Extended liver storage. Development and optimization of liver preservation and extracorporeal machine perfusion

This work focuses on the improvement of liver graft quality through the use of MP. It has been established that intracellular ATP is a significant measure of the cellular viability of an explanted organ. Chapter 1 examines the relationship between intracellular ATP and cellular viability at different time points of static cold storage. Both parameters were found to decline linearly as cold storage progresses. We went on to characterize the decline and recovery of ATP in a rat model of ex-vivo warm ischemia followed by subnormothermic machine perfusion and orthotopic liver transplantation (chapter 2). The use of MP at subnormothermic temperature (SNMP, 20oC) yielded improved graft survival, demonstrating feasibility of our rat model. Subsequently, a more clinically relevant porcine model was used (chapter 3), in which donation after cardiac death was followed by normothermic sanguineous machine perfusion, and reperfusion was simulated by NMP. Among other conclusions, this study confirms that ATP levels are a clinically relevant measure of viability. After demonstrating that our machine perfusion systems are capable of optimizing marginal organs, we wanted to examine whether machine perfusion could extend the duration of cold storage and whether MP is still effective after extended SCS. To this end, rat livers that had undergone various durations of cold storage were subjected to subnormothermic machine perfusion and then transplanted (chapter 4). Together, chapters 1, 2 and 4 establish a subnormothermic machine perfusion system tested under various clinically relevant circumstances, yielding several parameters that indicate graft viability during MP. We used this model as a tool to develop a novel strategy for liver preservation. In chapter 5, sub-zero non-freezing whole-organ preservation, or supercooling, was introduced, which can viably preserve rat livers at -6oC for extended periods of time. The previously established techniques of SNMP and liver transplantation enabled us to develop a rat model of supercooling preservation, which was verified with orthotopic transplantation yielding long-term survival. In chapter 6, we investigate the phenomenon of supercooling using molecular biological and cell culture techniques. Individual components of the protocol, as well as one of the agents used to facilitate supercooling were analyzed in an in vitro model of hepatocyte supercooling. In summary, in this thesis a new model of SNMP and rat liver transplantation is developed and applied to produce a novel liver preservation modality using supercooling preservation, which can significantly extend the current storage time for a rat liver.