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Decellularized cardiac extracellular matrix (ECM) has been introduced as a template for cardiac tissue engineering, providing the advantages of a prevascularized scaffold that mimics native micro- and macroarchitecture to a degree difficult to achieve with synthetic materials. Nonetheless, the decellularization protocols used to create acellular myocardial scaffolds vary widely throughout the literature. In this study we performed a direct comparison of three previously described protocols while introducing and evaluating a novel, specifically developed fourth protocol, by decellularizing whole rat hearts through software-controlled automatic coronary perfusion. Although all protocols preserved the macroarchitecture of the hearts and all resulting scaffolds could successfully be reseeded with C2C12 myoblasts, assessing their biocompatibility for three-dimensional in vitro studies, we found striking differences concerning …