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Vascularized composite allografts (VCAs) refer to en bloc heterogenous tissue that is transplanted to restore form and function after amputation or tissue loss. Rat limb VCA has emerged as a robust translational model to study the pathophysiology of these transplants. However, these models have predominately focused on hindlimb VCAs which does not translate anatomically to upper extremity transplantation, whereas the majority of clinical VCAs are upper extremity and hand transplants. This work details our optimization of rat forelimb VCA procurement and sub-normothermic machine perfusion (SNMP) protocols, with results in comparison to hindlimb perfusion with the same perfusion modality. Results indicate that compared to hindlimbs, rat forelimbs on machine perfusion mandate lower flow rates and higher acceptable maximum pressures. Additionally, low-flow forelimbs have less cellular damage than high-flow forelimbs based on oxygen uptake, edema, potassium levels, and histology through 2 hours of machine perfusion. These results are expected to inform future upper extremity VCA preservation studies. [ABSTRACT FROM AUTHOR]

Early recognition of the clinical signs of bloodstream infection in pediatric burn patients is key to improving survival rates in the burn unit. The objective of this study was to propose a simple scoring criteria that used readily available temperature, heart rate (HR) and mean arterial pressure (MAP) data to accurately predict bloodstream infection in pediatric burn patients. A retrospective chart review included 100 patients admitted to the pediatric burn unit for >20% total body surface area (TBSA) burn injuries. Each patient had multiple blood culture tests, and each test was treated as a separate and independent "infection event" for analysis. The time at each blood culture draw was time 0 for that event, and temperature, HR and MAP data was collected for 24 hours after the blood culture was drawn. "Infection events" included in this study had at least six complete sets of temperature, HR and MAP data entries. Median temperature, HR and MAP, as well as mean fever spikes, HR spikes and MAP dips, were compared between infection group (positive blood cultures) and control group (negative blood cultures). These vital sign fluctuations were evaluated individually and as a combination of all three as timely predictors of bloodstream infection. In addition, we tested the prediction of Gram-negative bacteria versus Gram-positive or fungi present in blood cultures. Patients in the infection group had significantly higher median temperatures (p<0.001), mean fever spikes (p<0.001) and mean HR spikes (p<0.001), compared to the control group. Using the combination scoring criteria to predict bloodstream infection, the strongest predictive values in the 24-hour timeframe had high sensitivity (93%) and specificity (81%). The predictive test metric based on vital sign spikes predicted Gram-negative bacteria, but with limited sensitivity (57%) and specificity (44%). A simple scoring criteria using a combination of fever spikes, HR spikes and MAP dips predicted bloodstream infection in pediatric burn patients, and can be feasibly implemented in routine clinical care. There is also potential to use the predictive metric to detect a few select organisms based on vital signs, however further work is necessary to enhance accuracy to levels that would allow consideration for clinical use. [ABSTRACT FROM AUTHOR]

Ischemia reperfusion injury (IRI) is a critical problem in liver transplantation that can lead to life-threatening complications and substantially limit the utilization of livers for transplantation. However, because there are no early diagnostics available, fulminant injury may only become evident post-transplant. Mitochondria play a central role in IRI and are an ideal diagnostic target. During ischemia, changes in the mitochondrial redox state form the first link in the chain of events that lead to IRI. In this study we used resonance Raman spectroscopy to provide a rapid, non-invasive, and label-free diagnostic for quantification of the hepatic mitochondrial redox status. We show this diagnostic can be used to significantly distinguish transplantable versus non-transplantable ischemically injured rat livers during oxygenated machine perfusion and demonstrate spatial differences in the response of mitochondrial redox to ischemia reperfusion. This novel diagnostic may be used in the future to predict the viability of human livers for transplantation and as a tool to better understand the mechanisms of hepatic IRI. [ABSTRACT FROM AUTHOR]

