ObjectiveTo establish a primary culture method for rapid and stable extraction of neonatal mouse cardiomyocytes based on Percoll discontinuous density gradient method in separating and purifying cardiomyocytes. MethodsThe ventricle of C57BL/6 suckling mouse was taken in sterile condition and digested with low concentration collagenase II. The cardiomyocytes were separated and purified using Percoll density gradient method by adjusting the temperature, digestion method, centrifugation time and rotation speed. The morphology of cardiomyocytes at different times was observed; with 0.4% trypan blue staining, the cell survival rate and cell numbers were tested; meanwhile, immunofluorescence staining was used to detect the expression of cardiac troponin I (cTnI), and to identify cardiomyocytes and their purity. ResultsThe five-time culture results showed that the average survival rate of cardiomyocytes was (93.12 ± 0.75)%, and the purity was (96.36 ± 0.60)%. The cells grew well, and could form adhesion and spontaneous beating. ConclusionThe method can obtain the primary cardiomyocytes of neonatal mouse with high yield, survival rate and purity, with a short time-consuming, and thus it is an ideal method to separate and purify the primary cardiomyocytes, and can meet the requirements of subsequent.

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