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Rodent models have been essential to understanding the immune‐mediated failure of aortic valve allografts (AVAs). Decellularization has been proposed to reduce the immunogenicity of AVAs. The objective of this study was to determine the most effective method to decellularize AVAs for use in a rat model. Three different decellularization techniques were compared in Lewis aortic valves. Detergent decellularization involved a series of hypotonic and hypertonic Tris buffers at 4°C for 48 h/buffer containing 0.5% Triton X‐100 followed by a 72 h washout in phosphate‐buffered saline. Osmotic decellularization was performed in similar manner to the detergent‐based technique except without the addition of Triton X‐100. Enzymatic decellularization consisted of trypsin/EDTA at 37°C for 48 h. Assessment was performed with light microscopy (H&E, Movat's pentachrome), immunohistochemistry for residual cellular …