ABSTRACT Nearly one-third of deceased donor livers are unused for transplant or other purposes. Many of these organs would be valuable for therapeutic and research applications if preservation times could be extended. Cryopreservation at ultralow temperatures (< -140°C) can enable indefinite organ storage. Previous attempts at organ cryopreservation have failed due to cellular and structural disruption caused by ice crystal formation. One promising approach that overcomes the limitations of conventional strategies is vitrification – that is, cooling organs so quickly that the water within the organ cannot undergo the phase transition from liquid to solid ice. With the help of cryoprotective agents (CPAs), the organ enters a stable glass-like state wherein the viable storage time is theoretically unlimited. The critical challenge, however, is rewarming without ice formation or cracking. If rewarming is too slow, ice crystals form; if rewarming is not uniform, thermal stress causes cracking. Hence, speed and uniformity of warming are essential. We have developed a novel rewarming approach termed “nanowarming” that achieves both objectives. Iron oxide nanoparticles are perfused throughout the vasculature of the organ along with CPA solutions. The organ can then be vitrified by cooling (an existing technology) and rewarmed as needed by placing it in a radiofrequency coil that induces heating in the nanoparticles and, therefore, from within the organ. In preliminary studies, for the first time we have shown that we can vitrify and nanowarm human sized (i.e. porcine) and rat livers, thereby avoiding ice formation or cracking and preserving viability and organ-level function. Following on this physical success, we propose here the first study to assess both transplantation and biological viability/function success of these nanowarmed organs. Our central hypothesis is that cryopreservation by vitrification and nanowarming will enable functional long-term whole human liver banking for transplant, therapeutic and biomedical research purposes. While our long-term goal is to develop a method for cryopreserving human livers for transplant, our goals for this project are to refine whole-liver preservation technology to 1) improve in vitro and in vivo functionality of preserved rat livers during normothermic perfusion and in transplant models, 2) determine the mechanisms of cellular stress, injury, and death resulting from liver cryopreservation and strategies for injury mitigation, and 3) provide further investigation of large animal (porcine) and human liver cryopreservation and rewarming. If successful, this approach could revolutionize how these precious resources are allocated and utilized for patient and societal benefit.

PROJECT NARRATIVE Nearly one-third of deceased donor livers ultimately go unused due to the inability to preserve them long enough. We aim to develop a novel technology for indefinitely preserving whole livers in an extremely low-temperature vitrified (glass-like) state and subsequently using nanotechnology to rewarm them on demand. If successful, this novel technology will enable the banking, or “off-the-shelf” availability, of livers for transplantation and other vital biomedical applications such as cell therapy, biomedical research, and drug discovery.