Introduction: Optimizing preservation of donor organs has the potential to dramatically improve the outcome of organ transplantation by diminishing the donor organ shortage, enabling near perfect global HLA matching, permitting recipient immune tolerance induction, and allow transplantation in an elective surgical setting. For livers, the current preservation standard, static cold storage at +5°C (SCS) allows for a maximum preservation time of 12 hours. Recently, it has been shown that rat livers can be preserved in a supercooled state at -6°C for 3 days with 100% survival after transplantation. The main goal of this study is to translate this supercooling protocol to human livers. We hypothesized that preservation duration of human livers can be extended from 12 to 24 hours, by storing the organ at -4°C in an ice-free, supercooled state, followed by recovery with sub-normothermic machine perfusion (SNMP).
Method: Human livers, rejected for transplantation, were recovered from conventional SCS using SNMP before they were loaded with the cryoprotectant agent (CPA) 3-O-methylglucose (3OMG) using SNMP. Subsequently, the liver was gradually cooled to 4°C and flushed with oxygenated UW supplemented with the CPAs polyethylene glycol (PEG), glycerol and trehalose. The livers were supercooled and stored for 20 hours at -4°C. After supercooling the CPAs were washed out and the livers were recovered during 3 hours of SNMP. Pre- and post-supercooling SNMP conditions were identical. Vascular resistance, blood gas parameters, electrolytes, urea, liver enzymes and bile production were measured every 30 minutes during pre- and post- supercooling SNMP. Bilateral wedge biopsies for the measurement of mitochondrial energy charge and conventional histology were also sampled throughout the protocol.
Results: Mitochondrial energy charge, a marker for transplant success, was similar  pre- and post-supercooling. Also, we observed no difference between arterial resistance and oxygen consumption comparing pre- and post-supercooling SNMP. Viable livers produced bile pre-supercooling and continued bile production post supercooling. Similarly, histology shows minimal necrosis, no edema and limited endothelial injury. We also measured common markers of liver damage, such as ALT and AST, which were slightly elevated post-supercooling, as compared to pre-SNMP conditions.
Conclusion: Preliminary results demonstrate the feasibility of storing whole human livers in the supercooled state to double the preservation duration, as compared to clinical standards. Moreover, we identified important bio-stabilizing agents and perfusion conditions which are critical for success.