Abstract: AIM:To explore the appropriate culture conditions and method for separating myocardial cells and cardiac stem cells (CSCs) from Sprague Dawley neonatal rat hearts and to study the effect of Danshensu (DS182) on the proliferation of the two kinds of cells. METHODS: Different culture mediums, digestive enzyme concentrations, digestion times, differential adhesion times and inhibitors of fibroblasts were selected to optimize the separation and purification methods of rat myocardial cells and CSCs. MTT assay, ELISA, FCM and other methods were used to determine the effect of DS182 on cell proliferation of rat myocardial cells and CSCs. RESULTS: Separation and purification conditions of the two kinds of cells were DMEM/F12 medium 1∶1, 100 ml/L FBS, 25 g/L trypsin, digestion time of 5 min for five times and the differential time of 120 min. MTT assay results showed that compared with those in the blank group, cell A value increased significantly when cells were exposed to the DS182 concentration of 78×10-4-1×10-1 mol/L at 12, 24, 48, 72 h after administration (P<0.05). ELISA results showed that cell OD value increased significantly when cells were exposed to the DS182 concentration of 3.9×10-4-1×10-1 mol/L at 24 h after administration (P<0.05). FCM results showed that the number of ckit+ increased by 27.0% when cells were exposed to the DS182 concentration of 0.1 mol/L at 24 h after administration. CONCLUSION: We improved the separation and purification methods of myocardial cells and CSCs. DS182 at certain concentration (78 × 10-4-1×10-1 mol/L) obviously promotes cell proliferation of myocardial cells and CSCs.