Cold Preservation Research Articles

(505) - Relationship between Grafts’ Quality, Glucose Consumption and IL-6 Production during Ex Vivo Lung Perfusion

The clustering of ischemia, hypoxia, and proinflammatory changes in transplanted lungs facilitate fibrosis, leading to bronchiolitis obliterans syndrome (BOS) following lung transplantation. We hypothesized this clustering can be underlying mechanism for cellular damage in lung grafts during ex vivo lung perfusion (EVLP) and investigated their correlation among suboptimal blood supply without bronchial circulation, high glucose consumption due to tissue hypoxia, and proinflammatory cytokine production in relation to lung graft function on EVLP. Using rat heart-lung blocks after 1 hour cold preservation, an acellular normothermic EVLP system was established for 4 hours. The evaluation included lung graft function, lung compliance (Cdyn), and vascular resistance (PVR) as well as circulating glucose, lactate and other biochemical markers in the perfusate. The main finding in this study was a strong positive correlation between glucose consumption during EVLP and perfusate IL-6 levels during EVLP (Fig.1A). Glucose consumption also had a positive correlation with the changes in Cdyn and PVR during EVLP. Interestingly, the lungs with higher glucose consumption above 40 mg/dl had significantly higher PVR and lower Cdyn as compared with those with lower glucose consumption below 40 mg/dl (Fig.1B). Higher glucose consumption noted in lung grafts during EVLP represent underlying tissue hypoxia which can augment proinflammatory changes, leading to compromised lung graft quality. This focus may help move forward the strategy using EVLP ultimately to contribute to improving transplant outcomes.

(370) - Prolonged Warm Ischemia Prevents Lung Allograft Acceptance in Lung Transplantation from Donation after Cardiac Death in the Mouse

s S143 investigated the role of Cyclosporine A (CyA) which is well known as an immunosuppressant drug, in prohibiting mitochondrial permeability transition and subsequent proinflammatory changes in lung grafts, leading to their improved graft quality on EVLP. Methods: Using rat heart-lung blocks after 1 hour cold preservation, an acellular normothermic EVLP system was established for 4 hours. CyA was added into perfusate for 1 μ M as final content. The evaluation included the routine lung graft function, compliance (Cdyn), and vascular resistance (PVR) as well as biochemical markers in the perfusate at multiple time points. After 4 hours EVLP, their proinflammatory and metabolic profiles were also assessed in lung grafts, including tissue ATP levels, mitochondrial biogenesis and hypoxia inducible factor (HIF)-1α expressions. Results: Lung grafts on EVLP with CyA exhibited significantly better functional and physiological parameters as compared to those without CyA (Fig.1A). CyA administration attenuated proinflammatory changes (Fig.1B) and prohibited glucose consumption during EVLP through mitigating mitochondrial dysfunction and promoting mitochondrial biogenesis in lung grafts (Fig.1C). CyA-treated lungs exhibited significantly attenuated expression of HIF-1α . Conclusion: CyA can be a potent contributor to better preconditioning lung grafts by protecting mitochondrial function during EVLP.

Oxygenated hypothermic machine perfusion after static cold storage improves endothelial function of extended criteria donor livers

