Abstract
Background . Tissue engineering involves at a minimum cells and scaffold. However, reports of generating human-sized 3-dimensional scaffolds with appropriate architecture are limited. We hypothesize that perfusion decellularization of cadaveric pig solid organs will effectively remove cellular components and DNA, potentially providing a sufficient starting point for whole solid-organ tissue engineering. Methods . Adult Yorkshire swine (~60kg) were euthanized after systemic heparinization. Heart, liver, and kidneys were removed and mounted on a perfusion apparatus. Cannulation was performed using either the aorta (heart), portal vein (liver), or renal artery (kidney). Saline at 25C was perfused to remove blood, followed by Sodium Dodecyl Sulfate (SDS) detergent perfusion until tissues were translucent and white in color (heart=40 liters; liver=19 liters; kidney=31 liters). SDS was followed by 2 liters of 1% Triton-X 100 to sequester remaining detergent via micelle formation and clearance. Decellularization was evaluated by histology (H&E, DAPI, IHC). Tissue DNA content post-decellularization was quantified using a Hoechst-33258 DNA assay. Results . By histology, heart, liver, and kidney lacked intracellular components and DAPI, but retained major ECM proteins (fibronectin, collagen, and laminin). DNA in decellularized tissue was <2.2% of cadaveric controls by wet weight for all organs (heart=16.0 ± 2.9 vs. control=762.6 ± 48.5 ng/mg; liver=3.6 ± 0.7 vs. control=1157.6 ±91.7 ng/mg; kidney=25.2 ± 1.7 vs. control=1380.0 ± 201.2 ng/mg). Conclusion . SDS-based perfusion decellularization of large animal organs is effective at removing cellular content and DNA. The remaining whole-organ scaffold provides a platform technology for a novel construct for tissue engineering of human sized bioartificial organs.
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