In Vitro Detection System to Evaluate the Immunogenic Potential of Xenografts.

Tissue antigenicity represents the main limitation for the use of xenografts in clinical practice. To eliminate xenoantigens and avoid graft rejection in human, decellularization is often used to remove all immunoreactive components from the extracellular matrix (ECM). After decellularization, acellular scaffolds are required to be investigated regarding the presence of antigens, but commonly used detection methods solely focus on known xenoantigens such as alpha Gal (Galα1,3-Galß1-4GlcNAc-R) or major histocompatibility complex-I (MHC-I). However, there are unknown xenoantigens that escape the standard methods. To evaluate the immunological potential of xenogenic tissues, new in vitro methods need to be developed. Therefore, we established a novel human serum-based approach, including dot blot, Western blot, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA). With these methods, we analyzed protein extracts and tissue samples of native and decellularized bovine carotid arteries. All methods verified an effective removal of potential immunogens from the ECM through decellularization, and relative quantification with ELISA showed that 99.9% (p < 0.01) of antigenic components were successfully eliminated. We compared our human serum-based methods with commonly used assays for the detection of alpha Gal and MHC-I. Our results showed highly increased sensitivity for xenoantigens using the human serum antibody pool. This novel in vitro detection system allows the direct determination of the immunogenic potential of xenografts and is a vast improvement in comparison to the methods used so far. That way, it is possible to optimize the decellularization process to prevent hyperacute graft rejection in patients.

Detergent-Based Decellularization of Bovine Carotid Arteries for Vascular Tissue Engineering.

Vascular diseases are an increasing health issue, and common alloplastic, allogenic or autologous vascular grafts show frequent complications. The aim of this study is to develop an acellular, xenogenic bypass-graft from a bovine carotid artery (BAC) using detergent-based protocols. We compared decellularization with sodium desoxycholate (DOA), 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps), sodium dodecyl sulfate (SDS), and Triton X100 and improved suitable methods by variation of concentration, buffer system, incubation time, temperature, rinsing, and flow rate. All processes were evaluated systematically based on cellular residues, biocompatibility, structural and mechanical integrity. Decellularization with SDS and Triton X100 was not sufficient for the removal of cellular components. We optimized protocols using 1% DOA and Chaps by a buffered system at 37 °C with extended decellularization and rinsing. Decellularization with DOA depleted DNA to 0.5 ± 0.1% and soluble proteins to 0.6 ± 0.2%. Using Chaps, DNA was reduced to 0.2 ± 0.2% and proteins to 0.6 ± 0.3%. The improved protocols eliminated RNA completely from the matrix, and no cytotoxic effects were detected. Mechanical and structural integrity of decellularized tissues was comparable to non-decellularized controls. Our method effectively removed cellular components from the extracellular matrix while preserving the structural and mechanical integrity of the tissue. Decellularized BACs could be a promising alternative for vascular replacement therapy.