The mammalian heart does not regenerate in vivo. The heart is, therefore, an excellent candidate for tissue engineering approaches and for the use of biosynthetic devices in the replacement or augmentation of defective tissue. Unfortunately, little is known about the capacity of isolated heart cells to re-establish tissue architectures in vitro. In this study, we examined the possibility that cardiac cells possess a latent organizational potential that is unrealized within the mechanically active tissue but that can be accessed in quiescent environments in culture. In the series of experiments presented here, total cell populations were isolated from neonatal rat ventricles and recombined in rotating bioreactors containing a serum-free medium and surfaces for cell attachment. The extent to which tissue-like structure and contractile function were established was assessed using a combination of morphological, physiological, and biochemical techniques. We found that mixed populations of ventricular cells formed extensive three-dimensional aggregates that were spontaneously and rhythmically contractile and that large aggregates of structurally-organized cells contracted in unison. The cells were differentially distributed in these aggregates and formed architectures that were indistinguishable from those of intact tissue. These architectures arose in the absence of three-dimensional cues from the matrix, and the formation of organotypic structures was apparently driven by the cells themselves. Our observations suggest that cardiac cells possess an innate capacity to re-establish complex, three-dimensional, cardiac organization in vitro. Understanding the basis of this capacity, and harnessing the organizational potential of heart cells, will be critical in the development of tissue homologues for use in basic research and in the engineering of biosynthetic implants for the treatment of cardiac disease.

No abstract

JSUMMARYIn vitro characteristics of cardiac cells cultured in simulated microgravity are reported.o Tissue culture methods performed at unit gravity constrain cells to propagate, differentiate, and interact in a two dimensional (2D) plane. Neonatal rat cardiac cells in 2D culture organize predominantly as bundles of cardiomyocytes with the intervening areas filled by non-myocyte cell types. Such cardiac cell cultures respond predictably to the addition of exogenous compounds, and in many ways they represent an excellent in vitro model system. The gravity-induced 2D organization of the cells, however, does not accurately reflect the distribution of cells in the intact tissue. We have begun characterizations of a three-dimensional (3D) culturing system designed to mimic microgravity. The NASA designed High-Aspect-Ratio-Vessel (HARV) bioreactors provide a low shear environment which allows cells to be cultured in static suspension. HARV-3D cultures were prepared on microcarrier beads and compared to control-2D cultures using a combination of microscopic and biochemical techniques. Both systems were uniformly inoculated and medium exchanged at standard intervals. Cells in control cultures adhered to the polystyrene surface of the tissue culture dishes and exhibited typical 2D organization. Cells in cultured in HARVs adhered to microcarrier beads, the beads aggregated into defined clusters containing 8 to 15 beads per cluster, and the clusters exhibited distinct 3D layers: myocytes and fibroblasts appeared attached to the surfaces of beads and were overlaid by an outer cell type. In addition, cultures prepared in HARVs using alternative support matrices also displayed morphological formations not seen in control cultures. Generally, the cells prepared in HARV and control cultures were similar, however, the dramatic alterations in 3D organization recommend the HARV as an ideal vessel for the generation of tissue-like organizations of cardiac cells in simulated microgravity.

No abstract