Harris, Amy G.Schot, VicoCarrabba, MicheleIacobazzi, DomingaGhorbel, Mohamed T.Armstrong, James P.K.Perriman, Adam W.Caputo, MassimoBiglino, GiovanniBartoli-Leonard, Francesca2026-03-282026-03-28PubMed:38367231ORCID:/0000-0003-2205-9364/work/209746241https://hdl.handle.net/1885/733808010Surgical treatment of pediatric congenital heart disease with tissue grafts is a lifesaving intervention. Decellularization to reduce immunogenicity of tissue grafts is an increasingly popular alternative to glutaraldehyde fixation. Here, we present a protocol to decellularize porcine right ventricular outflow tracts using a 3D printed flow chamber. We describe steps for 3D printing the flow rig, preparing porcine tissue, and using the flow rig to utilize shear forces for decellularization. We then detail procedures for characterizing the acellular scaffold. For complete details on the use and execution of this protocol, please refer to Vafaee et al.: https://doi.org/10.1002/term.2391This work was supported by the British Heart Foundation (AA/18/1/34219, CH/17/1/32804, and TA/F/21/210028) to M. Caputoand the British Heart Foundation PhD studentship (FS/4yPhD/F/20/34125) to A.G.H. J.P.K.A. acknowledges funding from a UKRI Future Leaders Fellowship (MR/V024965/1). Figures were created with BioRender.com (agreement number MB2625E5U3). We are grateful to all staff members of the University of Bristol Large Animal Surgical Facility and those at the University of Bristol Wolfson Bioimaging Facility. A.G.H. performed all experiments and analyses and drafted the manuscript. V.S. oversaw the 3D printing aspects and critically reviewed the manuscript. M. Carrabba, D.I. and J.P.K.A. provided guidance on the study and critically reviewed the manuscript. M.T.G. aided in the collection of porcine tissue and critically reviewed the manuscript. A.W.P. M. Caputo, and F.B.-L. supervised the study, aided in study design, and critically reviewed the manuscript. G.B. supervised the study, aided in study design, oversaw the 3D printing aspects, and critically reviewed the manuscript. All authors discussed the data and agreed to the final version of the manuscript. J.P.K.A. is a co-founder and shareholder of Impulsonics Ltd. a company seeking to automate cell culture processes. This work was supported by the British Heart Foundation ( AA/18/1/34219 , CH/17/1/32804 , and TA/F/21/210028 ) to M. Caputoand the British Heart Foundation PhD studentship ( FS/4yPhD/F/20/34125 ) to A.G.H. J.P.K.A. acknowledges funding from a UKRI Future Leaders Fellowship ( MR/V024965/1 ). Figures were created with BioRender.com (agreement number MB2625E5U3). We are grateful to all staff members of the University of Bristol Large Animal Surgical Facility and those at the University of Bristol Wolfson Bioimaging Facility.36en© 2024 The Author(s)ImmunologyTissue Engineering2024-02-1610.1016/j.xpro.2024.10289985185403419