1H NMR Assessment of Safe Triton X-100 Levels in Decellularized Rat Aortic Valve Tissue

Abstract

Decellularization of allograft heart valves has been proposed as a method to reduce the alloreactive immune response, which limits the durability of these tissues. Rat models are essential for preliminary studies of the immunogenicity of decellularized tissue, but concerns have arisen about the toxic effects of Triton X-100 commonly used in a number of decellularization protocols. The purpose of this study was to determine the optimal washout conditions of Triton X-100 following decellularization. Sprague-Dawley rat aortic valves were decellularized with a combination of hypotonic and hypertonic buffers, protease inhibitors, and gentle detergents (0.5% Triton X-100) followed by a washout in phosphate buffered saline (PBS) for variable time periods. Decellularized valves were allowed to equilibrate in ddH2O and residual levels of Triton X-100 were determined using proton nuclear magnetic resonance spectroscopy (1H-NMR). The majority of Triton X-100 was removed with a PBS washout time of 4 hours, yielding a...