Extended limb preservation employing an optimization strategy for stabilization.

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In collaboration with Prof. Brandacher at Johns Hopkins University we plan to improve upon the currently most used clinical method of limb preservation, namely hypothermia at 0 to +4°C. We will build upon our base preservation formulation, Unisol™, that has been shown to preserved whole large animals below +10°C for 6- 8h after total blood replacement with Unisol™ with normal functions upon return to physiological conditions. We have also shown that Unisol™ can maintain blood vessel function for at least 6 days at both -7°C and +4°C and that mouse hearts stored at 4°C for 18 hours in Unisol™ have a significantly faster return of heart function than hearts stored in the gold standard hypothermic heart preservation solution Celsior (HTK). Encouraged by these results we propose evaluation of Unisol supplemented with reagents targeting oxidation, apoptosis (enhancers of stress tolerance) and metabolism (metabolic rate inhibition) in 2 specific aims using human skeletal myoblasts and vascular endothelial cells to select optimal reagent concentrations in vitro and a rat forelimb transplant model to evaluate the best supplement formulations developed in the in vitro studies. The lead in vitro assay will be alamarBlue, however outcomes will be checked using alternative assays including trypan blue, live/dead stain and MTT assay. Apoptosis will be evaluated if significant losses of metabolic activity are observed 1 to 2 days after return to physiologic culture conditions. The in vivo studies will evaluate the best formulations from the in vitro studies over 3 days of hypothermic storage by transplantation model. Controls limbs will be preserved in Unisol™ and HTK. We anticipate that the supplemented formulation will maintain viability and function of limbs for up to 36 hours, a considerable improvement over all current practice methods. This innovation will not only increase storage time, it will also provide the opportunity for more closely matched recipients and potentially induction of tolerance. Furthermore, we are improving on the most tried and true method for hypothermic limb storage in clinical practice that is relatively inexpensive and easy to ship by air. Demonstration of ≥24 hours of hypothermic storage with >70% retention of limb functions will be considered to be a successful demonstration of feasibility for progression to a Phase II SBIR proposal for further evaluation in large animal models and ex vivo human limb evaluation post-preservation.