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Abstract Preserving organs at subzero temperatures with halted metabolic activity holds the potential to prolong preservation and expand the donor organ pool for transplant. Our group recently introduced partial freezing, a novel approach in high-subzero storage at -15 °C, enabling 5-day storage of rodent livers through precise control over ice nucleation and unfrozen fraction. However, increased vascular resistance and tissue edema suggested a need for improvements to extend viable preservation. Here, we describe an optimized partial freezing protocol with key optimizations, including an increased concentration of polyethylene glycol (PEG) to enhance membrane stability while minimizing shear stress during cryoprotectant unloading with an acclimation period and a maintained osmotic balance through an increase in bovine serum albumin (BSA). These approaches ensured the viability during preservation and recovery processes, promoting liver function and ensuring optimal preservation. This was evidenced by increased oxygen consumption, decreased vascular resistance, and edema. Ultimately, we show that using the optimized protocol, livers can be stored for 10 days with comparable vascular resistance and lactate levels to 5 days, outperforming the viability of time-matched static cold stored (SCS) livers as the current gold standard. This study represents a significant advancement in expanding organ availability through prolonged preservation, thereby revolutionizing transplant medicine.

Background: Liver donation after cardiac death (DCD) makes up a small percentage of the organs used in transplantation and poses a higher risk of graft loss compared to donation after brain death (DBD); this is a result of ischemia reperfusion for which the exact injury mechanisms are currently not fully understood. However, reperfusion injury has been shown to lead to necrosis as well as apoptosis through oxidative stress and mitochondrial dysfunction. In this work, we propose that use of the pro-survival, anti-apoptotic CEPT cocktail in post-ischemia normothermic machine perfusion (NMP) may improve recovery in rat livers subjected to extended durations of warm ischemia. Materials and Methods: Livers procured from male Lewis rats were subjected to 90 min of warm ischemia, followed by 6 h of NMP where they were treated either with the survival-enhancing anti-apoptotic cocktail (CEPT), the vehicle (DMSO) or the base media with no additives. Results: The CEPT-treated group exhibited lower expression of hepatic injury biomarkers, and improvement in a range of hepatocellular symptoms associated with the hepatic parenchyma, biliary epithelium and the sinusoidal endothelium, including recovery of bile secretion and lowered vascular resistance. Conclusions: This study's findings suggest apoptosis plays a more significant role in ischemia-reperfusion injury than previously understood, and provide useful insight for further investigation of the specific underlying mechanisms and development of novel treatment methods.

Ex vivo preservation of transplanted organs is undergoing spectacular advances. Machine perfusion is now used in common practice for abdominal and thoracic organ transportation and preservation, and early results are in favor of substantially improved outcomes. It is based on decreasing ischemia-reperfusion phenomena by providing physiological or sub-physiological conditions until transplantation. Alternatively, supercooling techniques involving static preservation at negative temperatures while avoiding ice formation have shown encouraging results in solid organs. Here, the rationale is to decrease the organ's metabolism and need for oxygen and nutrients, allowing for extended preservation durations. The aim of this work is to review all advances of supercooling in transplantation, browsing the literature for each organ. A specific objective was also to study the initial evidence, the prospects, and potential applications of supercooling preservation in Vascularized Composite Allotransplantation (VCA). This complex entity needs a substantial effort to improve long-term outcomes, marked by chronic rejection. Improving preservation techniques is critical to ensure the favorable evolution of VCAs, and supercooling techniques could greatly participate in these advances.