Cold Preservation Research Articles

Non-thermal treatment for the stabilisation of liquid food using a tubular cellulose filter from corn stalks

Thermal pasteurisation is used for the microbial stabilisation of foods, but this process compromises quality food properties. This study proposes a novel cold preservation method with filtering materials obtained from agricultural by-products. Nano/micro-porous cellulose materials from native corn stalks (NCS), and the material obtained after delignification, were used as filtering materials for the microbial stabilisation of water and orange juice. The optimisation study of NCS and corn stalk tubular cellulose material (CSTC) to remove yeast Saccharomyces cerevisiae and Lactobacillus casei from water in accordance with processing variables was firstly conducted. Optimisation tests demonstrated the influence of cell concentration, filter size and flow rate on filter removal yields. Then the continuous filtration of commercial orange juice was performed for 20 days to remove S. cerevisiae. The results showed that delignified CSTC was an effective filtering material for removing S. cerevisiae and L. casei from water, and it successfully treated commercial contaminated orange juice. Despite juice colour diminishing when the process started and after filter regeneration cycles with hot water, almost initial parameter values were obtained as the process continued. Therefore, the proposed filtering material is a promising low-cost alternative to existing thermal pasteurisation technologies for liquid foods.

Effect of immediate cold formalin fixation on phosphoprotein IHC tumor biomarker signal in liver tumors using a cold transport device

Phosphoproteins are the key indicators of signaling network pathway activation. Many disease treatment therapies are designed to inhibit these pathways and effective diagnostics are required to evaluate the efficacy of these treatments. Phosphoprotein IHC have been impractical for diagnostics due to inconsistent results occurring from technical limitations. We have designed and tested a novel cold transport device and rapid cold plus warm formalin fixation protocol using phosphoproteins IHC. We collected 50 liver tumors that were split into two experimental conditions: 2 + 2 rapid fixation (2 hours cold then 2 hour warm formalin) or 4 hour room-temperature formalin. We analyzed primary hepatocellular carcinoma (n = 10) and metastatic gastrointestinal tumors (n = 28) for phosphoprotein IHC markers pAKT, pERK, pSRC, pSTAT3, and pSMAD2 and compared them to slides obtained from the clinical blocks. Expression of pERK and pSRC, present in the metastatic colorectal carcinoma, were better preserved with the rapid processing protocol while pSTAT3 expression was detected in hepatocellular carcinoma. Differences in pSMAD2 expression were difficult to detect due to the ubiquitous nature of protein expression. There were only 3 cases expressing pAKT and all exhibited a dramatic loss of signal for the standard clinical workflow. The rapid cold preservation shows improvement in phosphoprotein preservation.

Renal Procurement: Techniques for Optimizing the Quality of the Graft in the Cadaveric Setting.

Kidney transplantation is the best treatment for end-stage renal disease. However, due to organ shortage, suboptimal grafts are increasingly being used. We carried out a review on the methods and techniques of organ optimization in the cadaveric setting. Donor care is the first link in a chain of care. Right after brain death, there is a set of changes, of which hormonal and hemodynamic changes are the most relevant. Several studies have been conducted to determine which drugs to administer, although in most cases, the results are not definitive. The main goal seems rather achieve a set of biochemical and hemodynamic objectives. The ischemia-reperfusion injury is a critical factor for kidney damage in transplantation. One of the ways found to deal with this type of injury is preconditioning. Local and remote ischemic preconditioning has been studied for various organs, but studies on the kidney are scarce. A new promising area is pharmacological preconditioning, which is taking its first steps. Main surgical techniques were established in the late twentieth century. Some minor new features have been introduced to deal with anatomical variations or the emergence of donation after circulatory death. Finally, after harvesting, it is necessary to ensure the best conditions for the kidneys until the time of transplantation. Much has evolved since static cold preservation, but the best preservation conditions are yet to be determined. Conservation in the cold has come to be questioned, and great results have appeared at temperatures closer to physiological.

