Subnormothermic Ex Situ Limb Perfusion Using Red Blood Cells and Plasma: Critical Component Analysis

The long-term objective of this application is to keep vascularized composite tissues viable at orbeyond 48 hours using ex-vivo perfusion systems. In this project, we used rat hind limb allograft transplantation model along with a miniaturized version of our custom-made ex-vivo perfusion system and tested red blood cell based perfusate solutions.Methods: Under SA1, we tested the efficacy of three plasma components in maintaining muscle force generation on limb allografts after 8 hours of SNT ex-vivo perfusion/transplantation. These components were separated based on their molecular weight (full, high molecular weight, and low molecular weight plasma) and mixed with red blood cells to perfuse limbs, which were then transplanted to another recipient orthotopically to observe limb survival as well as the long-term outcomes of muscle and nerve regeneration 3 months following transplantation. Outcome measures included analysis of the EMG data, muscle and nerve histology, and proteomic analysis of the perfusate. Results: Under SA1, recipients in the group perfused with RBC and HMW plasma proteins had severe reperfusion injury and did not survive beyond 3 days. In other groups, the addition of whole plasma did not appear to improve amplitude and latency on gastrocnemius and EDL muscles compared to immediately transplanted limbs. Groups perfused with RBC only and RBC with LMW plasma demonstrated excellent amplitude and latencies on all muscle groups with no significant difference compared to immediately transplanted limbs (p>0.05). The EDL muscle in these groups also generated similar tetanic and twitch forces compared to the immediate transplantation group(p>0.05). Under SA2, we used the best perfusate combination established under SA1 and perfused limbs following various ischemic intervals after the limb amputation and demonstrated protective effects of ex-situ perfusion up to 4 hours following ischemia compared to static cold storage.