Enhanced VCA Storage: A Pilot Study Demonstrating Supercooling in Orthotopic Rodent Hindlimb Transplantation

The field of vascularized composite allograft (VCA) transplantation has seen steady, rapid growth, with new innovations driving the evolution from experimental procedures to more standardized therapies. With this expansion comes challenges with graft allocation, preservation, and postoperative graft rejection. Here, we outline the first example of subzero nonfreezing (SZNF), supercooled storage of a whole rat hindlimb with orthotopic transplantation. Rat hindlimbs were procured, loaded, and supercooled for 48 hours at -4°C (n = 4), after which, they were recovered. The loading and recovery phase were performed using subnormothermic machine perfusion (SNMP) during which viability markers (glucose and oxygen consumption, lactate, and resistance) were tracked. Control limbs underwent static cold storage (SCS). After ex vivo validation, the model was piloted in a transplant model, comparing 48 hours of SZNF (n = 1), 48 hours of SCS (n = 1), and 72 hours of SCS (n = 1), which demonstrated no survival beyond postoperative day 4 in the SCS models, and survival until the end of study (postoperative day [POD] 28) in the SZNF model. This study demonstrates the promise of this model in future studies on long-term VCA preservation.