Ex Vivo Normothermic Perfusion, a Novel Method to Assess Pancreases after Preservation

Julien Branchereau1,2* and Etohan Ogbemudia1*, Kaithlyn Rozenberg1, Fungai Dengu1, Gabriella Hakim1, Flavia Neri1, Georg Ebeling1, Letizia Lo Faro1, James Hunter1, Rutger Ploeg1* and Peter Friend1*
 * "Authors contributed equally to this work"
 1-Nuffield Department of Surgical Sciences - University of Oxford
 2-Nantes Hospital University - France
 Introduction
 Static cold storage (SCS) of pancreases remains the current standard method for preservation prior to transplantation. Pulsatile hypothermic machine perfusion (HMP) is an emerging method that could potentially improve the preservation of pancreases to enhance graft function. This is based on personal extensive experience with HMP of pancreases in human, porcine allotransplantation and large non-human primate models. All models consistently showed preservation of pancreatic parenchyma on histological assessment for up to 24hours. To provide reassurance of organ viability prior to transplantation further investigations are necessary. We propose that normothermic perfusion (NMP) of pancreases after cold preservation allows necessary functional and physiological assessments.
 Method
 Porcine pancreases (3) were retrieved from the slaughterhouse after 30 minutes of warm ischaemia and were flushed. After 3 hours of cold ischaemia two pancreases were perfused by HMP (Wave machine; Waters Medical Systems) for 6 hours followed by 1 hour of NMP. One pancreas after 3 hours of CIT was placed on NMP for 2.5hours, this was the first pancreas NMP to assess feasibility of this technique.NMP was achieved by modification of the kidney assist device. Oxygenation was with 21% oxygen. The pancreases were cannulat ed via the aorta with free drainage of perfusate from the portal vein. NMP parameters for all pancreases were a pressure of 40mmHg and temperature of 37C. Perfusate was composed of red blood cells, plasma to provide a haematocrit of 25%, with additives of co-amoxiclav and 25,000IU of heparin. During the perfusions we collected serial perfusate samples for blood gas analysis and for insulin enzyme-linked immunosorbent assay (ELISA).
 Results
 The macroscopic appearance of the pancreases and the attached duodenum at the end of NMP appeared viable. Average resistance index during NMP was 0.62 ru (range 0.30 to 0.90 ru). Average flow rate was 77mls/min (range 53 to 100 mls/min). Throughout, the duration of NMP in the two pancreases perfused for one-hour lactate remain stable with no increase from baseline. In the one pancreas perfused for 2.5hours lactate was also stable in the first hour then increased by 50% during the last 1.5hours of perfusion. ELISA confirmed the presence of insulin in the perfusate for all three perfusions.
 Conclusion
 Normothermic perfusion is a feasible method to allow physiological and functional assessment of pancreases after cold preservation techniques encouraging us to further develop this model.

Maintaining the permanence principle for death during in situ normothermic regional perfusion for donation after circulatory death organ recovery: A United Kingdom and Canadian proposal.

There is international variability in the determination of death. Death in donation after circulatory death (DCD) can be defined by the permanent cessation of brain circulation. Post‐mortem interventions that restore brain perfusion should be prohibited as they invalidate the diagnosis of death. Retrieval teams should develop protocols that ensure the continued absence of brain perfusion during DCD organ recovery. In situ normothermic regional perfusion (NRP) or restarting the heart in the donor's body may interrupt the permanent cessation of brain perfusion because, theoretically, collateral circulations may restore it. We propose refinements to current protocols to monitor and exclude brain reperfusion during in situ NRP. In abdominal NRP, complete occlusion of the descending aorta prevents brain perfusion in most cases. Inserting a cannula in the ascending aorta identifies inadequate occlusion of the descending aorta or any collateral flow and diverts flow away from the brain. In thoracoabdominal NRP opening the aortic arch vessels to atmosphere allows collateral flow to be diverted away from the brain, maintaining the permanence standard for death and respecting the dead donor rule. We propose that these hypotheses are correct when using techniques that simultaneously occlude the descending aorta and open the aortic arch vessels to atmosphere.

