ABSTRACT. The technical feasibility of VCA transplantation was demonstrated 20 years ago and the initial clinical outcomes have been encouraging. However, the ethical calculus of lifelong immunosuppression does often preclude VCA transplantation. To avoid the side effects of long-term immunosuppression, the most promising approach is the induction of tolerance by mixed chimerism via bone-marrow co-transplantation. This is the only strategy that has shown efficacy in large animal and clinical studies of solid organ transplantation. However, current tolerance protocols in clinical trials require somewhere between 3 to 7 days to pre-condition the recipient, measured from initial donor graft recovery to recipient transplantation. Sidestepping the need for preconditioning to induce mixed chimerism does not appear easy to achieve and failed in a clinical VCA transplant case when attempted by one team. Contrary to kidneys, in VCA there is no possibility of living donation, making advance recipient conditioning clinically unrealistic. Therefore, a technology to extend graft preservation to bridge the time for recipient conditioning remains a critical necessity that would enable tolerance induction in VCA transplants. Our overall goal is to develop a high subzero preservation protocol that will prolong the viability of vascular tissues during preservation prior to transplantation, thereby creating a time window to implement tolerance induction protocols. The overarching hypothesis of this study is that alleviating osmotic shocks during cryoprotective agent (CPA) loading, reducing shear injury and toxicity to endothelial cells during loading and unloading CPAs, and bypassing uncontrolled ischemia during rewarming from subzero temperatures by nanowarming, will enable superior and extended preservation of limb allografts. This hypothesis has been formulated based on our prior data demonstrating success in supercooling of human livers showing proof of scalability; however initial attempts to directly adopt the same protocol for rat and pig limbs led to excessive edema and identification of extreme sensitivity to ischemic injury and vascular resistance compared to livers. The multi-institutional partnership assembled for this work brings together the unique expertise of two institutions: 1) subzero organ storage at the MGH Center for Engineering in Medicine & Surgery, and 2) the University of Minnesota Institute for Engineering in Medicine’s unique technology of nanowarming. Our expected contribution here is the development of a new preservation protocol for rat vascularized composite tissues that extends total storage duration from hours to days, and provides time to prepare the recipient to induce tolerance. This contribution is significant because the methods developed here could be the proof-of-concept for a protocol that is clinically practical for tolerance induction in VCA transplants, and could therefore enable their wide-spread use which is not poss...