Method for producing heterograft using peptide-n-glycosidase f

Abstract

The present invention relates to a method for producing a heterograft, comprising the steps of: (a) decellularizing porcine pericardial tissue; (b) treating the decellularized porcine pericardial tissue with alpha-galactosidase to remove a galactose-alpha-1,3-galactose (α-Gal) xenoantigen; (c) treating the porcine pericardial tissue from which the α-Gal xenoantigen has been removed, with peptide-N-glycosidase F to remove non-Gal xenoantigens, including N-glycolneuramic acid (Neu5Gc); and (d) treating the porcine pericardial tissue from which the α-Gal xenoantigen and the non-Gal xenoantigens have been removed, with glutaraldehyde and fixing same. Also, the present invention relates to a heterograft (e.g., a prosthetic heart valve) produced by the production method

Description

Method for producing xenograft tissue using peptide-N-glycosidase F

[0001] The present invention relates to a method for producing a xenograft using peptide-N-glycosidase F. Specifically, the present invention relates to a method for producing a xenograft in which galactose-alpha-1,3-galactose (α-Gal) xenoantigen and non-Gal xenoantigens including N-glycolneuramic acid (Neu5Gc) are removed by treating decellularized porcine pericardial tissue with alpha-galactosidase and peptide-N-glycosidase F.

[0002] In addition, the present invention relates to a xenograft (e.g., an artificial heart valve) manufactured according to the method for manufacturing the xenograft. Specifically, the present invention relates to a xenograft from which galactose-alpha-1,3-galactose (α-Gal) xenoantigen and non-Gal xenoantigens, including N-glycolneuramic acid (Neu5Gc), have been removed.

[0003] This application is based on the results of a research project supported by the Pediatric Cancer and Rare Disease Support Center.

[0004] Assignment ID: 23C-022-0000

[0005] Assignment Number: 23C-022-0100

[0006] Ministry Name: Pediatric Cancer and Rare Disease Support Center

[0007] Project Management (Specialist) Institution Name: Seoul National University Hospital

[0008] Research Project Name: Lee Kun-hee's Pediatric Cancer and Rare Disease Overcoming Project

[0009] Research Project Name: Pediatric Cardiovascular Regenerative Medicine Clinical Trial Using Advanced Tissue Engineering to Improve Treatment Outcomes for Pediatric Rare Diseases

[0010] Project implementing organization: Seoul National University Hospital

[0011] Research period: January 1, 2023 - December 31, 2025.

[0012] Tissue engineering is an innovative technology that artificially creates tissues or organs and transplants them into living organisms to enable their function. This technology ultimately aims to regenerate tissues or organs through the fusion of cells, biomaterials, and the biological environment, and holds significant potential for medical and clinical applications.

[0013] According to the "Report on Organ Donation and Transplantation Activities 2021" published by the Global Observatory on Donation and Transplantation (GODT), a collaboration between the World Health Organization (WHO) and the Spanish transplant organization ONT (Organization Nacional de Trasplantes), organ transplants have been steadily increasing every year, with approximately 144,302 organ transplants performed worldwide in 2021. This is an increase of approximately 11.3% compared to 2020. Looking at the number of transplants by organ, kidneys were the most common at 92,532, followed by livers at 34,694 and hearts at 8,409. This means that 380 organ transplants are performed worldwide every day, or 16 every hour. However, the number of people waiting for an organ transplant is much higher; in the United States, it has been reported that 17 people die each day waiting for an organ transplant. As of 2021, the number of organ transplants in Korea was 5,842. However, even in Korea, the number of organ transplants is woefully inadequate compared to the number of people waiting for transplants.

[0014] Despite the large number of patients waiting for organ transplants, not all donated organs are used. The most common reasons are damage to the organ (38.2%) and the inability to immediately find a donor (14.6%). For these reasons, approximately 2,700 donated kidneys are reportedly discarded each year in the United States. Furthermore, the number of discarded kidneys is increasing annually, from 12.7% of donated kidneys in 2002 to 17.9% in 2011. While damaged organs are unusable, the inability to immediately find a donor, even when there are no issues with the organ, can lead to discarding due to the organ's short storage time. Donated hearts and lungs can be stored for 4-6 hours, livers for 8-12 hours, and kidneys for 24-36 hours.

