase-F (c, c') were incubated on day 7 (A) and day 28 (B), and these samples were stained with von Willebrand factor (vWF). The fluorescence signal of vWF was measured using a fluorescence microscope. Cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) and imaged at 200x magnification.

[0038] Embodiments of the present invention are described in detail by way of example below. However, the scope of the present invention should not be interpreted as being limited by the following examples.

[0039] Examples

[0040] Tissue preparation and decellularization

[0041] Porcine pericardia was obtained from Taewoong Medical Inc. of South Korea. After removing fat and connective tissue from the surface of the porcine pericardia, the porcine pericardia tissue was trimmed. The tissue was sterilized with 0.1% peracetic acid (PAA) for 4 hours at room temperature, followed by washing with phosphate buffered saline solution (PBS) under agitation at room temperature. The porcine pericardia was decellularized using various solutions. Each decellularization process was performed on an orbital shaker (SHO-2D from Daehan Science) under an agitator (180 rpm).

[0042] First, porcine pericardial tissue was decellularized in a hypotonic solution at 4°C for 14 hours, decellularized in a hypotonic solution containing 0.25% sodium dodecyl sulfate (SDS) in 0.5% (v / v) Triton X-100 deionized water (diH2O) at 4°C for 24 hours while stirring, and then washed with distilled water at 4°C for 12 hours. After washing, porcine pericardial tissue was decellularized in an isotonic solution at 4°C for 48 hours. Porcine pericardial tissue was treated with phosphate-buffered saline (PBS) containing 30% polyethylene glycol 100 (PEG-100) and 1% antibiotic / antimycotic for 48 hours at 4°C using a stirrer. Finally, porcine pericardial tissue was treated with a hypertonic solution at 4°C for 3 hours. The porcine pericardial tissue was washed with phosphate-buffered saline (PBS) for 24 hours.

[0043] enzymatic digestion

[0044] Native porcine pericardia and decellularized porcine pericardia were treated with PNGase-F (#P0704L, 1000U, NEB) at concentrations of 500 unit / mL, 1000 unit / mL, 1500 unit / mL, and 2000 unit / mL. Additionally, native porcine pericardia and decellularized porcine pericardia were treated with recombinant α-galactosidase (G7163, Sigma) at concentrations of 0.1 unit / mL and 0.2 unit / mL. Native porcine pericardia was used as an untreated control or negative control. Natural porcine pericardium and porcine pericardium decellularized with SDS + Triton X-100(Tx) were treated with different enzymes (α-galactosidase, PNGase-F, α-galactosidase and PNGase). Thinly sliced ​​porcine pericardial tissues measuring 5×5 mm were placed in tubes containing isotonic solution and stirred at 4°C for 24 hours. After further enzymatic digestion of the thinly sliced ​​tissues at 37°C for 24 hours under stirring, the tissues were washed with phosphate-buffered saline (PBS) at room temperature under stirring (120 rpm). Finally, tissue samples were stored in phosphate-buffered saline (PBS) supplemented with 1% (v / v) penicillin / streptomycin and 400 µl / L amphotericin B for 24 hours at 4°C.

[0045] biomechanical analysis

[0046] After enzymatic digestion, all samples were stored at 4°C for 24 hours and then washed with PBS. Biomechanical analysis was performed on natural porcine pericardia with and without enzymatic treatment. According to the manufacturer's instructions, thinly sliced ​​samples were cut into approximately 5.0 × 20 mm (length × width, n=30) pieces, and tensile strength was measured based on a 5 mm width. The thickness of the porcine pericardial tissue was measured at three points on the sample pieces using a thickness gauge (Quick-Mini 700-117 from Mitutoyo, Kawasaki, Japan). Tensile strength was measured using a tensile strength measuring machine (68SC-5 from Instron, USA) [100N load cell] to evaluate whether there was a difference in maximum tensile strength. The final strength and deformation behavior of each sample were analyzed using three parameters: tensile strain, maximum force, and tensile displacement. Permeability characteristics were measured by applying saline solution to the pericardial tissue at a constant pressure of 100 mmHg for one hour and measuring the amount of infiltrated saline solution. For this test, the tissue was cut into rectangular pieces approximately 1 cm in size.

