Rapid and effective method to decellularize the human heart using a handmade sample-holder

Cells in vivo are surrounded by a specialized and intricate microenvironment mainly consisting of extracellular matrix (ECM). The ECM affects cell behavior and can support organ function and repair, with an appropriate architecture and a molecular composition unique for each tissue and specific for the role played by the resident cells. During the last decade, the increasing interest for decellularized ECM (d-ECM) as natural scaffold for cell delivery and tissue regeneration has boosted the search for the optimal protocol to decellularize the human myocardium. In this study we propose an innovative, reproducible and effective decellularization method to produce a well-preserved cardiac d-ECM. Cardiac samples harvested from the free wall of both ventricles of adult human hearts were scaled down to fit embedding cassettes and decellularized by immersion in a solution containing SDS, Triton X-100 and antibiotics under constant agitation. To avoid the random suspension within the beaker and the irregular and uncontrolled solution transport through the samples, a plexiglass sample-holder was specifically designed and hand-made to hold up to four cassettes in a steady position while equally exposing samples to decellularizing detergents. After decellularization, samples were fixed and processed for histological analyses or snap-frozen for molecular biology analyses. The effectiveness of the decellularization procedure was confirmed by the absence of nuclei at the Hematoxylin and Eosin staining and by the quantification of DNA content that resulted well below the accepted threshold of 50 ng/mg of dry tissue. Masson’s and Mallory’s trichrome stainings revealed the preservation of the three-dimensional architecture of cardiac d-ECM, while Van Gieson, Alcian Blue, Periodic Acid Schiff and Sirius Red stainings documented the retention of elastic fibers, glycosaminoglycans (GAGs), non-collagenous proteins and collagen, respectively. Further, immunohistochemistry revealed in the cardiac d-ECM the retention of proteins typical of the native cardiac ECM, like fibronectin, tenascin and laminin, while quantitative dye binding assay and specific protein array documented the retention of elastin and growth factors, respectively. These results prove that our method, combined with a hand-made sample-holder specifically designed for decellularization procedures under constant agitation, yields a cardiac d-ECM well preserved in its composition and architecture.