Attachment of Embryonic Stem Cells-derived Cardiomyocytes in CultiSpher-S Microcarriers by using Spinner Flask

Embryonic Stem (ES) cells have the ability to differentiate under in vitro conditions into cardiomyocytes. A transgenic α- myosin heavy chain (α-MHC+) ES cell line was generated, exhibiting puromycin resistance and expressing enhanced green fluorescent protein (EGFP) under control of the α-MHC+ promoter. A puromycin-resistant, EGFP-positive α-MHC+ cardiomyocyte population was isolated with over 92% purity. The cultivation of these cardiomyocytes, in macroporous gelatine microsphere beads in a spinner flask bioreactor has been studied. After we specified the most suitable agitationconditions and the optimal timeframes of cultivation, the average number of cultivated cells per microsphere was optimised. Our study shows that 80 % of microspheres were colonised by cardiomyocytes under optimal conditions. The results of the scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) showed that the population of the beads was not limited to the microcarrier surface, but also invaded the inner surfaces of the microspheres. The present findings demonstrate the successful culture of α-MHC+ cardiomyocytes in macroporous biodegradable microcarriers while maintaining the typical morphological and electrophysiological properties of cardiomyocytes. These findings suggest significant improvement in survival of grafted cardiomyocytes, thus helping overcome current limitations of cell replacement approaches