MV decellularization

Porcine MVs were collected from a local abattoir and transported to the laboratory on ice. After cleaning and trimming extraneous heart muscle, the valves were immersed in ddH₂O overnight at 4°C to induce hypotonic shock and cell lysis. The next day, the valves were treated with NaOH 0.05 M for 2 h, then incubated for 5 days under agitation, in a solution containing 0.2% SDS, 1% Triton X-100, 1% deoxycholic acid, and 0.4% EDTA, prepared in 20 mM Tris pH 7.4. To remove the detergents, the valves were rinsed 10 times for 15 min with ddH₂O, then treated with 70% ethanol for 20 min to reduce the bio-burden, and rinsed again 4 times for 15 min with ddH₂O. Nucleic acid removal was completed by incubation in a 720 mU/mL deoxyribonuclease, 720 mU/mL ribonuclease mixture in PBS for 2 days at 37°C. Finally, scaffolds were rinsed in ddH₂O (three times for 15 min), and incubated in 70% ethanol overnight at room temperature. The MV scaffolds were stored in sterile PBS with 1% protease inhibitors and 1% antibiotic/antimycotic (Pen-Strep) at 4°C.