Isolation and Purification of Adult Mouse Cardiomyocytes by Langendorff Perfusion and Gravity Sedimentation.

Cardiovascular diseases remain a major global health challenge, creating a critical need for reliable in vitro models. This study presents an optimized protocol for the isolation and culture of adult mouse cardiomyocytes to address limitations of low viability and non-cardiomyocyte contamination in existing methods. The protocol is based on retrograde Langendorff perfusion, utilizing heparin sodium for anticoagulation and employing collagenase type II alone (1.2 mg/mL) for digestion. Cardiomyocytes were purified by gravity sedimentation and cultured on laminin-coated surfaces. This method consistently yielded 8.75 ± 0.36 × 105 cardiomyocytes per heart with a high proportion of rod-shaped cells (91.2% ± 2.11%), and a high purity (the percentage of cTNT⁺DAPI⁺ cells relative to DAPI⁺ cells was approximately 97.47% ± 1.365%), which exhibited clear sarcomeric structures. By providing a standardized and reproducible method for obtaining high-quality adult cardiomyocytes, this protocol effectively bridges the gap between in vitro studies and pathophysiological relevance, thereby supporting advanced cardiovascular research.