[0015] Meanwhile, when heart valves using xenografts are transplanted into the human body, immune rejection reactions due to galactose-alpha-1,3-galactose (α-Gal) and N-glycolneuramic acid (Neu5Gc) can cause calcification and dysfunction of the valves. α-Gal can be removed by alpha-galactosidase treatment, but non-alpha-gal xenoantigens such as Neu5Gc cannot be removed, leading to a problem of reduced durability due to immune rejection reactions.

[0016] Korean Patent Publication No. 10-2012-0002379 discloses a method for producing a living tissue for xenotransplantation, comprising the steps of: decellularizing living tissue of a mammal other than a human; fixing the decellularized living tissue; and removing toxicity from the fixed living tissue. Furthermore, Korean Patent Publication No. 10-2022-0002048 discloses a method for producing an artificial valve, comprising the steps of: decellularizing a porcine heart valve with sodium dodecyl sulfate (SDS) or the like; inactivating a xenoantigen by treating with α-galactosidase; fixing by treating with glutaraldehyde; and detoxifying with glycine.

[0017] The present inventors, after extensive research to overcome the problems of prior art for preventing anti-calcification and tissue degeneration, have developed a method for producing xenograft tissue that can completely eliminate immune rejection and increase the durability of a xenograft valve by removing non-alpha-gal xenoantigens such as Neu5Gc by treating with peptide-N-glycosidase F.

[0018] The present invention aims to provide a method for producing a xenograft tissue in which galactose-alpha-1,3-galactose (α-Gal) xenoantigen and non-Gal xenoantigen including N-glycolneuramic acid (Neu5Gc) are removed by treating decellularized porcine pericardial tissue with alpha-galactosidase and peptide-N-glycosidase F.

[0019] In addition, the present invention aims to provide a xenograft tissue having improved durability and from which galactose-alpha-1,3-galactose (α-Gal) xenoantigen and non-galactose xenoantigen including N-glycolneuramic acid (Neu5Gc) are removed.

[0020] The present invention provides a method for producing a xenograft, comprising the steps of: (a) decellularizing porcine pericardial tissue; (b) treating the decellularized porcine pericardial tissue with peptide-N-glycosidase F (PNGase-F) to remove non-galactose xenoantigens including N-glycolneuramic acid (Neu5Gc); and (c) fixing the decellularized porcine pericardial tissue from which non-galactose xenoantigens have been removed with glutaraldehyde. In addition, the present invention provides a xenograft tissue from which non-galactose xenoantigens, including N-glycolneuramic acid (Neu5Gc), have been removed, manufactured according to the method for manufacturing the xenograft tissue.

[0021] In addition, the present invention comprises the steps of (a) decellularizing porcine pericardial tissue; (b) treating the decellularized porcine pericardial tissue with alpha-galactosidase to remove galactose-alpha-1,3-galactose (α-Gal) xenoantigen; (c) a step of removing non-galactose xenoantigens including N-glycolneuramic acid (Neu5Gc) by treating porcine pericardial tissue from which galactose-alpha-1,3-galactose (α-Gal) xenoantigens have been removed with peptide-N-glycosidase F (PNGase-F); And (d) a method for producing a heterograft, comprising a 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 the porcine pericardial tissue with glutaraldehyde. In addition, the present invention provides a xenograft tissue manufactured according to the method for manufacturing the above xenograft tissue, wherein galactose-alpha-1,3-galactose (α-Gal) xenoantigen and non-galactose xenoantigen including N-glycolneuramic acid (Neu5Gc) are removed.

[0022] In the method for producing a xenograft tissue of the present invention, porcine pericardium is decellularized using a hypotonic solution containing 0.5% Triton X-100 [10 mM Tris, 0.05% EDTA, 0.5 μg / mL Leupeptin, and 50 μg / mL neomycin] and 0.25% sodium dodecyl sulfate (SDS). In addition, decellularization is performed using at least one method selected from the group consisting of a method of repeatedly freezing and thawing to decellularize, a method of removing cells from the surface of an extracellular matrix (ECM) through stirring, a method of inducing cell lysis by creating micropores in a cell membrane through electroporation, a method of lysing cells by pressurization, and a method of using a supercritical fluid.

