To bridge the gap between 2D cell culture and tissue, various 3D cell culture approaches have been developed for the investigation of cardiac myocytes (CM) and fibroblasts (CF), but limitations still exist. The goal of this study was to develop a scaffold-free cardiac culture that could easily and inexpensively create many uniform cardiac microtissues that mimic the cellular distribution and functional behavior of CM and CF in tissue. Using micromolded non-adhesive agarose hydrogels containing 822 concave recesses (800 λm deep x 400 λm wide), we demonstrate that neonatal rat ventricular CM and CF, when seeded alone or in combination, self-assembled into viable (Live/Dead® stain) spherical-shaped microtissues. Importantly, when CM and CF were seeded simultaneously or sequentially, they self-sorted to be highly interspersed, reminiscent of their distribution in ventricular tissue, as shown by cell type-specific CellTracker
TM
or antibody labeling. Depending on their cellular composition, microtissues expressed extracellular matrix and calcium handling proteins (including SERCA2a), featured typical
I
K1
and
I
Ca-L
current densities and IV relationships, and formed functional cell-cell connections (as evidenced by spontaneous action potentials, contractions and connexin 43 expression). Microelectrode recordings and optical mapping showed characteristic triangular action potentials (AP) with a resting membrane potential of -66±7 mV (n=4) in spontaneously contracting CM microtissues. Under pacing, optically mapped AP duration at 90% repolarization and conduction velocity were 100±30 ms and 18.0±1.9 cm/s, respectively (n=5 each). The presence of CF led to a prolongation of both AP and calcium transients in CM:CF microtissues (1:1). Furthermore, cell-type-specific adenoviral gene transfer was achieved, with no impact on microtissue formation or cell viability. In conclusion, we have developed a novel scaffold-free cardiac 3D culture model, in which more than 800 homotypic (CM or CF) or heterotypic (CM:CF) microtissues can be formed in a 6-well with ease, analyzed functionally and subjected to cell-type-specific gene transfer, paving the way for future
in vitro
investigations of CM and CF behavior and their interactions in a 3D environment.