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:

contributed equally to this work.Abbreviations: ADP, adenosine diphosphate; AMP, adenosine monophosphate; ATP, adenosine triphosphate; CIT, cold ischemia time; DBD, donation after brain death; DCD, donation after circulatory death; FENa, fractional excretion of sodium; HBOC, hemoglobin-based oxygen carrier; HMP, hypothermic machine perfusion; NEDS, New England Donor Services; NEVKP, normothermic ex vivo kidney perfusion; NMP, normothermic machine perfusion; PRBC, packed red blood cells; QAS, quality assessment score; SCS, static cold storage; WIT, warm ischemia time.Normothermic machine perfusion presents a novel platform for pretransplant assessment and reconditioning of kidney grafts. Maintaining the metabolic activity of a preserved graft at physiologic levels requires an adequate oxygen supply, typically delivered by crystalloid solutions supplemented with red blood cells. In this study, we explored the feasibility of using a synthetic hemoglobin-based oxygen carrier (HBOC) in human kidney normothermic perfusion. Fourteen discarded human kidneys were perfused for 6 hours at a mean temperature of 37°C using a pressurecontrolled system. Kidneys were perfused with a perfusion solution supplemented with either HBOC (n = 7) or packed red blood cells (PRBC) (n = 7) to increase oxygencarrying capacity. Renal artery resistance, oxygen extraction, metabolic activity, energy stores, and histological features were evaluated. Throughout perfusion, kidneys from both groups exhibited comparable behavior regarding vascular flow (P = .66), oxygen consumption (P = .88), and reconstitution of tissue adenosine triphosphate (P = .057). Lactic acid levels were significantly higher in kidneys perfused with PRBC (P = .007). Histological findings were comparable between groups, and there was no evidence of histological damage caused by the HBOC. This feasibility experiment demonstrates that a HBOC solution can offer a logistically more convenient off-theshelf alternative to PRBC in normothermic machine perfusion of human kidneys. K E Y W O R D Sbasic (laboratory) research/science, kidney transplantation/nephrology, organ perfusion and preservation, organ procurement and allocation, tissue/organ engineering, translational research/science | 2815 ABURAWI et Al.

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.

Ex situ machine perfusion is a promising technology to help improve organ viability prior to transplantation. However, preclinical studies using discarded human livers to evaluate therapeutic interventions and optimize perfusion conditions are limited by significant graft heterogeneity. In order to improve the efficacy and reproducibility of future studies, a split-liver perfusion model was developed to allow simultaneous perfusion of left and right lobes, allowing one lobe to serve as a control for the other. Eleven discarded livers were surgically split, and both lobes perfused simultaneously on separate perfusion devices for 3 h at subnormothermic temperatures. Lobar perfusion parameters were also compared with whole livers undergoing perfusion. Similar to whole-liver perfusions, each lobe in the split-liver model exhibited a progressive decrease in arterial resistance and lactate levels throughout perfusion, which were not significantly different between right and left lobes. Split liver lobes also demonstrated comparable energy charge ratios. Ex situ split-liver perfusion is a novel experimental model that allows each graft to act as its own control. This model is particularly well suited for preclinical studies by avoiding the need for large numbers of enrolled livers necessary due to the heterogenous nature of discarded human liver research.

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

Contrast enhancement magnetic resonance imaging (CE-MRI) of synovial volume is the radiographic gold standard to quantify joint inflammation but cost limits use. Therefore, we examined if power Doppler-ultrasound (PD-US) outcomes of synovitis in tumor necrosis factor transgenic (TNF-Tg) mice correlate with CE-MRI. TNF-Tg mice underwent PD-US of their knees to measure the joint space volume (JSV) and PD volume (PDV), and the results were correlated with synovial volume determined by CE-MRI. Immunohistochemistry for CD31 was performed to corroborate the PD signal. Synovial volume strongly correlated with both JSV and PDV (p<0.01). CD31+ blood vessels were observed in inflamed synovium proximal to the joint surface, which corresponded to areas of intense PD signals. JSV and PDV are valid measures of joint inflammation that correlate with synovial volume determined by CE-MRI and are associated with vascularity. Given the emergence of PD-US as a non-quantitative outcome of joint inflammation, we find JSV and PDV to be feasible and highly cost-effective for longitudinal studies in animal models. Furthermore, given the increasing utilization of PD-US in standard clinical practice, JSV and PDV could be translated to better quantify joint flare and response to therapy in RA patients.

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Drug-drug-interactions (DDIs) occur when a drug alters the metabolic rate, efficacy, and toxicity of concurrently used drugs. While almost 1 in 4 adults now use at least 3 concurrent prescription drugs in the United States, the Non-alcoholic fatty liver disease (NAFLD) prevalence has also risen over 25%. The effect of NALFD on DDIs is largely unknown. NAFLD is characterized by lipid vesicle accumulation in the liver, which can progress to severe steatohepatitis (NASH), fibrosis, cirrhosis, and hepatic carcinoma. The CYP450 enzyme family dysregulation in NAFLD, which might already alter the efficacy and toxicity of drugs, has been partially characterized. Nevertheless, the drug-induced dysregulation of CYP450 enzymes has not been studied in the fatty liver. These changes in enzymatic inducibility during NAFLD, when taking concurrent drugs, could cause unexpected fatalities through inadvertent DDIs. We have, thus, developed an in vitro model to investigate the CYP450 transcriptional regulation in NAFLD. Specifically, we cultured primary human hepatocytes in a medium containing free fatty acids, high glucose, and insulin for seven days. These cultures displayed intracellular macro-steatosis after 5 days and cytokine secretion resembling NAFLD patients. We further verified the model’s dysregulation in the transcription of key CYP450 enzymes. We then exposed the NAFLD model to the drug inducers rifampicin, Omeprazole, and Phenytoin as activators of transcription factors pregnane X receptor (PXR), aryl hydrocarbon receptor (AHR) and constitutive androstane receptor (CAR), respectively. In the NAFLD model, Omeprazole maintained an expected induction of CYP1A1, however Phenytoin and Rifampicin showed elevated induction of CYP2B6 and CYP2C9 compared to healthy cultures. We, thus, conclude that the fatty liver could cause aggravated drug-drug interactions in NAFLD or NASH patients related to CYP2B6 and CYP2C9 enzymes.