An improved two-step method for extraction and purification of primary cardiomyocytes from neonatal mice

Primary mouse cardiomyocytes are essential tools for cardiovascular pharmacology research at the cellular and molecular levels, but their low viability and low purity have often caused challenges in previous studies. Hence, we developed an improved two-step method for extraction and purification of primary cardiomyocytes from neonatal mice. This method consisted of two steps: 1) isolation and pre-digestion of heart tissues from 1- to 3-day-old C57 neonatal mice and 2) extraction and purification of cardiomyocytes. The traditional method of primary mouse cardiomyocyte isolation was used as the control group to assess the extraction efficiency of cardiomyocytes by the two-step method, and the purity and viability of cardiomyocytes were evaluated by immunofluorescence staining and autonomous beating analysis, respectively. Compared with the control method, the two-step method enabled acquisition of more cells from mouse hearts (1.28 ± 0.11 × 106 vs 0.59 ± 0.15 × 106 cells/heart), and the resulting cells exhibited higher adherence rates and cell purity (93.25 ± 1.69% vs 73.62 ± 9.76%) after 48 h of culture. Moreover, the viability of cardiomyocytes was also evidently higher in the two-step group than in the control group (124.67 ± 10.50 vs 88.50 ± 6.61 beats/min). Compared with the traditional method, the two-step method exhibited significantly better efficiency in extraction of primary cardiomyocytes and yielded cells with greater purity and viability. The two-step method will likely become a standard method for studies based on primary mouse cardiomyocytes in the future.