DESCRIPTION (provided by applicant): This program targets the long-term preservation of vascularized composite tissues for both autologous- and allotransplantation (VCA), such as of e.g. muscle flaps, digits, faces and whole limbs, to radically improve restorative and reconstructive medicine. We have conceived an integrated two-pronged approach in which we will develop new nature inspired and further augmented stasis cocktails optimized for the critical phases of VCA preservation - preconditioning and protection (prior to storage), preservation (during storage) and revival- resuscitation-repair (after storage) - as wells as new devices purposefully designed for perfusion, storage and, evaluation and quality control before transplant decision. Our approach is based on using the best strategies employed by freeze-tolerant and hibernating animals in nature and then augmenting them with complementary strategies developed through recent scientific understanding, bioengineering principles and modern tools. Thus our preservation approach is unique and distinct from other approaches since we seek to accomplish storage times of several weeks to months using an unexplored, yet highly promising methodology. Importantly, our approach will not seek to solve all the problems needed for classical cryopreservation approaches, but rather be the first to develop preservation in a equilibrium state using high subzero storage temperatures (ranging from -5 to -20 °C) combined with programmed metabolic depression and enhanced stress tolerance pathways. These are temperatures and strategies used in nature by many species able to survive months in a state of `suspended animation,' with the whole animal, including every single organ being "banked" without injury. The technical objective of this Phase 1 proposal is to develop a non-toxic, multi-component, next generation cryostasis cocktail and protocol which in Phase 2 will be used for whole mammalian limb preservation with re- transplantation. We seek to develop a unique, holistic and non-toxic approach to salvage and preserve human (and eventually tissue engineered) VCA for unprecedented durations. Across three specific aims, in this first phase, we use skeletal muscle and vascular endothelial cells as a model with the central goal of creating a cocktail and protocol that enables high subzero cryopreservation and the development of a thermodynamically stable state, while actively suppressing metabolism and enhancing stress tolerance. Several of these sub-strategies should also help mitigate ischemia reperfusion injury and in other ways help with rewarming, revival and repair post storage. This proposal constitutes an important standalone project that should also lead to solutions for preservation of natural and engineered vital organs, as well as for traditional cell and tissue banking.

PUBLIC HEALTH RELEVANCE: 185,000 amputations are conducted each year in the US. Almost half are caused by trauma, disproportionally affecting young people. Furthermore, conservative estimates indicate that another 15,000 Americans suffer life-changing facial disfigurement annually. The combination of clinical need for vascularized complex allotransplants for these patients and increased surgical capabilities in hand, face and limb transplantation results in an ever-growing need for vascularized composite tissues. At the same time, the short current preservation times severely limit the number of these transplants that can be salvaged, constrain donor- recipient matching, and contribute to graft rejection in important ways. Our technology promises to overcome these issues by providing banks of vascularized complex allotransplants that can be provided to patients as needed with improved donor-recipient matching and graft outcomes.