[0023] In the method for producing a xenograft tissue of the present invention, when decellularized porcine pericardial tissue is treated with peptide-N-glycosidase F (PNGase-F), the level of lectin binding is significantly reduced in a concentration-dependent manner, which means that non-galactose xenoantigens including N-glycolneuramic acid (Neu5Gc) are removed. In other words, xenograft immunogenicity is effectively removed, and biomechanical stability and biocompatibility are maintained.

[0024] In the method for producing a xenograft tissue of the present invention, when decellularized porcine pericardial tissue is treated with alpha-galactosidase, galactose-alpha-1,3-galactose (α-Gal) xenoantigen can be removed.

[0025] According to the present invention, non-galactose carbohydrate xenoantigens that cause immune rejection reactions are safely removed, thereby enabling non-immunogenic living tissue transplantation with improved durability.

[0026] In addition, the present invention can be usefully utilized in bio-organ transplantation, cardiovascular valve transplantation, valve conduit transplantation surgery, etc., and can be applied as a living tissue that can be reconstructed when the heart, arteries, and aorta are damaged.

[0027] Figures 1a and 1b show the histology of decellularized porcine pericardium.

[0028] Figure 1a shows the results of hematoxylin & eosin (H&E) staining of native porcine pericardial tissue, longitudinally sectioned and non-decellularized. H&E staining was visualized using a microscope and showed complete decellularization without histological changes. Cell nuclei were stained with 4',6'-diamidino-2-phenylindole (DAPI). As a result of decellularization, a DAPI-negative fluorescent signal appeared in the decellularized tissue. Fluorescence was detected using a fluorescence microscope. The magnification used was ×200.

[0029] Figure 1b shows the quantification of DNA content in longitudinally sectioned porcine pericardial tissue, both decellularized and non-decellularized. DNA content was measured using a spectrophotometer. Decellularized tissue showed a significantly reduced DNA content compared to native tissue.

[0030] Figures 2a and 2b show the histological characterization of peptide-N-glycosidase F (PNGase-F) treated porcine pericardium.

[0031] Figure 2a shows the results of H&E staining of longitudinally sectioned porcine pericardial tissue treated with PNGase-F (0–2 units / ml). H&E staining was imaged using a microscope. The magnification used was ×200. No histological changes, such as collagen fiber pattern and structural loosening, were observed according to the PNGase-F treatment concentration.

[0032] Figure 2b shows scanning electron microscopy (SEM) images of porcine pericardium treated with PNGase-F (0, 1.0, and 2.0 unit / ml). The magnification used was ×200. The gaps between collagen fiber bundles showed similar collagen morphology and patterns in porcine pericardium treated with various concentrations of PNGase-F. PNGase-F treatment did not affect histomorphological changes.

[0033] Figures 3a and 3b show the biomechanical properties of PNGase-F treated porcine pericardium.

[0034] Figure 3a shows the uniaxial tensile analysis of porcine pericardium analyzed using a tensile tester. There were no significant differences in tensile stress, tensile displacement, and tensile strain at break between porcine pericardiums (n=30 each) treated with different concentrations (0–2 unit / ml) of PNGase-F.

[0035] Figure 3b shows the results of permeability analysis of porcine pericardium using a permeability tester. No significant differences in permeability were observed between porcine pericardiums (n=30 each) treated with different concentrations (0–2 units / ml) of PNGase-F.

[0036] Figures 4a and 4b show the results of lectin histochemistry of the porcine pericardium treated with PNGase-F. Figure 4a shows the results of staining the PNGase-treated (0–2 unit / ml) porcine pericardium with Jacalin, MALI, WGA, RCA, and GSL, and Figure 4b shows the results of staining the PNGase-treated (0–2 unit / ml) porcine pericardium with ECA, PNA, SBA, WFA, and DSL. Carbohydrate-binding lectins were stained red. The magnification used was ×200. Fluorescence imaging results showed that the group treated with PNGase-F showed lower fluorescence in proportion to the increasing concentration.

