Background <p>The atrioventricular node (AVN) plays a critical role in coordinating the sequential activation and contraction of the heart’s chambers by transmitting electrical impulses from the sinoatrial node (SAN) to the ventricles via the His-Purkinje system. Besides its primary function, the AVN can generate intrinsic pacemaker activity when the SAN fails, and it serves as an important pharmacological target for controlling ventricular rate in the case of atrial arrhythmias. Despite its clinical significance, detailed electrophysiological studies of the AVN have been challenging due to difficulties in isolating viable AVN cells. The dense cellular network and complex structure of the AVN hinder enzymatic digestion, often leading to low yield and poor cell viability. Traditional isolation methods—such as adapting SAN cell protocols or employing the Langendorff perfusion technique—are limited by inadequate enzymatic penetration and preferential perfusion of the ventricular region, which significantly hampers the yield and viability of AVN cells for electrophysiological studies. Therefore, it is necessary to develop an optimized method to isolate high-quality AVN cells.</p> Methods <p>A refined method that does not rely on the Langendorff technique was used to isolate AVN cells from adult mice. Immunofluorescent imaging was used to confirm the presence of HCN4-positive cells. Patch clamp techniques were employed to record action potentials and ionic currents in AVN cells. Intracellular Ca²⁺ transients and sarcomere length measurements were obtained using the IonOptix system.</p> Results <p>We have developed an improved non-Langendorff perfusion technique that combines targeted enzymatic digestion with enhanced perfusion of the AVN region. By cannulating and ligating the aorta and packing the perfusion cannula tip with gauze, we achieved uniform enzyme distribution throughout the AVN area. This innovation results in a high yield of viable AVN cells that maintain their electrophysiological properties, making them suitable for advanced analyses such as patch-clamp recordings and calcium transient measurements.</p> Conclusions <p>Our method provides a strong platform for investigating the physiological and pathological roles of the AVN. This approach has the potential to aid in the development of novel therapeutic strategies for atrioventricular conduction disorders.</p>