Jeffery Nielson, Brigham Young University
Engineering
Annually, 500,000 US inhabitants suffer from end-stage renal disease (ESRD). Allogeneic transplantation struggles with few donors and the high risk of organ rejection. Decellularized kidneys reseeded with autologous cells present a promising solution. Proposed decellularization methods require long times or high flow rates that may damage extracellular matrices’ (ECMs’) native architecture and lead to implantation thrombosis. We aim to optimize decellularization to preserve ECM integrity for subsequent recellularization and reimplantation.
We decellularized the kidneys through continual internal antegrade perfusion in a bioreactor with a series of “tonic cycles.” The brief cycles were alternated from hypertonic (with respect to cytoplasm concentrations) to hypotonic solutions, then to sodium dodecyl sulfate (SDS). The cycles repeated. Various combinations of flow rates, concentrations, temperatures, and detergents were compared. To determine the best method, we analyzed the decellularization rate and ECM structural integrity by testing the different decellularized kidneys via histology and Fluorescence based cell adhesion testing.
Continuously flowing the tonic cycle solutions antegrade in short, alternating intervals provided a faster, non-damaging decellularization process, when flow rates remained low. This method limited the kidneys to 4 hours of 0.5% SDS exposure, limiting SDS’s denaturing effects on ECM proteins. The total decellularization time decreased to a 10-hour average.
We discovered using osmotic stress, which aids in lysing cells and thereby minimizes SDS exposure (necessary for solubilizing lipids), as the best decellularization method to maintain ECM integrity. Variables important to decellularization included flow entry point, flow rate and pressure, surfactant and solution choices, thrombosis prevention during organ extraction, and surfactant saturation and age.
Decellularization functioned best with antegrade flow through kidneys’ vascular networks. A three-vessel bioreactor kept the solution fresh and the kidney properly submerged; it allowed alternation between tonic solutions. Our approach will optimally preserve kidneys’ architecture so appropriate cells can repopulate the kidneys before transplantation.