[0037] Figure 5 shows lectin binding to solubilized proteins from porcine pericardium treated with PNGase-F. To quantitatively measure the expression of WGA, ECA, GSL, RCA, MALI, WFA, and DSL, lectin binding assays were performed on porcine pericardium treated with PNGase-F (0–2 units / ml). PNGase treatment resulted in a significant inhibition of lectin binding levels in a concentration-dependent manner.

[0038] Figures 6a and 6b show the lectin histochemical results of decellularized porcine pericardium treated with PNGase-F. Figure 6a shows the results of staining PNGase-treated (0–2 unit / ml) decellularized porcine pericardium with Jacalin, MALI, WGA, RCA, and GSL, and Figure 6b shows the results of staining PNGase-treated (0–2 unit / ml) decellularized porcine pericardium with ECA, PNA, SBA, WFA, and DSL. Carbohydrate-binding lectins were stained red. The magnification used was ×200. The fluorescence signal of decellularization effectively reduced lectin expression. When PNGase-F treatment was added to decellularization, lectin expression was significantly reduced regardless of the PNGase-F concentration, demonstrating the synergistic effect of decellularization and PNGase-F treatment.

[0039] Figure 7a shows the results of lectin histochemistry staining (200x magnification) of porcine pericardium treated with peptide-N-glycosidase F using ricinus communis agglutinin I (RCA-I).

[0040] Figure 7b shows the results of lectin histochemistry staining (200x magnification) of porcine pericardium treated with peptide-N-glycosidase F using wheat germ agglutinin (WGA).

[0041] The present invention is described in detail by the following examples. However, the scope of the present invention should not be construed as being limited by the following examples.

[0042] Tissue preparation, decellularization, and sterilization of porcine pericardium

[0043] Porcine pericardium was purchased from Taewoong Medical Co., Ltd., Gimpo, Gyeonggi-do, South Korea. After removal of fat and connective tissue, the tissue was disinfected with 0.1% polyacrylic acid (PPA) for 4 h and then washed with phosphate-buffered saline (PBS) with agitation at room temperature (RT).

[0044] Decellularization of porcine pericardium

[0045] Porcine pericardial tissues were decellularized with hypotonic solution containing 0.5% Triton X-100 [10 mM Tris, 0.05% EDTA, 0.5 μg / ml Leupeptin, 50 μg / ml neomycin] (Sigma, USA) and 0.25% sodium dodecyl sulfate (SDS) in deionized water (diH2O) at 4°C for 24 h while stirring. The tissues were washed with distilled water for 72 h, and the solution was changed once daily using a stirrer at 4°C. After treatment with hypotonic solution containing 0.5% Triton X-100, the tissues were washed with distilled water for 12 h at 4°C. Subsequently, the tissues were decellularized with isotonic solution at 4°C for 48 h. Tissues were washed with phosphate-buffered saline (PBS) containing 30% polyethylene glycol 1000 and 1% antibiotic / antimycotic for 48 h at 4°C with stirring using a shaker. Finally, the tissues were treated with a hypertonic solution (200 mM Tris, 600 mM NaCl) for 3 h at 4°C. The tissues were washed with PBS for 24 h.

[0046] enzymatic digestion

[0047] Decellularized porcine pericardium was treated with PNGase-F (#P074L, 1000 U, NEB). After treatment with PNGase-F for 24 h at 37°C with agitation, the samples were rinsed with PBS at room temperature with agitation. Finally, the samples were stored in PBS supplemented with 1% (v / v) penicillin / streptomycin and 400 μL / L amphotericin B at 4°C for 24 h.

[0048] Histology

[0049] For hematoxylin & eosin (H&E) staining, porcine pericardial samples were fixed in 4% paraformaldehyde solution at 4°C for 24 hours, washed three times with PBS, dehydrated, and embedded in paraffin. Paraffin blocks were sectioned to 5 μm thickness, and the sections were stained with H&E and 4',6'-diamidino-2-phenylindole (DAPI). Samples were stained with DAPI to measure the removal of DNA structures. Stained sections were imaged using an inverted fluorescence microscope (DMI4000B, Leica, Germany). Stained images were obtained using the Application Suite Image Viewer (Leica, Germany).