[0047] Histological staining and lectin histochemical staining

[0048] For histological staining, natural porcine pericardium and porcine pericardium decellularized with SDS + Triton X-100(Tx) were treated with different enzymes (α-galactosidase, PNGase-F, α-galactosidase and PNGase), and thinly sliced ​​samples were fixed in para-formaldehyde at 4°C for 24 hours. Hematoxylin and eosin (H&E) and lectin staining were performed on histological sections (5 µm) of each sample. Histological sections were incubated overnight at 4°C using lectin (1:250).Isolectin B4 specific to α-galactosidase (IL-B4 / GSL-IB4) (Vector Laboratories #RL-1205), Wheat Germ Agglutinin (WGA) (Vector Laboratories #RL-1022), Datura Stramonium Lectin (DSL) (Vector Laboratories #B-1185), Castor Agglutinin-I (Ricinus Communis Agglutinin-I, RCA-I) (Vector Laboratories #RL-1082), Soybean Agglutinin (SBA) (Vector Laboratories #B-1015), Wisteria Floribunda Agglutinin (WFA) (Vector Laboratories #B-1355), Peanut Agglutinin (PNA) (Vector Laboratories #B-1075), The effects of enzymatic treatment on carbohydrate-binding lectins such as Maackia Amurensis Lectin-I (MAL-1) (Vector Laboratories #B-1315), Jacalin (Vector Laboratories #B-1155), and Erythrina Cristagalli Lectin (ECA) (Vector Laboratories #B-1145) were tested. The stained sections were additionally stained with avidin-linked Texas Red (Vector Laboratories #A-2006). The stained sections were washed three times with phosphate-buffered saline (PBS), and the stained sections were imaged using an inverted fluorescence microscope (Leica DMI4000B).A stained image was obtained using the Application Suite X Image Viewer from Leica, Germany.

[0049] lectin binding assay

[0050] Lectin binding assays were performed on porcine pericardial tissues with and without enzyme treatment (α-galactosidase, PNGase-F, α-galactosidase and PNGase), thinly sliced ​​samples were homogenized in RIPA lysis buffer (ATTO) and stored under frozen conditions. The tissue homogenates were coated twice onto 96-well plates (Corning) using coating buffer [Bio-Sesang Co., Ltd., Korea] and incubated overnight at 4°C. Afterward, each well was blocked for 1 hour at room temperature using a carbo-free blocking solution (Vector Laboratories #SP-5040-125). The wells were stirred and incubated for 3 hours at room temperature using primary lectin (1:500) diluted to 100 µl per well in the carbo-free blocking solution. After the first lectin incubation, the plates were washed three times with phosphate buffered saline containing 0.05% Tween 20 [phosphate buffered saline with Tween 20 (PBST), Biosesang Co., Ltd.]. Subsequently, the wells were incubated at room temperature for 30 minutes using streptavidin-conjugated peroxidase (1:100, Vector Laboratories #SA-S0004) in a blocking solution. After washing several times with phosphate buffered saline containing Tween 20 (PBST), the reaction was carried out using 3,3'-tetramethylbenzidine (TMB) solution (Thermo Scientifics #34028). The absorbance of lectin was measured at 450 nm using an ELISA reader (Spark's TECAN).

[0051] In vitro culture and expansion of human umbilical vein endothelial cells (HUVECs) and human adipose tissue-derived stem cells (ADSCs)

[0052] Human umbilical vein endothelial cells (HUVEC) (Lonza #2517A) were purchased from ATCC (PCS-100-010, USA). Human umbilical vein endothelial cells (HUVEC) were used as pooled primary cells that were frozen after the first subculture. Human umbilical vein endothelial cells (HUVEC) were cultured using Endothelial Growth Medium-2 BulletKit™ (EGM, Lonza CC-3162) in a humid environment of 37°C and 5% CO2. Human adipose tissue-derived stem cells (ADSC) (#7788115) were purchased from Thermo Scientifics, USA. Human adipose tissue-derived stem cells (ADSCs) were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS), transforming growth factor-beta 1 (TGF-β1, 2.5 ng / ml, Abcam's ab50036), and bone morphogenetic protein-4 (BMP-4, 2.5 ng / ml, Sigma's SRP6156) in a humid environment of 37°C and 5% CO2. The culture media for both cells were replaced every 3 days.

