Characteristics Of Mechanically-Conditioned, Substrate-Free Cardiac Cell Sheets

Heart failure remains a major cause of global morbidity and mortality. Since the benefits of heart transplantation are constrained by donor scarcity, and the ability of the heart to regenerate following infarction is limited, cell-based therapies have emerged as alternative treatments for the repair of damaged heart tissue. One method, myocardial cell sheet tissue engineering, detaches cultured cells from substrates without disrupting intercellular junctions critical for myocardial function. However, the challenges of generating mechanically strong and synchronously contractile cardiac sheets remain. In this study we characterized substrate-free cardiac cell sheets generated using a partial-lift method, whereby a portion of a cell monolayer is detached from the substrate. These cell sheets are dominated by cell-cell interactions and decoupled from cell-substrate interactions, but remain amenable to biological and chemical perturbations, and more importantly, mechanical conditioning and characterization. We show that lifted cardiac sheets exhibit significant changes in the distribution of cytoskeletal and junctional proteins, and that junctional expression of these proteins is enhanced by mechanical conditioning. Results further demonstrate that the mechanical strength and cohesion of these cell sheets depend on cytoskeletal and cell-cell junctional protein integrity. We also examined the microrheological characteristics, contraction frequency and calcium signaling of the cardiac cell sheets, which are critical for their potential clinical applications. Our results showed that mechanical conditioning the cell sheets alters several of these properties, with potential benefits to their function. This work represents a first systematic examination of mechanical conditioning on cardiac cells with primarily intercellular interactions. This method offers an unprecedented way to study cell junctions when substrate interactions are no longer dominant. The information gained from this study will help advance our understanding of cell-cell interactions and improve cardiac cell sheet biomechanical properties for tissue regeneration, and particularly heart repair.