Formation of three-dimensional fetal myocardial tissue cultures from rat for long-term cultivation

Three-dimensional cardiomyocyte cultures offer new possibilities for the analysis of cardiac cell differentiation, spatial cellular arrangement, and time-specific gene expression in a tissue-like environment. We present a new method for generating homogenous and robust cardiomyocyte tissue cultures with good long-term viability. Ventricular heart cells prepared from fetal rats at embryonic day 13 were cultured in a scaffold-free two-step process. To optimize the cell culture model, several digestion protocols and culture conditions were tested. After digestion of fetal cardiac ventricles, the resultant cell suspension of isolated cardiocytes was shaken to initialize cell aggregate formation. In the second step, these three-dimensional cell aggregates were transferred onto a microporous membrane to allow further microstructure formation. Autonomously beating cultures possessed more than 25 cell layers and a homogenous distribution of cardiomyocytes without central necrosis after 8 weeks in vitro. The cardiomyocytes showed contractile elements, desmosomes, and gap junctions analyzed by immunohistochemistry and electron microscopy. The beat frequency could be modulated by adrenergic agonist and antagonist. Adenoviral green fluorescent protein transfer into cardiomyocytes was possible and highly effective. This three-dimensional tissue model proved to be useful for studying cell-cell interactions and cell differentiation processes in a three-dimensional cell arrangement.