Enhanced supercooling for extending non-freezing preservation in preclinical porcine and human donor livers

ABSTRACT. One in five liver patients on the transplant waitlist die before a new liver is found, while many more patients are never listed but could benefit from liver replacement. While “bridge-to-transplant” technologies (ventricular assist devices, dialysis) have transformed the outlook for heart and kidney failure, no such treatment exists for patients with liver failure. Current liver sharing limits (about 500 miles) are based on short preservation durations (6-10 hours). Extending preservation to a few days would enable nationwide donor-recipient matching, allowing many livers that are not transplanted today (e.g., subsets of extended criteria donor livers have been shown to offer substantial survival benefits) to be offered to the patients who most need them. Current standard practice involves the vascular flush of livers with cold storage solution and subsequent transport on ice, to maintain viability via hypothermic static storage. While research has shown that lower temperatures are needed for viable, extended storage durations, storage of organs at or below 0°C risks ice formation (and significant damage) within the organ. However, it is possible to supercool water-based solutions beyond 0°C while remaining liquid in a metastable state. Published work from our group has shown that supercooling livers below 0°C can yield several fold increases in cold storage time. For example, simple supercooling has achieved 100% survival after three days with storage at -6°C in a rat transplant model. While our group has also supercooled and stored human livers at -4°C for a cumulative 27 hours, a significant improvement over current limitations in cold ischemic storage (6-10 hours), further extending the storage duration or decreasing the storage temperature pose additional challenges at this scale. The metastable nature of supercooled solutions can still result in stochastic ice nucleation and damaging crystal growth, a risk which scales with increasing organ size. The innovation here embodies development of novel techniques for a “next generation”, non-frozen preservation paradigm, dubbed “Enhanced Supercooling”. This will enable practically stable ice-free storage of clinical-size livers (pig and human) at high subzero temperatures (effective in nature but not yet studied extensively for human organ preservation). For this direct to Phase 2 proposal, building on our successful Phase 1 equivalent work, in partnership with Harvard/MGH and Johns Hopkins Univ, we develop methods for Enhanced Supercooling of human size livers via: 1) exploration of strategies for longer duration, lower temperature supercooling via (i) air- liquid interface sealing with immiscible oil, (ii) biocompatible synthetic ice modulators to inhibit heterogeneous ice nucleation; 2) enhancement of subzero cooling for ice-free preservation by freezing point depression (FPD) guided stable near equilibrium non-frozen storage, with biocompatible cryoprotective agents; 3) optimization of enhanced supercooling and/or FPD in pig livers in a pilot study of 3-5day preservation with liver orthotopic allotransplantation, and 4) validation of enhanced supercooling of human livers donated for research. Our goal is to achieve ice free storage of supercooled clinical scale livers at -6 to -12°C, or lower, for more than 72 hours.