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The objective of this study is to evaluate the closure of a transgastric natural orifice transluminal endoscopic surgery (NOTES) access using a multilayer extracellular matrix (ECM) occluder in a canine model.Mongrel dogs (n=4) underwent a transgastric NOTES peritoneoscopy and the gastrotomy was closed by deploying a 2-sided ECM occluder. Animals were killed at 7 days (n=2) and 8 weeks (n=2) for macroscopic and microscopic assessment.All procedures were completed without any complications. No air leaks were detected immediately after the procedure and at 48 hours postoperatively. At 7 days, ECM appeared to be resolved and mild mucosal inflammation was found at the site of gastrotomy. At 8 weeks, the gastrotomy site was macroscopically and microscopically covered with a normal-appearing gastric mucosa. There was an absence of inflammatory cells and no evidence of the ECM.The ECM occluder is safe and effective in this “proof-of-concept” preclinical model.