Sustained Myocardial Viability Following a 32-Hour Ex Vivo Preservation: The Impact of Intermittent Cold Storage and 24-Hour Normothermic Perfusion

Introduction: Normothermic ex vivo heart perfusion (NEHP) shows promise for extended organ preservation, yet clinical translation requires hearts to tolerate sequential cold ischemic periods inherent to hub-and-spoke logistics. We present a cold-storage NEHP protocol culminating in orthotopic transplantation to validate the complete preservation-to-transplant pathway.
Methods: Juvenile Yorkshire pigs served as heart donors (n=3). Hearts underwent a 4-hour initial cold storage (C1) in HTK (4–10°C), then reanimated on NEHP in Langendorff mode (Fig. 1A) with continuous plasma exchange and 22-kDa hemofiltration. The experimental protocol is outlined in Fig. 1B. Assessments included hemodynamics, echocardiography, and video-based kinematic analysis (contractility, biaxial strain) at T1, T12, and T24 hours in left-atrial working mode. After 24-hour NEHP, hearts underwent a second 4-hour cold storage (C2) period followed by reperfusion (RF). Two hearts (CS1 and CS2) were reperfused via ex-vivo perfusion and one heart (TXP) was reperfused via orthotopic heart transplantation and observed for 4 hours.
Results: All hearts completed 24 hours of NEHP with stable MAP (30–42 mmHg), coronary resistance (0.1–0.3 mmHg/mL/min), and oxygen consumption (Fig. 2i, A–D). CS1 and CS2 maintained stable normalized contractility throughout perfusion (T1→T24: +0.4% and +74.5%, respectively; Fig. 2ii, B). At reperfusion following two cold preservation blocks, CS1 demonstrated robust reanimation with a 56.7% increase in contractility and 24.2% improvement in circumferential strain (Fig. 2ii, D), with echocardiography improving from severely depressed to mildly depressed LV function. CS2 maintained strong contractility at RF (+66.0% from T1) with preserved compliance (Fig. 2ii, A). Left-atrial working mode confirmed adequate pressures in both subjects at reperfusion (Fig. 2i, E). In the transplant arm, the heart completed the full preservation sequence (5h17m C1, 24h NEHP, 4h19m C2; warm ischemia 64 min) and was implanted orthotopically. The graft was weaned from CPB within 100 minutes. Post-bypass hemodynamics were sustained for over 2 hours with minimal pressor requirement at 90 minutes: mean arterial pressure (MAP) 45–73 mmHg, and PA mean 20–30 mmHg (Fig. 2i, E). Post-transplant kinematics confirmed robust functional recovery, with contractility increasing 40.4% and longitudinal and circumferential strain improving 112% and 76% from T24, respectively (Fig. 2ii).
Conclusions: Porcine hearts remained viable through a complete 32-hour preservation sequence incorporating dual cold ischemic insults and 24-hour NEHP, with successful orthotopic transplantation confirming post-transplant functional recovery. This work provides critical preclinical evidence supporting extended NEHP with integrated cold transport as a pathway toward regional organ rehabilitation centers and expanded donor heart utilization.