Background: Lack of oxygen and biomechanical stimulation during static cold storage (SCS) of donor livers compromises endothelial cell function. This study investigated the effect of 2 hours of end-ischemic oxygenated hypothermic machine perfusion (HMP) on endothelial cell function of extended criteria donor (ECD) livers. Method: Sixteen human livers that were declined for transplantation were transported to our center using conventional static cold storage (SCS; 4oC) and subjected to normothermic machine perfusion (NMP) to assess viability and function. Six livers underwent 2 hours oxygenated HMP (12°C) after SCS and prior to NMP. Ten control livers underwent NMP immediately after SCS. Endothelial cell function was assessed by quantification of mRNA expression encoding for transcription factor Krüppel-like-factor-2 (KLF-2), endothelial nitric oxide synthase (eNOS), and thrombomodulin (TM) using real-time PCR. Nitric oxide (NO) production (nitrite/nitrate levels) and release of thrombomodulin (TM) into the perfusion fluid were determined during NMP. Results: In livers that underwent end-ischemic HMP, mRNA expression of KLF-2 (p = 0.04), eNOS (p = 0.03) and TM (p = 0.03) increased significantly during NMP. This response was not observed in the control livers. In parallel, NO levels in the perfusate increased during NMP of livers that first underwent HMP, but not in control livers. Moreover, at the end of NMP cumulative TM release into the perfusate was significantly higher in control livers compared to livers first subjected to HMP (p = 0.03). Conclusion: A short period of 2 hours oxygenated HMP restores endothelial cell function and integrity after conventional static cold preservation and subsequent reoxygenation of ECD livers.

Effective Hypothermic Storage of Human Pluripotent Stem Cell-Derived Cardiomyocytes Compatible With Global Distribution of Cells for Clinical Applications and Toxicology Testing.

To fully explore the potential of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), efficient methods for storage and shipment of these cells are required. Here, we evaluated the feasibility to cold store monolayers and aggregates of functional CMs obtained from different PSC lines using a fully defined clinical-compatible preservation formulation and investigated the time frame that hPSC-CMs could be subjected to hypothermic storage. We showed that two-dimensional (2D) monolayers of hPSC-CMs can be efficiently stored at 4°C for 3 days without compromising cell viability. However, cell viability decreased when the cold storage interval was extended to 7 days. We demonstrated that hPSC-CMs are more resistant to prolonged hypothermic storage-induced cell injury in three-dimensional aggregates than in 2D monolayers, showing high cell recoveries (>70%) after 7 days of storage. Importantly, hPSC-CMs maintained their typical (ultra)structure, gene and protein expression profile, electrophysiological profiles, and drug responsiveness. The applicability of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) in the clinic/industry is highly dependent on the development of efficient methods for worldwide shipment of these cells. This study established effective clinically compatible strategies for cold (4°C) storage of hPSC-CMs cultured as two-dimensional (2D) monolayers and three-dimensional (3D) aggregates. Cell recovery of 2D monolayers of hPSC-CMs was found to be dependent on the time of storage, and 3D cell aggregates were more resistant to prolonged cold storage than 2D monolayers. Of note, it was demonstrated that 7 days of cold storage did not affect hPSC-CM ultrastructure, phenotype, or function. This study provides important insights into the cold preservation of PSC-CMs that could be valuable in improving global commercial distribution of hPSC-CMs.

Efficacy of cold preservation solution containing desferrioxamine in protecting rat hearts

Objective To evaluate the efficacy of cold preservation solution containing desferrioxamine(DFO)in protecting the rat hearts. Methods Pathogen-free male Sprague-Dawley rats, weighing 300-350 g, were used in the study.Thirty-two isolated rat hearts were equally and randomly divided into control group(C group)and DFO group using a random number table.Hearts of rats were stored for 6 h in 4 ℃ histidine-tryptophan-ketoglutarate(HTK)solution in group C. DFO was added to HTK solution(DFO concentration 100 μmol/L), and hearts of rats were stored for 6 h in 4 ℃ HTK solution in group DFO.At 6 h of cold storage, creatine kinase and lactate dehydrogenase activities in the cold preservation solution were determined.Myocardial specimens were obtained from the apex, cut into sections which were stained with haematoxylin and eosin, and examined under a microscope.The pathological changes of myocardial tissues were scored.The content of malondialdehyde in myocardial tissues was determined using thiobarbituric acid method, and hypoxia-inducible factor-1α protein and mRNA expression in myocardial tissues was detected by real-time reverse transcriptase polymerase chain reaction and Western blot. Results Compared with group C, the creatine kinase and lactate dehydrogenase activities in the cold preservation solution, malondialdehyde content in myocardial tissues, and pathological scores were significantly decreased, and the expression of hypoxia-inducible factor-1α protein and mRNA expression in myocardial tissues was significantly up-regulated in group DFO(P<0.05). Conclusion Cold preservation solution containing DFO can protect the rat hearts effectively, and the mechanism is related to up-regulation of the expression of hypoxia-inducible factor-1α. Key words: Deferoxamine; Cryopreservation; Myocardial ischemia