[0050] DNA content quantification

[0051] For DNA content quantification, tissue lysates were used after homogenization. DNA content was measured using the QiAmp DNA Mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The isolated DNA pellet was measured using a NanoDrop UV-VIS spectrophotometer (Thermo Fisher), and the absolute amount of DNA (ng) was quantified based on the total tissue mass (mg) used.

[0052] Scanning electron microscopy

[0053] High-resolution images of porcine pericardial tissue were obtained using scanning electron microscopy (SEM). Native porcine pericardial tissue, either treated or untreated with PNGase-F, was examined for surface features and processed for SEM. Samples were fixed in 4% paraformaldehyde for 24 hours at room temperature and prepared for SEM. Images were captured using a SIGMA 500 scanning electron microscope (Carl Zeiss, Germany).

[0054] Mechanical stability test

[0055] After storing the tissues at 4°C for 24 hours, they were rinsed with PBS. Mechanical analysis tests were performed in tensile oscillation mode using a tension film clamp. According to the manufacturer's instructions, the tissues were cut into rectangular pieces measuring approximately 5.0 mm × 10 mm (length × width, n = 30). The tissue thickness was measured at three points on the pieces using a thickness gauge (Quick-Mini 700-117; Mitutoyo, Kawasaki, Japan). Tensile properties were performed using a tensile tester (68SC-5, INSTRON, USA) [100 N load cell] to evaluate whether there was a difference in the ultimate tensile strength of each sample. The ultimate tensile strength and failure strain were evaluated from the recorded stress-strain curves. The stress-strain behavior of each specimen was analyzed using parameters such as ultimate tensile strength, failure strain, maximum force, transfer strain, and E-modulus. Permeability properties were measured by measuring the permeation volume of saline solution and applying a constant pressure of 100 mmHg to the pericardial tissue for 1 hour. For this test, the tissue was cut into pieces approximately 1 cm 2The tissues were cut into rectangular pieces (n=8). Permeability tests were performed to measure the differences in the degree of penetration, elongation, and gaps in the collagen fiber bundles within the tissue. Permeability tests of pericardial tissues were performed using Universal Analysis 2000.

[0056] Immunofluorescence analysis

[0057] For lectin staining, native porcine pericardium, decellularized porcine pericardium, and decellularized and PNGase-F-treated porcine pericardium samples were fixed in 4% paraformaldehyde at 4°C for 24 h. Tissue sections were incubated overnight at 4°C with lectins (1:250). Isolectin B4 (IL-B4 / GSL-IB4), specific for α-galactose residues; wheat germ agglutinin (#RL-1022, Vector Laboratories Inc); Datura stramonium lectin (DSL) (#B-1185, Vector Laboratories Inc); Ricinus communis agglutinin I (RCA-I) (#RL-1082, Vector Laboratories Inc); soybean agglutinin (SBA) (#B-1015, Vector Laboratories Inc); The effect of PNGase treatment was analyzed on carbohydrate-binding lectins such as wisteria lectin (WFA) (#B-1355, Vector Laboratories Inc); peanut agglutinin (PNA) (#B-1075, Vector Laboratories Inc); Macia amurensis lectin I (MAL I) (#B-1315, Vector Laboratories Inc); jacalin (#B-1155, Vector Laboratories Inc); and Erythrina christchagalli lectin (ECA) (#B-1145, Vector Laboratories Inc). The stained sections were further stained with avidin-conjugated Texas Red (#A-2006; Vector Laboratories Inc.). The sections were washed twice with PBS, and the stained sections were imaged using an inverted fluorescence microscope (DMI4000B, Leica, Germany). The stained images were obtained using Application Suite X Image Viewer (Leica).

[0058] Lectin binding assay

[0059] For lectin binding analysis, native and PNGase-treated porcine pericardium samples were lysed in radioimmunoprecipitation assay (RIPA) protein extraction solution (ATTO). The lysed tissue samples were incubated with coating buffer (Biosesang) overnight at 4°C, and then each well was blocked with carbohydrate-free blocking solution (#SP-5040-125, Vector Laboratories Inc.) for 30 min at room temperature. Each well was incubated with carbohydrate-free blocking solution containing primary lectin (1:500) for 3 h at room temperature. After primary lectin incubation, streptavidin-conjugated peroxidase (1:10000, #SA-S0004, Vector Laboratories Inc.) was added to each well together with carbohydrate-free blocking solution for 30 min at room temperature. After washing several times with PBS containing 0.05% Tween 20 (Nyahum), the reaction was carried out with 3,3',5,5'-tetramethylbenzidine (TMB) solution (#34028, Thermo Scientific). The absorbance of the lectin was measured at 450 nm using an ELISA reader (Tecan, spark).