[0053] Modification of porcine pericardial tissue

[0054] For recellularization, modifications were performed on natural porcine pericardium, decellularized porcine pericardium, and decellularized porcine pericardium treated with α-galactosidase and PNGase. Porcine pericardium samples were cut into approximately 1×1 cm (length×width, n=3) pieces, and the thinly sliced ​​porcine pericardium samples were coated with a fibrin mesh (P- or DP-Fb) on a 12-well plate. The samples were washed with 0.05 M Tris-HCl buffer (TB), modified by heparin attachment overnight at 4°C, then the fibrin mesh + heparin (P- or DP-Fb + H) samples were washed with phosphate-buffered saline (PBS), incubated with vascular endothelial growth factor (VEGF) (VEGF 165 from GenScript, USA, 100 ng / mL in PBS) at room temperature for 2 hours, and rinsed with phosphate-buffered saline (PBS).

[0055] Human umbilical vein endothelial cells (HUVEC) and human adipose tissue-derived stem cells (ADSC) inoculated onto modified porcine pericardium

[0056] For the endothelialization of porcine pericardia, all porcine pericardial samples were inoculated bilaterally with ADSCs and HUVECs. Approximately 1 × 10⁶ per well in a 12-well plate. 5 ADSCs (3rd passage) were inoculated onto the top of all modified porcine pericardial samples (P- or DP-Fb + H) (Day 0). The inoculated samples were inverted, and ADSCs were inoculated onto the bottom of the samples in DMEM medium supplemented with 2% FBS, TGF-β1, and BMP-4 (Day 2). After inoculating ADSCs on both sides, approximately 2 × 10⁶ cells were inoculated per well on a 12-well plate. 5 After inoculating the upper portion of porcine pericardial samples with HUVEC cells (6th generation) on day 16, approximately 2 × 10⁶ cells were inoculated per well on a 12-well plate. 5HUVEC (6th generation) cells were inoculated into the lower part of porcine pericardial samples (day 18). Porcine pericardial samples were incubated in EGM-2 medium supplemented with TGF-β1 (2.5 ng / ml) and BMP-4 (2.5 ng / ml), and these porcine pericardial samples were cultured until day 28.

[0057] Immunofluorescence staining of recellularized porcine pericardium

[0058] Natural porcine pericardium, decellularized porcine pericardium, and decellularized porcine pericardium treated with α-galactosidase and PNGase were inoculated with ADSCs and HUVECs, fixed in a 4% para-formaldehyde solution, and sectioned into 5 µm pieces on a microtome. The sections were paraffin-embedded and treated for hematoxylin and eosin (H&E) staining and immunofluorescence (IF) staining. Immunofluorescence (IF) staining was performed using the embedded sections, followed by deparaffinization. Tissue samples were incubated at 95°C for 10 minutes using antigen retrieval buffer (10 mM citrate buffer, pH 6.0). Subsequently, the tissue sections were blocked using 5% blocking serum. Tissue sections were incubated overnight at 4°C using primary antibodies against vimentin (Abcam’s ab16700, dilution ratio 1:200), calponin (Abcam’s ab227661, dilution ratio 1:100), fibronectin (Abcam’s F0916, dilution ratio 1:100), and CD31 (Abcam’s ab182981, dilution ratio 1:2000), and washed three times with PBST. Subsequently, tissue samples were incubated for 2 hours using an anti-rabbit secondary antibody conjugated with Alexa Fluor 488 (Invitron A11008, dilution ratio 1:500) and an anti-mouse secondary antibody conjugated with Alexa Fluor 488 (Invitron AB150113, dilution ratio 1:500), and rinsed 3 times with PBST. Nuclei were counterstained for 1 minute using 4',6-diamidino-2-phenylindole (DAPI) (Invitron, dilution ratio 1:1000).Sample sections were washed three times with PBST, and the stained sample sections were imaged using an inverted fluorescence microscope (Leica DMI4000B, Germany). Stained images were obtained using the Application Suite X Image Viewer from Leica, Germany.

