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 residual Triton
X-100 concentration of 7.95 × 10–6 ± 3.66 × 10–6 M. Furthermore, no significant change in Triton X-100 concentration
was observed after the 4-hour wash time (p < 0.001) and this concentration of Triton X-100 has previously
been shown to be not toxic to cells.
The optimal PBS washout time for removal of residual Triton X-100 is 4 hours. These findings ameliorate the
concerns about the potentially toxic effect of Triton X-100 leached out of decellularized allograft heart valves, thus
permitting the ongoing studies of immunogenicity of decellularized tissues in a rat model. This provides additional
information that will aid in finding the ideal cardiac valve replacement. Ongoing studies are needed to determine
the immunogenicity and physical properties of these decellularized tissues.