Analysis of Myoglobin Stability and Bacterial Community Diversity in Mutton Chop Rolls During Cold Preservation.

The nutritional value of mutton chop rolls is gradually recognized by people, but it is easy to cause microbial contamination during storage, leading to spoilage and shortening of storage time. The bacterial diversity of mutton chop rolls in differentcoldpreservation time was analyzed to explore the main pathogens of spoilage of mutton chop rolls. At the same time, the oxidative state of myoglobin and the change of mitochondrial Metmyoglobin (MMb) Reduction Ability (MRA) in different coldpreservation were studied. It lays a foundation for further study on the mechanism of meat color stabilization of mutton chop rolls during coldpreservation. A total of 10,123,180 effective Tags were obtained from three samples with different cold preservation time by high throughput sequencing. The relative abundance of Pseudomonas was the highest in the samples refrigerated for 8days, Acinetobacter, Brochothrix and Lactobacillales showed the highest relative abundance in the samples refrigerated for 4days, which were closely related to the deterioration of mutton chop rolls and color deterioration. With the increase of cold preservation time, Oxymyoglobin (OMb) content decreased and Metmyoglobin (MMb) content increased. MRA was negatively correlated with MMb. The content of NADH was extremely significant difference with OMb and MMb. At the same time, the content of NADH was a significant difference with MRA. This study provides theoretical basis for prolonging the shelf life, maintaining meat color stability, improving the quality of mutton chop rolls. And it also plays a certain role in promoting the production and consumption of chilled meat.

A prospective, randomized, single-blind, multicentre, phase III study on organ preservation with Custodiol-N solution compared with Custodiol\xae solution in organ transplantation (kidney, liver and pancreas)

BackgroundOrgan preservation before transplantation is still a challenge. Both the University of Wisconsin and Bretschneider’s histidine-tryptophan-ketoglutarate (HTK; Custodiol®) solution are standard for liver, kidney and pancreas preservation. Organ preservation with both solutions is comparable; recently, however, Custodiol® solution has been modified to Custodiol-N according to the needs of today. Thus, our study was defined to study its effect in clinical transplantation.MethodsPatients undergoing kidney transplantation (n = 412) (including approximately 30 combined kidney–pancreas) or liver transplantation (n = 202) receive grafts that have been cold stored in either Custodiol® or Custodiol-N to demonstrate noninferiority of Custodiol-N regarding both graft function and graft injury after transplantation.DiscussionPreclinical data have clearly shown that Custodiol-N is superior to Custodiol® in cold static organ preservation via mechanisms including inhibition of hypoxic cell injury, cold-induced cell injury and avoidance of adverse effects during warm exposure to the solution. Further clinical safety data on Custodiol-N for cardioplegia are available. Thus, this study was designed to compare Custodiol® with Custodiol-N for the first time in a prospective, randomized, single-blinded, multicentre, phase III clinical transplantation trial.Trial registrationEudra-CT, 2017–002198-20. Registered on 28 November 2018.

Reconstruction surgery case report: ex vivo surgery and auto lung transplantation for pulmonary artery sarcoma

This is a report on the lung autotransplant techniques for the treatment of intimal sarcoma of pulmonary artery (PA). A 58-year-old man was diagnosed with PA sarcoma occluding the right main PA and peripherally extending into the right interlobar PA. First, the tumor was completely removed by pulmonary endarterectomy combined with right pneumonectomy under cardiopulmonary bypass. A following ex vivo evaluation of the explanted right lung revealed that the right lower PA was intraluminally intact, so the right lower lobe was used for reimplantation after being flushed with cold organ preservation solution on a back table. The patient was safely weaned from cardiopulmonary bypass following transplantation. His early post-transplant course was uneventful. He died of tumor recurrence at the distal left PA and liver and bone metastasis 14 months after surgery.