[0059] Histological and biomechanical characteristics of enzyme-treated decellularized porcine pericardium

[0060] Porcine pericardial tissue was treated by a multi-step method using hypotonic, isotonic, and hypertonic solutions according to a decellularization protocol using 0.25% SDS + Triton X-100 (see Figs. 1 and 2). Individual samples were treated with or not treated with PNGase (greater than 0 unit / mL and less than 2000 unit / mL) in the absence or presence of α-galactosidase (0.1–0.2 unit / mL) for 24 hours. Fibrous structures, such as collagen fiber patterns and structural loosening, and the presence of cells were investigated using H&E staining; in all cases, normal collagen structures similar to those of natural porcine pericardial tissue were observed regardless of enzyme concentration (see Figs. 3 and 4).

[0061] Biomechanical properties were measured using uniaxial tensile and permeability tests. As a result of the tensile stress and permeability tests, there were no significant differences between natural porcine pericardial tissue, porcine pericardial tissue treated with different concentrations (0-1000 unit / mL) of PNGase-F, porcine pericardial tissue treated with different concentrations (0.1-0.2 unit / mL) of α-galactosidase, and porcine pericardial tissue treated with both PNGase-F and α-galactosidase (see Fig. 5). This suggests that the effect of PNGase-F / α-galactosidase treatment concentrations and combinations does not affect the biomechanical properties of porcine pericardium.

[0062] Lectin Histochemistry and Lectin Binding Analysis of Enzyme-Treated Decellularized Porcine Pericardium

[0063] Carbohydrate components such as lectins play an important role in xenotransplantation, and elevated lectin levels are closely associated with immune rejection. Through lectin expression, we investigated the effects of enzymatic treatment with PNGase-F and α-galactosidase on carbohydrate removal in natural porcine pericardium. Natural porcine pericardial tissue and porcine pericardial tissue treated with various concentrations of PNGase-F, α-galactosidase, or PNGase-F and α-galactosidase were stained with Jacalin, MAL-I, WGA, RCA-I, GSL, ECA, PNA, SBA, WFA, and DSL. Each enzymatic treatment showed a decrease in lectin expression with increasing enzyme concentration, which implies effective removal of carbohydrates in lectin histochemistry. In addition, when α-galactosidase and PNGase-F were treated together, a more significant decrease in lentin expression was observed, which means that both α-Gal epitopes and non-α-Gal epitopes were removed more effectively (see Fig. 6).

[0064] To quantitatively measure the expression of MAL-I, WGA, RCA-I, GSL, ECA, PNA, SBA, WFA, and DSL, lectin binding assays were performed on natural porcine pericardial tissue, porcine pericardial tissue treated with PNGase-F (0-1000 unit / mL), porcine pericardial tissue treated with α-galactosidase (0.1-0.2 unit / mL), and porcine pericardial tissue treated with α-galactosidase (0.1 unit / mL) + PNGase-F (1000 unit / mL). Treatment with PNGase-F significantly inhibited lectin binding levels in a concentration-dependent manner. When α-galactosidase was simultaneously treated at the same concentration as PNGase-F, a synergistic effect of reducing lectin binding was observed compared to treatment with PNGase-F alone (see Figure 7).

[0065] After applying an additional decellularization protocol, immunohistochemical staining revealed that the group treated with α-galactosidase 0.1 unit / mL / PNGase-F 1000 unit / mL exhibited the most effective removal of lectin binding levels (see Fig. 8).

[0066] In vitro evaluation of recellularized porcine pericardium via co-culture of human adipose tissue-derived stem cells (ADSCs) and human umbilical vein endothelial cells (HUVECs)

[0067] Histological observations of ADSCs and HUVECs inoculated into modified porcine pericardial tissue

