Progress of cardiac tissue engineering: primary and passaged culturing of cardiomyocytes from human adult ventricular myocardium in vitro

AIM:This study describes a method of isolating, culturing, purifying, subculturing, and identifying noncontracting ventricular myocytes grown from myocardium removed from valvular heart disease patients during corrective cardiac surgery. METHODS:Minced myocardium was digested in trypsin and collagenase. The isolated cells were cultured in Iscove's modified Dulbecco's medium.The cardiomyocytes were purified by differential attachment technique. Subculturing was done by 0.125 % trypsin and 0.01 % ethylenediamine tetraacetic acid (EDTA) digestion and transferring of the cells to new culture dishes containing the above culture medium. The myocardial sarcomeric actin and myoglobin were identified by immunofluorescence antibody staining. Myofibrils and mitochondria were visible by electron microscopy. RESULTS:The ratio of viable cells was 99 % identified by trypan staining. The ratio of attachment cells was 95 % after 24 h in culture. The cultured human adult heart cells were roundness- shaped, rod- shaped, shuttle- shaped, ellipse- shaped, star- shaped and bifurcate- shaped with non- contractility. The myocardial actin and myoglobin were identified by immunocytochemistry antibody staining and immunofluorescence antibody staining. The ultrastructure of cells was similar to that of the cardiac tissue in vivo by electron microscopy. Human adult heart cells after 30 d of primary culturing and after 15 d of passaged culturing were growed well. CONCLUSION:The method for isolating and culturing human adult heart cells is successful and reliable. The cultured cardiomyocytes promise to be useful in the study of normal and abnomal aspects of the cell and molecular biology of the heart.