BACKGROUND:The cardiomyocytes obtained by traditional culture methods exhibit poor purity and viability, cells grow in clusters, the connection between cells is less, and most of cells beat separately. OBJECTIVE: To obtain high-purity cardiomyocytes presenting with a sheet-like growth by modified culture method. METHODS: Myocardial tissues were digested using 0.1% trypsin at 4℃ overnight, and then digested repeatedly through the addition of type Ⅱ collagenase, and finally were isolated and purified by differential adhesion method combined with chemical inhibitors. The morphology of cardiomyocytes was observed under inverted phase contrast microscope. The purity of cardiomyocytes was identified by cardic troponin T antibody after cultured for 48 hours. The beating frequency of cardiomyocytes was recorded every two days for 10 consecutive days. RESULTS AND CONCLUSION:The cardiomyocytes adhered within 24 hours of culture, distributed in clusters at 72 hours and grew in sheets at 5-7 days of culture. The survival rate of cardiomyocytes was 96% and the purity identified by immunofluorescence was over 97%. There was no significant difference in the beating frequency at different time. These results suggest that the high-purity and high-viability cardiomyocytes are obtained by the modified culture method, which is an effective method for culturing neonatal cardiomyocytes.