[0068] First, decellularized porcine pericardia were treated with or not treated with 2000 unit / mL PNGase-F for 24 hours in the absence or presence of α-galactosidase (0.1 unit / mL). Natural porcine pericardia samples, decellularized porcine pericardia samples, and α-galactosidase / PNGase-F treated decellularized porcine pericardia samples were coated with fibrin mesh + heparin (Fb + H) and incubated with VEGF (Fb + H + VEGF). Subsequently, human ADSCs were inoculated, followed by HUVECs. The modified porcine pericardia samples were incubated on days 7 and 28 and stained with H&E. Tissue structure was preserved in all natural porcine pericardia samples, decellularized porcine pericardia samples, and α-galactosidase / PNGase-F treated decellularized porcine pericardia samples. Cell infiltration was observed in the groups treated with or not treated with α-galactosidase / PNGase-F after decellularization. In the decellularized tissue, cells were observed to be aligned at the outer edge of the tissue on day 7 and infiltrating into the tissue on day 28. In the group treated with α-galactosidase / PNGase-F, it was observed that cells had already infiltrated the tissue before day 7 and that a significantly large number of cells were distributed within the tissue before day 28 (see Fig. 9).

[0069] Immunofluorescence staining in ADSCs and HUVECs inoculated into modified porcine pericardial tissue

[0070] Natural porcine pericardial samples, decellularized porcine pericardial samples, and α-galactosidase / PNGase-F-treated decellularized porcine pericardial samples were incubated for 7 and 28 days, and these samples were stained with vimentin, a marker for mesenchymal cells. Vimentin-positive cells were present on the surface of the porcine pericardial tissue on day 7. On day 28, a significant number of cells were observed to have aligned on the surface of the pericardial tissue and then infiltrated deep into the tissue. In particular, cells infiltrated the tissue more rapidly in the decellularized samples treated with α-galactosidase / PNGase-F, which implies accelerated recellularization (see Fig. 10).

[0071] Calponin is an intermediate marker indicating cell differentiation into vascular smooth muscle cells. On day 7 of culture, minimal staining was observed. On day 28, calponin-positive cells began to appear on the surface of the porcine pericardium. In decellularized samples treated with α-galactosidase / PNGase-F, calponin-positive signals were observed within the tissue, suggesting that infiltrating cells differentiate into smooth muscle cells (see Fig. 11).

[0072] Fibronectin is a major component of the extracellular matrix (ECM) and plays a role in promoting cell adhesion and regeneration. Strong fibronectin staining was observed, and stronger signals were detected in the tissue in the decellularization group cultured for 28 days compared to the decellularization group cultured for 7 days, and stronger signals were detected in the tissue in the decellularization group treated with α-galactosidase / PNGase-F (see Fig. 12).

[0073] CD31 is a platelet endothelial cell adhesion molecule-1 (PECAM-1) protein found in cells or tissues; it is primarily expressed in endothelial cells and some immune cells and plays a crucial role in angiogenesis, cell-cell adhesion, and the soft tissue response. As with the staining of other markers, a strong CD31 signal was observed in the tissues of the decellularized group treated with α-galactosidase / PNGase-F on day 28, suggesting that new blood vessels are forming in the recellularized grafts (see Figures 13 and 14).

[0074] Comprehensive evaluation

[0075] Decellularized xenograft scaffolds withstand hemodynamic stress and recellularize with host cells in vivo. Histological analysis of the xenografted valve revealed that valvular interstitial cells (VICs) and valcular endothelial cells (VECs) infiltrated the valve cusp. VECs form a non-thrombotic monolayer on the valve surface that plays a crucial role in the valve's hemodynamic properties. VICs are involved in the remodeling of the valve's extracellular matrix, ensuring tissue durability and growth characteristics. A significant number of myofibroblast-like VICs positive for α-smooth muscle actin (α-SMA) and vimentin were observed. This demonstrates the potential of the xenograft valve to recellularize and differentiate in vivo via the bloodstream.

[0076] However, when decellularized xenograft valve tissue is transplanted into host cells and recellularized, the absence or delayed influx of cells capable of restoring or remodeling the ECM can lead to tissue damage or degeneration resulting from valve-damaged tissue during the valve cycle. In situ recellularization has been less successful in clinical settings compared to preclinical studies. While recellularization of decellularized grafts is observed to a limited extent, in some cases, they have been shown to be more inflammatory than phenotypically suitable valve cells. Therefore, a strategy to induce recellularization by inoculating the surface of decellularized valve tissue in vitro with mesenchymal stem cells (MSCs) is being proposed. If the rate of recellularization is further enhanced in vivo, or if the recellularization process is accelerated by transplanting xenograft valves that have been pre-recellularized in vitro, there is a possibility of generating valve tissues with superior biocompatibility and regenerative properties within the human body.

