Hydrogel Assisted Double Molding of 3D-Print Enables Prestress Regulation of Micro-Heart Muscle Physiology

Abstract: Tissue engineered in-vitro models are an essential tool in biomedical research. Tissue geometry is a key determinant of function, but controlling geometry of micro-scale tissues remains a challenge. We developed a new double molding approach that allows precise replication of high-resolution stereolithographic prints into poly(dimethylsiloxane), facilitating rapid design iterations and highly parallelized sample production. Hydrogels are used as an intermediary mold, and gel mechanical properties including crosslink density predict replication fidelity. We leveraged this approach to study the effects of geometry on the electrophysiology of miniaturized heart muscles engineered from human induced pluripotent stem cells (iPSC). Geometries predicted to increase tissue prestress globally affected cardiomyocyte structure and tissue electrophysiology. Strikingly, pharmacologic studies revealed a prestress threshold is required for sodium channel function. Analysis of RNA and protein levels suggest electrophysiology changes were related to post-transcriptional and potentially post-translational changes of the gap-junction protein Connexin 43 and the sodium channel Nav1.5.