There is a significant organ shortage in the field of liver transplantation, partly due to a high discard rate of steatotic livers from donors. These organs are known to function poorly if transplanted but make up a significant portion of the available pool of donated livers. This study demonstrates the ability to improve the function of steatotic rat livers using a combination of ex situ machine perfusion and a "defatting" drug cocktail. After 6 hours of perfusion, defatted livers demonstrated lower perfusate lactate levels and improved bile quality as demonstrated by higher bile bicarbonate and lower bile lactate. Furthermore, defatting was associated with decreased gene expression of pro-inflammatory cytokines and increased expression of enzymes involved in mitochondrial fatty acid oxidation. Rehabilitation of marginal or discarded steatotic livers using machine perfusion and tailored drug therapy can significantly increase the supply of donor livers for transplantation. [ABSTRACT FROM AUTHOR]

There continues to be a significant shortage of donor livers for transplantation. One impediment is the discard rate of fatty, or steatotic, livers because of their poor post-transplant function. Steatotic livers are prone to significant ischemia-reperfusion injury (IRI) and data regarding how best to improve the quality of steatotic livers is lacking. Herein, we use normothermic (37°C) machine perfusion in combination with metabolic and lipidomic profiling to elucidate deficiencies in metabolic pathways in steatotic livers, and to inform strategies for improving their function. During perfusion, energy cofactors increased in steatotic livers to a similar extent as non-steatotic livers, but there were significant deficits in anti-oxidant capacity, efficient energy utilization, and lipid metabolism. Steatotic livers appeared to oxidize fatty acids at a higher rate but favored ketone body production rather than energy regeneration via the tricyclic acid cycle. As a result, lactate clearance was slower and transaminase levels were higher in steatotic livers. Lipidomic profiling revealed ω-3 polyunsaturated fatty acids increased in non-steatotic livers to a greater extent than in steatotic livers. The novel use of metabolic and lipidomic profiling during ex situ normothermic machine perfusion has the potential to guide the resuscitation and rehabilitation of steatotic livers for transplantation. [ABSTRACT FROM AUTHOR]

Cellular rejection of liver transplant allografts remains a concern despite immunosuppressant use. Existing transplant biomarkers are often not sensitive enough to detect acute or chronic rejection at an early enough stage to allow successful clinical intervention. We herein developed a cell-based sensor that can potentially be used for monitoring local events following liver transplantation. Utilizing a machine perfusion system as a platform to engraft the cells into a donor liver, we effectively established the biocompatibility of the biosensor cells and confirmed efficient delivery of cells distributed throughout the organ. This work proves an innovative concept of integrating synthetic reporter cells ex vivo into organs as a transplant-within-a-transplant during functional organ preservation with a vision to use cell biosensors as a broad way to monitor and treat tissue transplants. [ABSTRACT FROM AUTHOR]

Background: Liver transplantation plays a pivotal role in the treatment of patients with end-stage liver disease. Despite excellent outcomes, the field is strained by a severe shortage of viable liver grafts. To meet high demands, attempts are made to increase the use of suboptimal livers by both pretransplant recovery and assessment of donor livers. Here we aim to assess hepatic injury in the measurement of routine markers in the post-ischemic flush effluent of discarded human liver with a wide warm ischemic range. Methods: Six human livers discarded for transplantation with variable warm and cold ischemia times were flushed at the end of preservation. The liver grafts were flushed with NaCl or Lactated Ringer’s, 2 L through the portal vein and 1 L through the hepatic artery. The vena caval effluent was sampled and analyzed for biochemical markers of injury; lactate dehydrogenase (LDH), alanine transaminase (ALT), and alkaline phosphatase (ALP). Liver tissue biopsies were analyzed for ATP content and histologically (H&E) examined. Results: The duration of warm ischemia in the six livers correlated significantly to the concentration of LDH, ALT, and ALP in the effluent from the portal vein flush. No correlation was found with cold ischemia time. Tissue ATP content at the end of preservation correlated very strongly with the concentration of ALP in the arterial effluent (P<0.0007, R2 = 0.96). Conclusion: Biochemical injury markers released during the cold preservation period were reflective of the duration of warm ischemic injury sustained prior to release of the markers, as well as the hepatic energy status. As such, assessment of the flush effluent at the end of cold preservation may be a useful tool in evaluating suboptimal livers prior to transplantation, particularly in situations with undeterminable ischemic durations. [ABSTRACT FROM AUTHOR]

