OBJECTIVEThis study investigates construction of cardiac muscle cell-porous collagen scaffold complex in a bioreactor so as to unveil the possibility of generating 3-dimensional cardiac muscle tissue under the environment that mimics microgravity in vitro.

METHODS1-2-day old neonatal rat cardiac muscle cells were isolated by sequential digestion and pre-plating methods, then seeded onto porous collagen scaffold. The cell-collagen complex was transferred into rotary cell culture system (RCCS) and incubated for 7 days. Cells cultured in 75 ml flasks and constructs cultures on plates served as control. Morphological changes of the cells were observed by light microscope and metabolic rate was recorded. Ultrastructure of the cells growing in porous collagen was observed by transmission electron microscopy. Content of total DNA and protein in the newly-formed tissue were analyzed. H-E and anti-sarcomeric alpha-actin stains were performed in comparison with native cardiac muscle.

RESULTSThe isolated cardiac muscle cells adhered to the bottom of the flasks 24 hours after plating and began to beat spontaneously. When incubated for 7 days in RCCS, cell-collagen constructs of form a continuous outer tissue layer containing cells aligned with each other. The cell population in the interior of the construct was less in density than the outer part. Transmission electron microscopy demonstrated that subcellular elements characteristic of cardiac myocytes were in the outermost layer of constructs. A strongly positive stains of anti-sarcomeric alpha-actin suggested presence of cell population of differentiated cardiac myocytes in these constructs. Construct biomass was not significantly different from that in neonatal rat ventricle and approximately 40% of that in adult rat ventricles. Construsts in plates contained a few of cells which were less than those in RCCS. Metabolic activity of cells cultured in RCCS was higher than that in flasks and plates.

CONCLUSIONSDissociated cardiac muscle cells cultured on 3-dimensional scaffolds in RCCS under favorable conditions can form engineered constructs with structural and functional features resembling those of native cardiac tissue.