A Simplified Langendorff-Based Method for Mouse Cardiac Myocyte Isolation

Isolation of adult mouse ventricular myocytes is essential for studying cardiac physiology and cellular function. Traditional methods commonly rely on Langendorff perfusion systems, which provide continuous retrograde coronary perfusion but require specialized equipment and can be complex to operate. Here, we describe a simplified Langendorff-based protocol that uses a syringe pump-driven system to achieve constant-flow retrograde aortic perfusion during enzymatic digestion. The setup incorporates an inline heater for precise temperature control and uses widely available laboratory components, enabling consistent delivery of digestion enzymes. This approach maintains stable perfusion despite changes in coronary resistance and reduces variability associated with conventional gravity-driven systems. The protocol yields high-quality adult ventricular myocytes suitable for downstream functional analyses, including electrophysiology, contractility, and calcium imaging. Compared with traditional systems, this method is more accessible, reduces technical complexity, and improves reproducibility, facilitating adoption in laboratories without dedicated isolated-heart perfusion infrastructure. Key features • Accessible cardiomyocyte isolation without dedicated Langendorff apparatus, suitable for laboratories with limited perfusion infrastructure. • Constant-flow perfusion overcomes enzyme delivery variability caused by changing coronary resistance during tissue digestion. • Inline heating enables rapid, precise temperature control without water-jacket systems, reducing setup complexity and contamination risk. • Optimized for producing calcium-tolerant adult ventricular myocytes for electrophysiology, contractility, and calcium imaging studies. Alternative methods may be more appropriate for non-cardiomyocyte populations.