[0060] Histology and DNA content of decellularized porcine pericardium

[0061] Porcine pericardial tissue was processed using a novel decellularization protocol using 0.25% SDS and Triton X-100 through a multistep method using hypotonic, isotonic, and hypertonic buffer solutions, and stained with H&E. Staining results showed complete decellularization without histological changes compared to native porcine pericardium. Cell nuclei were stained with DAPI. The decellularized porcine pericardium showed a DAPI-negative signal when observed under a fluorescence microscope (Fig. 1a).

[0062] Additionally, the DNA content was quantified using a spectrophotometer, and a significant decrease in the DNA content of the decellularized porcine pericardial tissue was observed compared to the native tissue (Fig. 1b).

[0063] Histological analysis of PNGase-F-treated porcine pericardium using SEM.

[0064] Porcine pericardium was treated with different concentrations of PNGase-F (0–2.0 units / ml) and stained with H&E. Histological characteristics, such as collagen fiber pattern and structural loosening, were not altered, and the concentration of PNGase-F treatment was not reflected in histological changes (Fig. 2a).

[0065] High-resolution images using SEM showed that the gaps between collagen fiber bundles in porcine pericardium treated with different concentrations (0–2.0 unit / ml) of PNGase-F showed similar collagen morphology and patterns. PNGase-F treatment concentration did not affect histological changes (Fig. 2b).

[0066] Biomechanical analysis of porcine pericardium treated with PNGase-F

[0067] The biomedical properties were measured by uniaxial tensile tests and permeability tests. There was no significant difference in the tensile stress at break between native porcine pericardium (n=30) and porcine pericardium treated with different concentrations of PNGase-F (0.5–2.0 unit / ml, each n=30). There was no significant difference in the tensile displacement at break between native porcine pericardium (n=30) and porcine pericardium treated with different concentrations of PNGase-F (0.5–2.0 unit / ml, each n=30). There was no significant difference in the tensile strain at break between native porcine pericardium (n=30) and porcine pericardium treated with different concentrations of PNGase-F (0.5–2.0 unit / ml, each n=30) (Fig. 3a). In addition, a permeability test was performed to evaluate the mechanical properties according to the PNGase treatment concentration. Permeability tests revealed no significant differences between native porcine pericardium (n=6) and porcine pericardium treated with different concentrations of PNGase-F (0.5–2.0 unit / ml, respectively; n=6 each) (Fig. 3b). These results suggest that the effect of PNGase treatment concentration was not reflected in the biochemical properties of porcine pericardium.

[0068] Lectin immunochemical and lectin binding analysis of PNGase-F-treated porcine pericardium.

[0069] In xenotransplantation, lectins as carbohydrate components and increased lectin levels are highly correlated with immune rejection. Using lectin expression, the effect of enzymatic digestion with PNGse-F on carbohydrate removal was investigated. Longitudinally dissected native and PNGase-F-treated porcine pericardium samples were stained with 10 lectins, including Jakalin, MAL-I, WGA, RCA-I, GSL, ECA, PNA, SNA, DSL, and WFA. Fluorescence imaging of the lectins showed that PNGase-F-treated porcine pericardium exhibited lower fluorescence than native porcine pericardium. Furthermore, the PNGase-F-treated group showed lower fluorescence in proportion to increasing concentrations (0 to 2 units / ml) (Figures 4a and 4b).

[0070] To quantitatively measure the expression of WGA, ECA, GSL, RCA, MAL-I, WFA, and DSL, a lectin binding assay was performed on pig pericardium treated with PNGase-F (0–2 unit / ml). PNGase-F treatment resulted in a significant inhibition of lectin binding levels in a concentration-dependent manner (Fig. 5). Furthermore, PNGase-F treatment at a concentration of 2.0 unit / ml showed the greatest decrease in lectin fluorescence signal, suggesting that a high concentration of PNGase-F is most effective in removing xenoantigens.