Increased and safe utilization of high-risk donor livers for transplantation after ex situ resuscitation and assessment using sequential hypo- and normothermic machine perfusion

Background: Despite persistent donor organ shortage, a high number of donor livers is currently not used for transplantation. We aimed to increase the number of transplantable livers by resuscitating and assessing hepatobiliary viability of initially declined high-risk livers using a protocol of end-ischemic sequential ex situ hypothermic and normothermic machine perfusion. Method: In this prospective clinical trial, all nationwide declined livers were eligible for inclusion (Netherlands Trial Registry NTR5972). The protocol consisted of one hour hypothermic oxygenated perfusion (10°C) for resuscitation, one hour of controlled oxygenated rewarming, and subsequent normothermic machine perfusion (NMP) for viability testing. A perfusion fluid containing a hemoglobin-based oxygen carrier was used for all temperature phases. During the first 150 min of NMP, viability of liver and biliary tree was assessed, using the following criteria: perfusate lactate <1.7mmol/L, pH 7.35-7.45, cumulative bile production >10mL and biliary pH>7.45. Livers meeting these criteria were secondary accepted for transplantation. All recipients gave written informed consent. Primary endpoint was safety and feasibility, as reflected by a 3-months graft survival rate of at least 80%. Results: Between August 2017 and October 2018, 16 livers underwent machine perfusion after an average of 288 (241-480) min of static cold preservation. All livers were derived from donation after circulatory death donors, with a median age of 63 (range 42-82) years. During NMP, all livers cleared lactate and produced sufficient bile volume, but in 5 cases biliary pH remained <7.45. The 11 (69%) livers that met all viability criteria were successfully transplanted, increasing the number of deceased donor liver transplants by 20%. Patient and with graft survival at 3 months was 100%. Conclusion: Sequential hypo- and normothermic machine perfusion enabled resuscitation and selection of initially declined high-risk donor livers. This method offered a valuable tool to safely increase the number of transplantable livers by 20%.

Solar PV power driven cold room storage for Saharan rural area

The use of renewable energy sources is usually a reliable alternative in rural areas and developing countries, where the gridline does not exist or is at a great distance. The present work is to study the possibility of feeding the tomatoes preservation cold room storage in the Sahara region driven by solar photovoltaic generator. An energy-efficient design has been achieved by analytical calculation. A PV field calculation approach yielded the following results: for a storage capacity of about 1 ton of tomatoes, the cooling capacity is 2.5 kW. The use of the design unit gave us in terms of electricity requirements 27 kWh/day by the GPV generator at Adrar province. Electrical power demands can be obtained using an alternative solution, which appears in Vapor Compression Cycles driven by PV modules. This technology considered as promoting concept for the south of Algeria investment.

ScRNA-seq assessment of the human lung, spleen, and esophagus tissue stability after cold preservation

BackgroundThe Human Cell Atlas is a large international collaborative effort to map all cell types of the human body. Single-cell RNA sequencing can generate high-quality data for the delivery of such an atlas. However, delays between fresh sample collection and processing may lead to poor data and difficulties in experimental design.ResultsThis study assesses the effect of cold storage on fresh healthy spleen, esophagus, and lung from ≥ 5 donors over 72 h. We collect 240,000 high-quality single-cell transcriptomes with detailed cell type annotations and whole genome sequences of donors, enabling future eQTL studies. Our data provide a valuable resource for the study of these 3 organs and will allow cross-organ comparison of cell types.We see little effect of cold ischemic time on cell yield, total number of reads per cell, and other quality control metrics in any of the tissues within the first 24 h. However, we observe a decrease in the proportions of lung T cells at 72 h, higher percentage of mitochondrial reads, and increased contamination by background ambient RNA reads in the 72-h samples in the spleen, which is cell type specific.ConclusionsIn conclusion, we present robust protocols for tissue preservation for up to 24 h prior to scRNA-seq analysis. This greatly facilitates the logistics of sample collection for Human Cell Atlas or clinical studies since it increases the time frames for sample processing.

