Culturing of ventricle cells at high density and construction of engineered cardiac cell sheets without scaffold

In natural heart tissue, cell density is about 1.0 x 108/cm3, and the cell metabolism is very active. Therefore, culturing heart cells in 3-dimensions at high density and construction of engineered cardiac tissue in vitro is very difficult. The aim of this study was to simulate 3-dimensional culturing of cardiac cells and pursue a novel method to construct engineered cardiac tissue in vitro. The isolated neonatal rat ventricle cells were cultured at a high seeding density of 1 x 10(6)/cm2. The cells at high density metabolized actively; the glucose consumption and lactic acid production of ventricle cells were much greater than those of fibroblasts cultured at the same density. The pH value of the culture medium of ventricle cells consistently decreased more rapidly. These cultured ventricle cells contained vascular endothelial cells, cardiomyocytes, and smooth muscle cells that appeared close to each other, and had overlapping nuclei and plenty of extracellular collagen. The cells at high density were treated with 0.2% trypsin to construct engineered cardiac cell sheets without scaffold. The engineered cardiac cell sheets could beat and roll up spontaneously, each sheet was 3 to 5 cells thick, and contained abundant cardiomyocytes and extracellular collagen. In conclusion, cells cultured at high-density in vitro grew well in a 2-dimensional culturing environment, formed "quasi 3-dimension" culturing, and engineered cardiac cell sheets comprised of several layers of cells were constructed. This study provides some guidance for cardiac tissue engineering and a novel method to construct engineered cardiac tissue without scaffold.