The inability to preserve vascular organs beyond several hours contributes to the scarcity of organs for transplantation 1,2. Standard hypothermic preservation at +4°C 1,3 limits liver preservation to less than 12 hours. Our group previously showed that supercooled ice free storage at −6°C can extend viable preservation of rat livers 4,5 However, scaling supercooling preservation to human organs is intrinsically limited because of volume-dependent stochastic ice formation. Here, we describe an improved supercooling protocol that averts freezing of human livers by minimizing favourable sites of ice nucleation and homogeneous preconditioning with protective agents during machine perfusion. We show that human livers can be stored at −4°C with supercooling followed by subnormothermic machine perfusion, effectively extending the ex vivo life of the organ by 27 hours. We show that viability of livers before and after supercooling is unchanged, and that after supercooling livers can withstand the stress of simulated transplantation by ex vivo normothermic reperfusion with blood. The absence of technology to preserve organs for more than a few hours is one of the fundamental causes of the donor organ shortage crisis 1-3. Subzero preservation has the potential to extend the organ storage limits 1-5 , as the metabolic rate halves for every 10°C Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Liver transplantation (LT) provides the only effective therapy for end-stage liver disease. Unfortunately, only two-thirds of the approximately 13,000 patients on the waitlist receive a life-saving LT. 1 Efforts to alleviate the organ shortage include increasing living donation, optimizing allocation models, 2 and improving utilization of grafts from extended-criteria donors (ECD), such as those from older donors and donation after circulatory death (DCD), or those with macrosteatosis, abnormal liver function tests (LFTs), or significant alcohol or drug use history. 3 These livers are often declined because poor post-transplant function and graft survival rates as low as 20% pose too great a risk for the recipient. 4,5 Interestingly, a landmark European clinical trial of normothermic machine perfusion (NMP) demonstrated a 50% lower rate of organ discard with NMP than with cold storage (CS), in spite of longer warm ischemic and total preservation times. Availability of functional metrics during NMP likely increased surgeon confidence in these grafts, a judgment subsequently supported by the finding that more aggressive acceptance of organs in the NMP arm

Loss of hepatocyte viability and metabolic function after cryopreservation is still a major issue. Although vitrification is a promising alternative, it has generally been proven to be unsuitable for vitrification of large cell volumes which is required for clinical applications. Here we propose a novel bulk droplet (3 to 5 mm diameter) vitrification method which allows high throughput volumes (4 ml/min), while using a low pre-incubated CPA concentration (15% v/v) to minimize toxicity and loss of cell viability and function. We used rapid (1.25 s) osmotic dehydration in order to concentrate a low pre-incubated intracellular CPA concentration ahead of vitrification, without the need of fully equilibrating toxic CPA concentrations. We compared direct post-preservation viability, long-term viability and metabolic function of bulk droplet vitrified, cryopreserved and fresh hepatocytes. Simulations and cooling rate measurements confirmed an adequate concentration of the intracellular CPA concentration (up to 8.53 M) after dehydration in combination with high cooling rates (960 to 1320°C/min) for successful vitrification. Compared to cryopreserved hepatocytes, bulk droplet vitrified hepatocytes had a significantly higher viability, directly after preservation and after one day in culture. Moreover, bulk droplet vitrified hepatocytes had evidently better morphology and showed significantly higher metabolic activity than cryopreserved hepatocytes in long term collagen sandwich cultures. In conclusion, we developed a novel bulk droplet vitrification method of which we validated the theoretical background and demonstrated the feasibility to use this method to vitrify large cell volumes. Moreover, we showed that this method results in improved hepatocyte viability and metabolic function as compared to cryopreservation.

Improving organ preservation and extending the preservation time would have game-changing effects on the current practice of organ transplantation. Machine perfusion has emerged as an improved preservation technology to expand the donor pool, assess graft viability and ensure adequate graft function. However, its efficacy in extending the preservation time is limited. Subzero organ preservation does hold the promise to significantly extend the preservation time and recent advances in cryobiology bring it closer to clinical translation. In this review, we aim to broaden the perspective in the field from a focus on these individual technologies to that of a systems engineering. This would enable the creation of a preservation process that integrates the benefits of machine perfusion with those of subzero preservation, with the ultimate goal to provide on demand availability of donor organs through organ banking.