Insulation Sheath Materials for Cold Preservation

The article studies one of the aspects of the heat-insulating multifunctional shells use - the cold preservation. Such task is relevant for cold storages, workshops and warehouses, in which low temperatures constant maintenance is necessary, for sports facilities (for example, ice rinks and ski facilities). In all cases of application, the main goal is to achieve the economic efficiency, which is expressed in functional results (preservation of the material that breaks down at positive temperatures) and in energy savings. The use of polyethylene-based shells, which are connected by a lock joint, is substantiated. The experimental studies results of the material given in the article shows that in terms of resistance to low and alternating temperatures, operational durability, tensile strength and water absorption, this material and insulating shells based on it are quite applicable for insulation of cold-saving objects. The experience of using heat, vapor and moisture insulating shells based on polyethylene foam with the equipment of frameless and frame structures, sports facilities, as well as for the preservation of snow at ski resorts is also provided in the article.

Hydrogen sulphide-induced hypometabolism in human-sized porcine kidneys.

BackgroundSince the start of organ transplantation, hypothermia-forced hypometabolism has been the cornerstone in organ preservation. Cold preservation showed to protect against ischemia, although post-transplant injury still occurs and further improvement in preservation techniques is needed. We hypothesize that hydrogen sulphide can be used as such a new preservation method, by inducing a reversible hypometabolic state in human sized kidneys during normothermic machine perfusion.MethodsPorcine kidneys were connected to an ex-vivo isolated, oxygen supplemented, normothermic blood perfusion set-up. Experimental kidneys (n = 5) received a 85mg NaHS infusion of 100 ppm and were compared to controls (n = 5). As a reflection of the cellular metabolism, oxygen consumption, mitochondrial activity and tissue ATP levels were measured. Kidney function was assessed by creatinine clearance and fractional excretion of sodium. To rule out potential structural and functional deterioration, kidneys were studied for biochemical markers and histology.ResultsHydrogen sulphide strongly decreased oxygen consumption by 61%, which was associated with a marked decrease in mitochondrial activity/function, without directly affecting ATP levels. Renal biological markers, renal function and histology did not change after hydrogen sulphide treatment.ConclusionIn conclusion, we showed that hydrogen sulphide can induce a controllable hypometabolic state in a human sized organ, without damaging the organ itself and could thereby be a promising therapeutic alternative for cold preservation under normothermic conditions in renal transplantation.

Therapeutic Properties of Mesenchymal Stem Cell on Organ Ischemia-Reperfusion Injury.

The shortage of donor organs is a major global concern. Organ failure requires the transplantation of functional organs. Donor’s organs are preserved for variable periods of warm and cold ischemia time, which requires placing them into a preservation device. Ischemia and reperfusion damage the organs, due to the lack of oxygen during the ischemia step, as well as the oxidative stress during the reperfusion step. Different methodologies are developed to prevent or to diminish the level of injuries. Preservation solutions were first developed to maximize cold static preservation, which includes the addition of several chemical compounds. The next chapter of organ preservation comes with the perfusion machine, where mechanical devices provide continuous flow and oxygenation ex vivo to the organs being preserved. In the addition of inhibitors of mitogen-activated protein kinase and inhibitors of the proteasome, mesenchymal stem cells began being used 13 years ago to prevent or diminish the organ’s injuries. Mesenchymal stem cells (e.g., bone marrow stem cells, adipose derived stem cells and umbilical cord stem cells) have proven to be powerful tools in repairing damaged organs. This review will focus upon the use of some bone marrow stem cells, adipose-derived stem cells and umbilical cord stem cells on preventing or decreasing the injuries due to ischemia-reperfusion.

Heavy Water (D2O) Containing Preservation Solution Reduces Hepatic Cold Preservation and Reperfusion Injury in an Isolated Perfused Rat Liver (IPRL) Model.

