Human Recellularization for Xenoantigen-Free Decellularized Cardiac Xenografts

Removal of major xenoantigens of the Galα1-3Gal (α-Gal) epitope and the nonhuman sialic acid <i>N</i>-glycolylneuraminic acid (Neu5Gc) is essential to eliminate xenoimmunogenicity and optimize recellularization for cardiac xenografts. The aim of this study was to evaluate the safety and efficacy of α-galactosidase for removal of α-Gal xenoantigen and peptide <i>N</i>-glycosidase F (PNGase-F) for removal of non-α-Gal xenoantigen combined with optimal decellularization, and the potential of <i>in vitro</i> recellularization was assessed with coculturing human mesenchymal stem cells and human umbilical vein endothelial cells for major xenoantigen-free cardiac xenografts. We investigated the biomechanical properties and efficacy for xenoantigen removal with expression of carbohydrate-binding lectins in porcine pericardia decellularized and treated with α-galactosidase and PNGase-F. There were no histological changes depending on α-galactosidase and PNGase-F treatment. There was no difference in tensile stress, tensile displacement, tensile strain at break, and permeability test following enzymatic treatments. Both enzyme-treated xenografts were stained with Jacalin, Maackia amurensis lectin I, wheat germ agglutinin, Ricinus communis agglutinin, Griffonia simplicifolia lectin (GSL), erythrina cristagalli lectin, peanut agglutinin, soybean agglutinin, Wisteria floribunda lectin, and Datura stramonium lectin and showed synergistic effects for low fluorescence qualitatively and quantitatively. The enzymatic treatments for decellularization significantly reduced lectin expression, demonstrating the synergistic effect of both enzymes and decellularization. <i>In vitro</i> recellularization for decellularized and both enzymes-treated xenografts was assessed with vimentin, calponin, fibronectin, and CD31 staining. Stronger signals were detected in decellularized xenografts, and decellularized xenografts treated with both enzymes showed significantly faster mesenchymal cell infiltration into the tissue, leading to accelerated recellularization. We have successfully produced major xenoantigen-free scaffolds by demonstrating the safety and the synergistic effect of α-galactosidase and PNGase-F treatments and proved effective recellularization for the xenoantigen-free scaffolds not previously reported in the literature.