Subzero preservation of human organs has been an elusive goal for many decades. The major complication hindering successful subzero preservation is the formation of ice at temperatures below freezing. Supercooling, or subzero non-freezing, preservation completely avoids ice formation at subzero temperatures. We previously showed that rat livers can be viably preserved three times longer by supercooling compared to hypothermic preservation at +4°C. Scalability of supercooling preservation to human organs was intrinsically limited due to volume dependent stochastic ice formation at subzero temperatures however we adapted the rat preservation approach so it could be applied to larger organs. Here we describe a supercooling protocol that averts freezing of human livers by minimizing air liquid interfaces as favourable sites of ice nucleation and preconditioning with cryoprotective agents to depress the freezing point of the liver tissue. Human livers are homogenously preconditioned during multiple machine perfusion steps at different temperatures. Including preparations, the protocol takes 31 hours to complete. Using this protocol human livers can be stored free of ice at −4°C which substantially extends the ex vivo

The global shortage of donor organs has made it crucial to deeply understand and better predict donor liver viability. However, biomarkers that effectively assess viability of marginal grafts for organ transplantation are currently lacking. Here, we showed that hepatocytes, sinusoidal endothelial, stellate, and liver-specific immune cells were released into perfusates from Lewis rat livers as a result of cold ischemia and machine perfusion. Perfusate comparison analysis of fresh livers and cold ischemic livers showed that the released cell profiles were significantly altered by the duration of cold ischemia. Our findings show for the first time that parenchymal cells are released from organs under non-proliferative pathological conditions, correlating with the degree of ischemic injury. Thus, perfusate cell profiles could serve as potential biomarkers of graft viability and indicators of specific injury mechanisms during organ handling and transplantation. Further, parenchymal cell release may have applications in other pathological conditions beyond organ transplantation.

The limited preservation duration of organs has contributed to the shortage of organs for transplantation. Recently, a tripling of the storage duration was achieved with supercooling, which relies on temperatures between −4 and −6 °C. However, to achieve deeper metabolic stasis, lower temperatures are required. Inspired by freeze-tolerant animals, we entered high-subzero temperatures (−10 to −15 °C) using ice nucleators to control ice and cryoprotective agents (CPAs) to maintain an unfrozen liquid fraction. We present this approach, termed partial freezing, by testing gradual (un)loading and different CPAs, holding temperatures, and storage durations. Results indicate that propylene glycol outperforms glycerol and injury is largely influenced by storage temperatures. Subsequently, we demonstrate that machine perfusion enhancements improve the recovery of livers after freezing. Ultimately, livers that were partially frozen for 5-fold longer showed favorable outcomes as compared to viable controls, although frozen livers had lower cumulative bile and higher liver enzymes.

Pumping new life into old ideas: Preservation and rehabilitation of the liver using ex situ machine perfusionRecent advances in machine perfusion technology have reinvigorated the field of liver transplantation with the possibilities of vastly improving the efficiency and safety of the life-saving procedure. With this improved preservation technology, transplant surgeons are now able to use previously untransplantable donor livers without significantly compromising patient outcomes. Early clinical studies demonstrate the ability to extend preservation times and assess a graft's potential viability using normothermic machine perfusion, in addition to restoring the energy supply in donor livers by supporting metabolism through circulation of vital nutrients and blood-based oxygen carriers. Future endeavors for surgeons and scientists should focus on improving criteria to assess viability, optimizing protocols for perfusion research, investigating mechanisms of poor graft viability, and targeting these mechanisms with novel therapies to improve graft function prior to transplantation. Long-term goals include extending preservation times on the scale of days to weeks, enabling long-distance organ sharing, and establishing regional organ perfusion centers to streamline the procurement, perfusion, and transplantation process.