Background: Heavy water (D2O) has many biological effects due to the isotope effect of deuterium. We previously reported the efficacy of D2O containing solution (Dsol) in the cold preservation of rat hearts. Here, we evaluated whether Dsol reduced hepatic cold preservation and reperfusion injury. Methods: Rat livers were subjected to 48-hour cold storage in University of Wisconsin (UW) solution or Dsol, and subsequently reperfused on an isolated perfused rat liver. Graft function, injury, perfusion kinetics, oxidative stress, and cytoskeletal integrity were assessed. Results: In the UW group, severe ischemia and reperfusion injury (IRI) was shown by histopathology, higher liver enzymes leakage, portal resistance, and apoptotic index, oxygen consumption, less bile production, energy charge, and reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio (versus control). The Dsol group showed that these injuries were significantly ameliorated (versus the UW group). Furthermore, cytoskeletal derangement was progressed in the UW group, as shown by less degradation of α-Fodrin and by the inactivation of the actin depolymerization pathway, whereas these changes were significantly suppressed in the Dsol group. Conclusion: Dsol reduced hepatic IRI after extended cold preservation and subsequent reperfusion. The protection was primarily due to the maintenance of mitochondrial function, cytoskeletal integrity, leading to limiting oxidative stress, apoptosis, and necrosis pathways.

Portable Microfluidic Biosensing System for Real-Time Analysis of Microdialysate in Transplant Kidneys.

Currently, there is a severe shortage of donor kidneys that are fit for transplantation, due in part to a lack of adequate viability assessment tools for transplant organs. This work presents the integration of a novel wireless two-channel amperometric potentiostat with microneedle-based glucose and lactate biosensors housed in a 3D printed chip to create a microfluidic biosensing system that is genuinely portable. The wireless potentiostat transmits data via Bluetooth to an Android app running on a tablet. The whole miniaturized system is fully enclosed and can be integrated with microdialysis to allow continuous monitoring of tissue metabolite levels in real time. We have also developed a wireless portable automated calibration platform so that biosensors can be calibrated away from the laboratory and in transit. As a proof of concept, we have demonstrated the use of this portable analysis system to monitor porcine kidneys for the first time from organ retrieval, through warm ischemia, transportation on ice, right through to cold preservation and reperfusion. The portable system is robust and reliable in the challenging conditions of the abattoir and during kidney transportation and can detect clear physiological changes in the organ associated with clinical interventions.

Beneficial effects of the novel marine oxygen carrier M101 during cold preservation of rat and human pancreas.

Ischaemia impairs organ quality during preservation in a time‐dependent manner, due to a lack of oxygen supply. Its impact on pancreas and islet transplantation outcome has been demonstrated by a correlation between cold ischaemia time and poor islet isolation efficiency. Our goal in the present study was to improve pancreas and islet quality using a novel natural oxygen carrier (M101, 2 g/L), which has been proven safe and efficient in other clinical applications, including kidney transplantation, and for several pre‐clinical transplantation models. When M101 was added to the preservation solution of rat pancreas during ischaemia, a decrease in oxidative stress (ROS), necrosis (HMGB1), and cellular stress pathway (p38 MAPK)activity was observed. Freshly isolated islets had improved function when M101 was injected in the pancreas. Additionally, human pancreases exposed to M101 for 3 hours had an increase in complex 1 mitochondrial activity, as well as activation of AKT activity, a cell survival marker. Insulin secretion was also up‐regulated for isolated islets. In summary, these results demonstrate a positive effect of the oxygen carrier M101 on rat and human pancreas during preservation, with an overall improvement in post‐isolation islet quality.

