Quantification of α-Gal Antigen Removal in the Porcine Dermal Tissue by α-Galactosidase

Abstract

The α-Gal (Galα1,3-Galβ1-4GlcNAc-R) epitope, the major xenoantigen, is the first barrier in a porcine-to-man tissue and organ xenotransplantation. The elimination or reduction of the α-Gal epitopes is therefore an important step for a successful xenotransplantation. The present study is to evaluate the α-Gal elimination in the porcine skin with α-galactosidase treatment, and to assess two methods (immunohistochemistry and inhibition ELISA) that may be used in quality control for quantifying the extent of the α-Gal elimination. Enzymatic cleavage in a single-step process is extremely efficient and affordable at eliminating the α-Gal epitope even in a tissue as dense as the porcine dermis. The cost of enzymatic cleavage is found to be less than US$7 for a 10 × 10 cm piece of porcine skin (0.5 mm thick) or about US$140 for 100 g of 3-dimensional soft tissues. After enzymatic cleavage, the α-Gal-positive immunostaining was essentially undetectable in enzyme-treated porcine skin. The inhibition rate constant of the monoclonal anti-Gal antibody M86 binding to α-Gal-bovine serum albumin in ELISA was reduced from 15.0 ± 4.3 (n = 10) to 6.1 ± 2.6 (n = 7) after enzyme treatment, in comparison to 4.4 ± 1.8 (n = 9) background inhibition of decellularized human skin (the ultimate negative control), which demonstrates ∼ 84% elimination of α-Gal epitopes in treated porcine skin. To examine the suitability of two detection methods for the routine quality control application, comparative studies were made with control and enzyme-treated porcine skin, porcine skin from the α-Gal knockout animal, as well as decellularized human skin. The data show that the traditional immunohistochemistry and, to a less extent, the inhibition ELISA with further modifications can be used as quality control tools in the production and selection of biocompatible bioprosthetic devices. The biological evaluation of enzyme-treated porcine skin is ongoing with a small animal model and a nonhuman primate model.

A potential dermal substitute using decellularized dermis extracellular matrix derived bio-ink

“…It also plays a pivotal role in tissue morphogenesis and organ development, thus providing the most suitable environment for the cells [7][8][9]. Through physical, chemical and biochemical methods, dECM is prepared by preserving the basic structural and functional ECM proteins of tissue and removing the cellular components that might cause immune rejection or inflammatory responses [10][11][12]. Although various studies have been focused on 3D constructs based on the use of dECM, there is a general lack of research on changes in gene expression within the 3D construct.…”

In Vitro Regeneration of Decellularized Pig Esophagus Using Human Amniotic Stem Cells

“…Study has shown that treatment of an enzyme a-galactosidase on xenogeneic scaffolds could eliminate a-Gal epitopes from the animal tissue. 12 According to a recent report, when decellularized porcine a-Gal knockout lungs and decellularized lung containing a-Gal epitope were recellularized with human lung cells, there was no difference found in terms of cell growth and proliferation in both the lung recellularization. 13 Commercial glutaraldehydefixed heart valve transplantation exhibits anti-gal antibody immune reactions in humans and non-human primates.…”

Biomechanical comparison of xenogeneic bone material treated with different methods

“…The rationale of enzyme digestion is that it can be used to digest and destroy cell membranes. The enzyme α‐Gal glycosidase acts in a specific manner against the α‐Gal antigen and has been applied extensively to remove the α‐Gal antigen in porcine skin and heart valves . Trypsin is a non‐specific enzyme, which is used as a decellularization agent in cell harvesting protocols.…”