Normothermic machine perfusion has previously been demonstrated to restore damaged warm ischemic livers to transplantable condition in animal models. However, the mechanisms of recovery are unclear, preventing rational optimization of perfusion systems and slowing clinical translation of machine perfusion. In this study, organ recovery time and major perfusate shortcomings were evaluated using a comprehensive metabolic analysis of organ function in perfusion prior to successful transplantation. Two groups, Fresh livers and livers subjected to 1 hr of warm ischemia (WI) received perfusion for a total preservation time of 6 hrs, followed by successful transplantation. 24 metabolic fluxes were directly measured and 38 stoichiometrically-related fluxes were estimated via a mass balance model of the major pathways of energy metabolism. This analysis revealed stable metabolism in Fresh livers throughout perfusion while identifying two distinct metabolic states in WI livers, separated at t = 2 hrs, coinciding with recovery of oxygen uptake rates to Fresh liver values. This finding strongly suggests successful organ resuscitation within 2 hrs of perfusion. Overall perfused livers regulated metabolism of perfusate substrates according to their metabolic needs, despite supraphysiological levels of some metabolites. This study establishes the first integrative metabolic basis for the dynamics of recovery during perfusion treatment of marginal livers. Our initial findings support enhanced oxygen delivery for both timely recovery and long-term sustenance. These results are expected to lead the optimization of the treatment protocols and perfusion media from a metabolic perspective, facilitating translation to clinical use. [ABSTRACT FROM AUTHOR]

With over 110,000 patients waiting for organ transplantation, the current crisis in organ transplantation is based on a lack of donors after brain-death (DBD). A very large alternative pool of donor organs that remain untapped are the donors after cardiac death (DCD), recovered after cardiac activity has ceased and therefore sustained some ischemic injury. Machine perfusion has been proposed as a novel modality of organ preservation and treatment to render such cadaveric organs, and in particular livers, transplantable. Two key issues that remain unaddressed are how to assess whether a DCD liver is damaged beyond repair, and whether machine perfusion has rendered an injured organ sufficiently viable for transplantation. In this work, we present a metabolic analysis of the transient responses of cadaveric rat livers during normothermic machine perfusion (NMP), and develop an index of ischemia that enables evaluation of the organ ischemic injury level. Further, we perform a discriminant analysis to construct a classification algorithm with >0.98 specificity to identify whether a given perfused liver is ischemic or fresh, in effect a precursor for an index of transplantability and a basis for the use of statistical process control measures for automated feedback control of treatment of ischemic injury in DCD livers. The analyses yield an index based on squared prediction error (SPE) as log(SPE) >1.35 indicating ischemia. The differences between metabolic functions of fresh and ischemic livers during perfusion are outlined and the metabolites that varied significantly for ischemic livers are identified as ornithine, arginine, albumin and tyrosine. [ABSTRACT FROM AUTHOR]

Supercooling preservation holds the potential to drastically extend the preservation time of organs, tissues and engineered tissue products, and fragile cell types that do not lend themselves well to cryopreservation or vitrification. Here, we investigate the effects of supercooling preservation (SCP at -4oC) on primary rat hepatocytes stored in cryovials and compare its success (high viability and good functional characteristics) to that of static cold storage (CS at +4oC) and cryopreservation. We consider two prominent preservation solutions a) Hypothermosol (HTS-FRS) and b) University of Wisconsin solution (UW) and a range of preservation temperatures (-4 to -10 oC). We find that there exists an optimum temperature (-4oC) for SCP of rat hepatocytes which yields the highest viability; at this temperature HTS-FRS significantly outperforms UW solution in terms of viability and functional characteristics (secretions and enzymatic activity in suspension and plate culture). With the HTS-FRS solution we show that the cells can be stored for up to a week with high viability (~56%); moreover we also show that the preservation can be performed in large batches (50 million cells) with equal or better viability and no loss of functionality as compared to smaller batches (1.5 million cells) performed in cryovials.