Perfusion-based delivery of an ice recrystallization inhibitor in rat liver tissue

Extending the storage of donated livers beyond the current 4-6 h via freezing could significantly improve transplant availability. However, mitigating freezing injury in complex organs remains challenging because conventional cryoprotective agents (CPAs) are difficult to perfuse uniformly and several biologically important injury mechanisms are not well controlled. Ice recrystallization, where larger ice crystals grow at the expense of smaller ones, is one such mechanism. To address this, we evaluated two ice recrystallization inhibitors (IRIs), N-(2-fluorophenyl)-C6-azido-D-gluconamide (2FA) and N-(2-chlorophenyl)-D-gluconamide (4CLA), for hepatocompatibility and tissue permeation during a 4 h portal vein perfusion of rat livers. Hepatic function assessments showed no general toxicity for either IRI. However, 4CLA-perfused livers exhibited biliary stasis and lacked bile production. Conversely, 2FA maintained bile production and, in post-perfusion biopsies subjected to freezing, successfully reduced ice crystal growth within 200 μm of portal venules. These findings demonstrate that 2FA is a highly promising candidate for supporting long-term liver preservation in the frozen state.