2484 Intravenous Acetaminophen Associated With Hepatotoxicity in an Orthotopic Liver Transplant Patient

INTRODUCTION: Intravenous acetaminophen (IV APAP) is a commonly used post-operative analgesic. In adults, IV APAP administered at therapeutic doses of less than 4 g over 24 hours is considered safe. Five to ten percent of APAP is metabolized into N-acetyl-p-benzoquinone imine (NAPQI), the toxic metabolite responsible for APAP hepatotoxicity. NAPQI is inactivated when conjugated to tripeptide glutathione. CASE DESCRIPTION/METHODS: A 69 year old male with a history of secondary biliary liver cirrhosis status post orthotopic liver transplantation in 2006 and newly diagnosed prostate adenocarcinoma was admitted to the Urology service for planned robotic-assisted prostatectomy. He underwent uncomplicated robotic-assisted prostatectomy, requiring no fluid resuscitation, vasopressor support or blood products. Post-operatively, vital signs remained normal with no episodes of hypotension. He was started on IV APAP 1000 mg every 6 hours for 36 hours, followed by PO APAP 650 mg every 6 hours for post-operative pain. On post-operative day 2, AST was 2751 IU/L, ALT 3447 IU/L, TBili 1.9 mg/dL and INR 1.5 (Figure 1). All orders for acetaminophen were stopped. Liver Doppler ultrasound was normal. A serum acetaminophen level drawn 10 hours after the last IV APAP dose was &lt;15 ug/mL. Work up for infectious etiologies was negative. The patient was started on IV NAC therapy. Following completion of IV NAC therapy, AST was 986 IU/L, ALT 2340 IU/L, TBili 1.9 mg/dL, and INR 1.4. The patient was discharged home on post-operative day 4. Labs during his outpatient Hepatology follow-up appointment 4 days after discharge were AST 62 IU/L, ALT 426 IU/L, TBili 1.3 mg/dL, and INR 1.1. DISCUSSION: This is the first case of acute liver toxicity in a post-OLT patient who received therapeutic doses of IV APAP. There was no evidence of hypotension or APAP overdose in the electronic medical records. All other etiologies for acute liver toxicity were ruled out, including shock liver, infection and vascular compromise. It is possible APAP metabolism is altered in transplant livers compared to native livers. A study on 13 liver transplant patients concluded APAP metabolism was transiently altered in orthotopic livers following liver transplantation, resulting in enhanced NAPQI production. It is possible transplant livers carry less glutathione stores due to cold storage preservation and post-transplant reperfusion, rendering them more susceptible to hepatotoxicty from NAPQI concentrations that a native liver would tolerate.

Succinate accumulation drives ischaemia-reperfusion injury during organ transplantation.

During heart transplantation, storage in cold preservation solution is thought to protect the organ by slowing metabolism; by providing osmotic support; and by minimising ischaemia-reperfusion (IR) injury upon transplantation into the recipient1,2. Despite its widespread use our understanding of the metabolic changes prevented by cold storage and how warm ischaemia leads to damage is surprisingly poor. Here, we compare the metabolic changes during warm ischaemia (WI) and cold ischaemia (CI) in hearts from mouse, pig, and human. We identify common metabolic alterations during WI and those affected by CI, thereby elucidating mechanisms underlying the benefits of CI, and how WI causes damage. Succinate accumulation is a major feature within ischaemic hearts across species, and CI slows succinate generation, thereby reducing tissue damage upon reperfusion caused by the production of mitochondrial reactive oxygen species (ROS)3,4. Importantly, the inevitable periods of WI during organ procurement lead to the accumulation of damaging levels of succinate during transplantation, despite cooling organs as rapidly as possible. This damage is ameliorated by metabolic inhibitors that prevent succinate accumulation and oxidation. Our findings suggest how WI and CI contribute to transplant outcome and indicate new therapies for improving the quality of transplanted organs.

Simple Transportation of Genetically Engineered Mice via Cold Storage Techniques.

Live genetically modified (GM) mice are often shipped in transportation cages. However, the shipment of live mice is sometimes associated with difficulties. Recently, we developed an alternative means to transport GM mice via cold storage techniques for sperm and embryos. Cold storage of the cauda epididymis, the male reproductive organ for storing mature sperm, can maintain the fertility of sperm in cold preservation medium for 10 days. The sperm can then be used to produce embryos or pups via in vitro fertilization and embryo transfer. Conversely, cold-stored two-cell embryos maintain developmental ability for 4days. The embryos can be used to produce pups via embryo transfer. Cold transport techniques are being increasingly employed to create local and global networks between research institutes for the shipment of GM mice. In this text, we described a cold transport technique for sperm and two-cell embryos in mice.