[0077] In the case of in vitro recellularization, it is essential to use an appropriate cell source and provide conditioning signals to the bioreactor to induce cell proliferation and differentiation. Various cells have been studied for cardiac valve tissue engineering, and among these cells, the inventors utilized mesenchymal stem cells (MSCs). Mesenchymal stem cells (MSCs) possess self-renewal and differentiation capabilities and can be readily obtained from various tissues such as bone marrow, adipose tissue, placenta, and umbilical cord. Mesenchymal stem cells (MSCs) interact with surrounding tissues, differentiate into tissue-specific cells, reduce inflammatory cytokines, and promote new angiogenesis. The inventors induced recellularization using human adipose tissue-derived mesenchymal stem cells (MSCs) as the cell source. The process of attaching mesenchymal stem cells (MSCs) to a scaffold is facilitated through the interaction of specific cell integrins with various ECM proteins. The inventors enhanced cell adhesion, proliferation, and differentiation by coating decellularized porcine pericardium with fibrin. In addition, the inventors further promoted recellularization by using heparin and VEGF.

[0078] Fibronectin mediates the interaction of various cells with the extracellular matrix, which plays a crucial role in cell adhesion, migration, growth, and differentiation. When decellularized tissues treated with α-galactosidase / PNGase-F were co-cultured with mesenchymal stem cells (MSCs) and HUVECs, the expression of fibronectin was elevated, suggesting that the MSCs infiltrated the tissues more rapidly and abundantly. The inventors confirmed that the use of fibrin and heparin-VEGF in the co-culture of HUVECs and ADSCs can promote increased cell adhesion for the recellularization of decellularized scaffolds.

[0079] Through vimentin staining, the inventors confirmed that mesenchymal stem cells (MSCs) migrate into tissues and the activation of stromal cells is initiated, which signifies the start of tissue remodeling. In particular, myofibroblasts may exhibit vimentin positivity, which suggests that they represent the early stages of ECM remodeling and tissue recovery. However, to confirm whether these cells are in the early stages of myofibroblasts, it is necessary to check for the co-expression of α-SMA positivity.

[0080] Calponin staining suggested that mesenchymal stem cells (MSCs) interacted with the surrounding ECM and transformed into myofibroblasts exhibiting a mature smooth muscle cell phenotype. Calponin expression significantly increased after 4 weeks of co-culture of MSCs and HUVECs on tissues treated with α-galactosidase / PNGase-F. This implies that pluripotent stem cells undergo recellularization at the valve site and differentiate into a mature cell phenotype.

[0081] However, excessive activation of myofibroblasts can lead to tissue fibrosis and valve sclerosis, and may result in long-term functional impairment.

[0082] When the graft achieves sufficient endothelialization within the human body and therapeutic angiogenesis occurs, the incidence of thrombosis is reduced. Additionally, paracrine signaling promotes recellularization and enables differentiation into enhanced tissue structures. Co-culture of HUVECs and MSCs is effective for the rapid endothelialization of the vascular matrix. Xenografts recellularized using MSCs can modulate the host immune response, thereby enabling effective tissue remodeling until successful ECM replacement is achieved, delaying the biodegradation process, and extending tissue survival.

[0083] Compared to recent TRICOL (Triton, RNase, Cholate, and Lipase) decellularization based on osmotic shock, detergent, and nuclease treatment, the inventors' decellularization protocol using SDS and Triton X-100 via a multi-step method using hypotonic, isotonic, and hypertonic solutions was developed without nucleases and has been successfully commercialized due to its economic feasibility and mass production capabilities. Recently, decellularization and subsequent recellularization have focused on obstacles and unresolved issues that need to be overcome, particularly regarding optimal tissue engineering. Glycans with α-Gal and Neu5Gc at their ends are known to play a major role in xenoimmunogenicity and ultimately lead to transplant rejection. Decellularization protocols, combined with enzymatic deglycosylation, focus on reducing xenoimmunogenicity and removing α-Gal epitopes, and decellularized xenografts treated with PNGase-F result in mechanical stability along with potentially reduced immunogenicity.

