Background

The isolation of intact adult cardiac myocytes is a foundational technique in cardiac research, enabling direct investigation of cellular structure and function under controlled experimental conditions. The isolated heart preparation described by Oscar Langendorff in 1897 [1], and methodologically refined by others [2,3], forms the basis of the modern “Langendorff” method [4]. In this preparation, the excised heart is rapidly cannulated via the ascending aorta and perfused ex vivo while suspended in a temperature-controlled chamber. Retrograde perfusion through the aorta allows controlled delivery of physiological solutions through the intact coronary vasculature while preserving myocardial architecture [1]. Its further adaptation in the 1970s for enzymatic dissociation established the Langendorff method as the standard approach for isolating viable cardiac myocytes [5,6], a status it has maintained for more than five decades [7–9]. Langendorff perfusion enables enzymatic delivery through the coronary circulation while minimizing mechanical trauma to the myocardium by retrograde aortic perfusion. In this configuration, flow delivered through the cannulated proximal aorta fills the aortic root, generating pressure that causes coaptation of the aortic cusps and closure of the valve. Aortic valve closure prevents retrograde ventricular filling and directs perfusate into the coronary circulation via the coronary ostia at the base of the root [4]. This technique is particularly well-suited to rodents and other small-animal models, in which the aorta is sufficiently large for reliable cannulation yet small enough to allow economical use of dissociation enzymes [10]. For larger hearts, perfusing a coronary branch and excising the region is more practical than perfusing the whole heart [10].

Langendorff systems operate under either constant-pressure or constant-flow perfusion regimes [8]. Constant-pressure setups are typically gravity-fed from a reservoir of perfusate and exhibit variable coronary flow as vascular resistance changes during digestion. As enzymatic dissociation reduces resistance, coronary flow increases, providing a useful indicator of digestion-progression that is commonly monitored by changes in drip rate [10,12–15]. Because coronary vascular resistance dynamically influences the pressure–flow relationship [16], variations in flow under constant-pressure perfusion may result in variable delivery of enzyme, particularly as resistance drops during digestion [10,15]. Variability in extracellular matrix composition and fibrosis degree across strain, age, and pathological conditions ostensibly will also influence myocardial resistance and thereby perfusion flow dynamics [15].

In contrast, constant-flow systems employ a fixed volumetric perfusion rate while perfusion pressure varies in response to downstream resistance. Careful selection of the flow rate is important: if set too low, inadequate coronary perfusion may occur; if excessive, supraphysiological perfusion pressures can invert the aortic valve cusps and divert perfusate into the ventricular cavity rather than the coronary circulation. Many traditional constant-flow Langendorff setups rely on peristaltic pumps as well as dedicated perfusion rigs incorporating water-jacketed glassware, bubble traps, recirculation loops, and continuous carbogen or oxygen gassing of bicarbonate-buffered solutions [5,6,9,12]. The complexity of specialized apparatus, combined with the technical expertise required for rapid aortic cannulation, has been criticized as limiting accessibility. In response, simplified “Langendorff-free” methods have been developed [17–20].

Langendorff-free methods, sometimes referred to as “injection methods,” are based on direct injection of perfusate into the ventricular lumen [18–21]. In these approaches, the aorta is clamped, and buffer is forced into the coronary circulation in an antegrade manner, flowing through the aortic valve into the coronary ostia. Although injection-based methods are touted as requiring less technical skill than aortic cannulation, they nonetheless depend on accurate clamp placement and precise ventricular injections into the right ventricular lumen, followed by multiple injections into the left ventricular lumen. These procedures demand operator precision and introduce distinct risks. Repeated ventricular punctures, particularly in the small and thin-walled mouse heart, introduce the risk of rupture or buffer leakage. Preservation of aortic integrity is also critical, as damage can compromise effective coronary perfusion. These risks may be exacerbated in structurally fragile or diseased hearts or those exhibiting pathological remodeling.

Moreover, injection methods rely on non-physiological alkaline buffers (pH ~7.8) for optimal cell yield [17–19], the basis for which is speculative rather than mechanistically established [18]. Cardiomyocyte function is sensitive to pH, and extracellular alkalinization directly influences intracellular pH via established acid–base transport mechanisms, including Na⁺/H⁺ exchange and lactate/H⁺ transport [22,23]. Because intracellular pH must be tightly maintained within a narrow physiological range (~7.15–7.25) to preserve excitation–contraction coupling and calcium homeostasis, even modest deviations can alter Ca²⁺ handling and myofilament sensitivity [24–27]. Furthermore, extracellular pH fluctuations regulate autophagy, a process central to cellular viability and metabolic control [28]. Exposure to alkaline conditions during isolation may influence post-isolation cellular physiology and subsequent functional readouts. For studies requiring transcriptomic or functional characterization of the full spectrum of cardiac cell types, including fibroblasts, endothelial cells, and immune cells, non-perfusion enzymatic digestion approaches have been developed that enable isolation of viable cells across all major cardiac populations, as demonstrated by single-cell sequencing studies of both healthy and diseased myocardium [29,30].

Taken together, these considerations highlight the need for a method that preserves the physiological fidelity and controlled delivery afforded by constant-flow coronary perfusion while eliminating the specialized equipment and technical expertise required by traditional Langendorff systems. The protocol described here addresses this need through a simplified, syringe pump–driven Langendorff approach. It retains the critical advantages of aortic cannulation and continuous retrograde perfusion while implementing them within a minimal and accessible apparatus optimized for the isolation of adult mouse ventricular myocytes.