[0071] Lectin histochemistry of porcine pericardium decellularized and further treated with PNGase-F.

[0072] Decellularized porcine pericardium treated with PNGase-F (0–2.0 unit / ml) was stained with Jacalin, MAL-I, WGA, RCA, GSL, ECA, PNA, SBA, WFA, and DSL. Decellularization effectively reduced the fluorescent signals of all lectin expression compared to native tissue, but did not completely remove all lectins from the porcine pericardium. In contrast, treatment with PNGase-F in combination with decellularization completely eliminated the fluorescent signals of all lectin expression compared to decellularization alone (Figs. 6a and 6b).

[0073] Although we observed a significant reduction in lectin expression at high concentrations of PNGase-F treatment, treatment with PNGase-F in combination with decellularization completely abolished all lectin expression even at low concentrations, regardless of the PNGase-F concentration, demonstrating the synergistic effect of decellularization and PNGase-F treatment (Figs. 6a and 6b). These results indicate that PNGase-F treatment completely abolished carbohydrate-binding lectin levels in xenopericardium.

[0074] Evaluation of the stability of heterologous tissues - testing for mechanical properties

[0075] Xenografts require glutaraldehyde fixation for stability, sterility, and immune response reduction. Furthermore, decellularization is essential to eliminate immunogenicity. Furthermore, even decellularization cannot remove alpha-Gal, which causes immune rejection, so treatment with alpha-galactosidase is required to remove alpha-Gal. Furthermore, even if alpha-Gal, which causes immune rejection, is removed, PNGase-F treatment is still necessary to remove non-Gal.

[0076] To evaluate the stability of the xenografts, mechanical properties were tested for porcine pericardial tissues of Group 1 (fixation by glutaraldehyde treatment), Group 2 (decellularization and fixation by glutaraldehyde), Group 3 (decellularization, alpha-Gal removal by alpha-galactosidase treatment, and fixation by glutaraldehyde treatment), and Group 4 (decellularization, non-Gal removal by PNGase-F treatment, and fixation by glutaraldehyde treatment). The results are presented in Tables 1 and 2 below.

[0077] Tensile strain (displacement) Gauge length (mm) Tensile stress at break (MPa) Tensile strain (displacement) at break (%) Young's modulus (MPa) Group 1 (mean ± standard deviation) 34 ± 00.11875 ± 0.04675 401158.59375 ± 0.1293 1913143 ± 44.84982 Group 2 (mean ± standard deviation) 34 ± 0026125 ± 0.1520228 1958.65375 ± 0.106 176445 197.835 ± 46.02085 Group 3 (mean ± standard deviation) 34 ± 00.15625 ± 0.0640190458.59375 ± 0.163778623227.0363 ±123.9649 Group 4 (mean ± standard deviation) 34 ± 00.33 ±0.50623611165.9575 ±12.63201661241.6513 ±140.6558

[0078] Maximum force (N)Tensile strain (displacement) at maximum force (%)Tensile displacement at maximum force (mm)Time at maximum force (sec)Tensile stress at maximum force (MPa)Group 1 (Mean ± Standard Deviation)10.01625 ±1.87056767840.43375 ±5.42683134413.74625 ±1.8453258.3025 ±1.10947918.70625 ±3.361324Group 2 (Mean ± Standard Deviation)10.67125 ±1.69956198432.23375 ±4.68087047910.96 ±1.5926636.345 ±0.49241820.04625 ±4.022275Group Group 3 (mean ± standard deviation) 12.66 ± 3.24 95 23 0 4 2 3 1.70 6 25 ± 7.80 5 12 16 16 10.78 ± 2.65 27 6 3 6.52 ± 1.59 19 8 2 0.91 ± 8.93 6 0 13 Group 4 (mean ± standard deviation) 12.10 37 5 ± 4.32 7 0 6 0 0 2 3 1.37 8 75 ± 9.28 15 14 4 7 4 10.66 75 ± 3.15 5 0 6 2 6.455 ± 1.89 5 6 9 9 2 1.23 12 5 ± 7.79 2 4 8 7

[0079] Evaluation of the effectiveness of xenografts - Lectin immunohistochemistry staining Lectin immunohistochemistry staining was performed on porcine pericardial tissues of Group 1 (fixation by glutaraldehyde treatment), Group 2 (decellularization and fixation by glutaraldehyde), Group 3 (decellularization, alpha-Gal removal by alpha-galactosidase treatment, and fixation by glutaraldehyde treatment), and Group 4 (decellularization, non-Gal removal by PNGase-F treatment, and fixation by glutaraldehyde treatment) to evaluate the effectiveness of xenografts.

