PROJECT SUMMARY In spite of significant effort to expand the pool of organ donors, there remains a severe gap between the supply of organs and the number patients in need of a living saving transplant. The persistence and sheer magnitude of this issue suggests the need for a disruptive new approach that can fundamentally change the paradigm of organ donation. We propose to achieve this paradigm shift by developing new organ perfusion technology that can transform donor eligibility criteria to allow for donation after death in an uncontrolled setting outside of the hospital. At current, only 0.7% of organ donors that die in a given year will have their organs actually recovered. A major contributing factor to this low percentage is the requirement that donors must die in a controlled hospital setting to facilitate rapid organ recovery that ensures <1 hr of warm ischemia. If this tolerable warm ischemic time could be expanded to ~3 hrs or more, this would make it logistically feasible to allow for donation after death in uncontrolled settings such as cardiac arrest on the way to the hospital. This could dramatically increase the number of donor organs available, particularly following sudden cardiac arrest. We have recently shown in two independent studies of human kidney and pig brain, that ex vivo organ perfusion with the appropriate therapeutic perfusate can access a previously unappreciated resilience to ischemic injury. Based on this, we hypothesize that an appropriately designed perfusion system will be capable of restoring stable renal function ex vivo in kidneys after at least ~3 hrs of warm ischemia. To this end, we have developed a novel perfusion approach which combines an acellular cytoprotective perfusate paired with a custom perfusion system capable of continuous physiologic monitoring and real-time integrated organ support. Instead of red blood cells, our perfusate uses polymerized hemoglobin to enable oxygen delivery under a range of perfusion temperatures and avoid the complications of hemolysis. In this proposal, we will refine and test the capacity of our novel approach to mitigate two critical modes of failure following 3 hrs of warm ischemia: 1) Severe ATP depletion with related disruption of the electron transport chain (ETC); and 2) Dysfunctional inflammation induced by regulated necrosis after reperfusion. Successful completion of this project will establish new technology with the potential to transform our definition of what constitutes an eligible organ donor.