[0084] In the development of tissue-engineered heart valves, strategies for recellularizing the entire valve, including a decellularized, xenoantigen-free scaffold, face numerous challenges. The present invention presents the potential to promote the recellularization of xenovascular vascular tissue prior to transplantation. While the inventors confirmed the degree of recellularization through immunohistochemical staining, it is necessary to further quantify this process and to further quantify the degree of differentiation and reconstruction using various additional markers. Furthermore, measuring ECM proteins associated with tissue reconstruction is important for elucidating the processes regarding tissue changes. It is necessary to evaluate the immune response of recellularized tissue in vivo and investigate how recellularized tissue can maintain growth and differentiation under stress conditions.

[0085] In conclusion, the inventors successfully produced a scaffold free of heterogenic antigens by demonstrating the safety and synergistic effects of α-galactosidase and PNGase-F, and demonstrated effective recellularization of the scaffold free of heterogenic antigens.

Claims

1. (a) A step of decellularizing porcine pericardia tissue; (b) a step of removing galactose-alpha-1,3-galactose (α-Gal) xenoantigen by treating decellularized porcine pericardial tissue with α-galactosidase; (c) a step of treating porcine pericardial tissue from which galactose-alpha-1,3-galactose (α-Gal) xenoantigens have been removed with peptide-N-glycosidase F to remove non-Gal xenoantigens, including N-glycolylneuramic acid (Neu5Gc); (d) a step of modifying α-galactosidase and peptide-N-glycosidase F-treated decellularized porcine pericardial tissue by coating it with fibrin, heparin, and vascular endothelial growth factor (VEGF); and (e) A method for producing a non-immunogenic xenograft tissue with improved durability and regenerative capacity, comprising a recellularization step of co-culturing human adipose-derived stem cells (ADSC) and human umbilical vein endothelial cells (HUVEC) in the decellularized porcine pericardial tissue modified in step (d) above.

2. A method for producing non-immunogenic xenograft tissue with improved durability and regenerative capacity, wherein, in claim 1, the decellularization of step (a) is performed using sodium dodecyl sulfate (SDS) and Triton X-100.

3. A method for producing a non-immunogenic xenograft tissue with improved durability and regenerative capacity, wherein, in claim 1, step (b) and step (c) are performed simultaneously.

4. A method for producing an immunogenic xenograft tissue with improved durability and regenerative capacity, wherein, in claim 1, step (e) involves culturing human adipose-derived stem cells (ADSC) followed by human umbilical vein endothelial cells (HUVEC); or culturing human adipose-derived stem cells (ADSC) and human umbilical vein endothelial cells (HUVEC) simultaneously.

5. A method for producing a non-immunogenic xenograft tissue with improved durability and regenerative capacity, wherein, in claim 1, prior to step (d), the porcine pericardial tissue from which galactose-alpha-1,3-galactose (α-Gal) xenoantigen and non-Gal xenoantigen have been removed is further comprising the step of fixation by treating the porcine pericardial tissue with glutaraldehyde; and the step of detoxifying the fixed porcine pericardial tissue by treating it with glycine.

6. A non-immunogenic xenograft tissue with improved durability and regenerative capacity, manufactured according to the manufacturing method of paragraph 1, A non-immunogenic xenograft tissue with improved durability and regenerative capacity, from which galactose-alpha-1,3-galactose (α-Gal) xenoantigens and non-Gal xenoantigens including N-glycolylneuramic acid (Neu5Gc) have been removed and which has been recellularized.

7. In paragraph 6, an immunogenic xenograft tissue with improved durability and regenerative capacity, in which the expression of vimentin, a marker of mesenchymal cells, is increased.

8. In paragraph 6, an immunogenic xenograft tissue with improved durability and regenerative capacity, in which the expression of calponin, a marker of differentiation into vascular smooth muscle cells, is increased.

9. In paragraph 6, an immunogenic xenograft tissue with improved durability and regenerative capacity, wherein the expression of fibronectin, which promotes cell adhesion and regeneration as a major component of the extracellular matrix (ECM), is increased.

10. In paragraph 6, an immunogenic xenograft tissue with improved durability and regenerative capacity, in which the expression of CD31, which plays an important role in angiogenesis, intercellular adhesion, and inflammatory response as a major component of endothelial cells, is increased.

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