[0080] The results of lectin histochemistry staining of peptide-N-glycosidase F-treated porcine pericardium using ricinus communis agglutinin I (RCA-I) are shown in Fig. 7a (200x magnification). Porcine pericardium treated with peptide-N-glycosidase F was stained with ricinus communis agglutinin I (RCA-I) and counterstained with 4',6-diamido phenylindole (DAPI). Carbohydrate-binding lectins were stained red, whereas cell nuclei were stained blue.

[0081] In addition, the results of lectin histochemistry staining of porcine pericardium treated with peptide-N-glycosidase F using wheat germ agglutinin (WGA) (200x magnification) are shown in Fig. 7b. Porcine pericardium treated with peptide-N-glycosidase F was stained with wheat germ agglutinin (WGA) and counterstained with 4',6-diamido phenylindole (DAPI). Carbohydrate-binding lectins were stained red, whereas cell nuclei were stained blue.

[0082] In groups 1, 2, and 3 that were not treated with peptide-N-glycosidase F, non-alpha-gal (non-Gal) xenoantigens were not removed, but in group 4 that was treated with peptide-N-glycosidase F, non-Gal xenoantigens were removed.

Claims

1. (a) A step of decellularizing porcine pericardial tissue; (b) a step of treating decellularized porcine pericardial tissue with peptide-N-glycosidase F (PNGase-F) to remove non-galactose xenoantigens including N-glycolneuramic acid (Neu5Gc); and (c) A method for producing a heterograft, comprising a step of fixing decellularized porcine pericardial tissue from which non-galactose xenoantigen has been removed by treating it with glutaraldehyde. 2.(a) Step of decellularizing porcine pericardial tissue; (b) a step of treating decellularized porcine pericardial tissue with alpha-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) xenoantigen has been removed with peptide-N-glycosidase F (PNGase-F) to remove non-galactose xenoantigens including N-glycolneuramic acid (Neu5Gc); and (d) A method for producing a heterograft, comprising a 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 the porcine pericardial tissue with glutaraldehyde.

3. In paragraph 1 or 2, A method for producing a xenograft tissue, wherein the decellularization of the above step (a) is performed by decellularizing the porcine pericardium using a hypotonic solution containing 0.5% Triton X-100 [10 mM Tris, 0.05% EDTA, 0.5 μg / mL Leupeptin, and 50 μg / mL neomycin] and 0.25% sodium dodecyl sulfate (SDS).

4. In paragraph 1 or 2, A method for producing a xenograft tissue, wherein the decellularization in step (a) above is performed using at least one method selected from the group consisting of a method of decellularizing by repeatedly freezing and thawing, a method of removing cells from the surface of an extracellular matrix (ECM) through stirring, a method of inducing cell lysis by creating micropores in a cell membrane through electroporation, a method of lysing cells by pressurization, and a method of using a supercritical fluid.

5. In paragraph 1 or 2, A method for producing a xenograft tissue, comprising an additional step of detoxifying the fixed porcine pericardial tissue by treating it with glycine.

6. A heterograft manufactured according to the manufacturing method of paragraph 1, Xenograft tissue from which non-galactose xenoantigens, including N-glycolneuramic acid (Neu5Gc), have been removed.

7. A heterograft manufactured according to the manufacturing method of paragraph 2, A xenograft tissue in which galactose-alpha-1,3-galactose (α-Gal) xenoantigen and non-galactose xenoantigens, including N-glycolneuramic acid (Neu5Gc), have been removed.

8. In paragraph 6 or 7, The above xenograft tissue is an artificial heart valve.

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