Impact of time interval between donor brain death and cold preservation on long-term outcome in lung transplantation

Brain death (BD) is associated with various systemic responses and a cascade of inflammatory reactions. It is still unknown how the time interval from BD to cold preservation (CP) affects outcome after lung transplantation (LTx). This report investigates the impact of the time interval from BD to CP on long-term outcome in LTx. We reviewed 250 consecutive recipients who underwent LTx at our institution between January 2000 and December 2011. In Group I (n = 212), the time interval from BD to CP was <24 h, and in Group II (n = 38) >24 h. Cox proportional hazard regression analysis was performed to determine the risk factors affecting survival. The median time from BD to CP was 18.6 h (range 9-65). The rate of postoperative complications was comparable (P = 0.8). The 30-day mortality rate was 7.5% in Group I and 0% in Group II. The 5-year survival rate was better in Group II [70% (95% CI: 48.5-83.8%)] than in Group I [66% (95% CI: 58.3-72.5%)] without statistical significance (P = 0.3). Intraoperative extracorporeal membrane oxygenation (ECMO) use was identified as a significant risk factor for survival [HR = 1.7, (95% CI: 1.1-2.6), P = 0.01]. In our cohort, the time interval from BD to CP had no impact on long-term outcome after LTx.

Lifor Solution: An Alternative Preservation Solution in Small Bowel Transplantation.

Background and Objectives. The intestinal mucosa is extremely sensitive to ischemia. Better intestinal preservation is the first step to improve the results of intestinal transplantation. The aim of the study is to investigate the effect of cold Lifor solution on preservation of swine small bowel. Methods. Swine ileum segments (200 cm) were allotransplanted heterotopically after 9-hour cold storage with UW solution (group 1, n = 6), with Lifor solution (group 2, n = 6), or without storage (group 3, n = 6), respectively. After cold storage, mucosal adenosine triphosphate (ATP) concentrations and histopathologic analysis after preservation were performed. At day 7 after the transplantation, intestinal absorptive function was also observed. Results. After 9 h cold preservation, pathological changes, the content of ATP in the intestinal mucosa, and the intestinal absorptive function after transplantation in group 2 were similar to those of group 1. Conclusion. The effect of cold storage of swine small bowel with Lifor solution is similar to that of UW solution. It may provide additional rationale for further exploration of Lifor as an alternative preservation solution in small bowel transplantation.

Donor pretreatment with carbon monoxide prevents ischemia/reperfusion injury following heart transplantation in rats.

Because inhaled carbon monoxide (CO) provides potent anti-inflammatory and antioxidant effects against ischemia reperfusion injury, we hypothesized that treatment of organ donors with inhaled CO would decrease graft injury after heart transplantation. Hearts were heterotopically transplanted into syngeneic Lewis rats after 8 hours of cold preservation in University of Wisconsin solution. Donor rats were exposed to CO at a concentration of 250 parts per million for 24 hours via a gas-exposure chamber. Severity of myocardial injury was determined by total serum creatine phosphokinase and troponin I levels at three hours after reperfusion. In addition, Affymetrix gene array analysis of mRNA transcripts was performed on the heart graft tissue prior to implantation. Recipients of grafts from CO-exposed donors had lower levels of serum troponin I and creatine phosphokinase; less upregulation of mRNA for interleukin-6, intercellular adhesion molecule-1, and tumor necrosis factor-α; and fewer infiltrating cells. Although donor pretreatment with CO altered the expression of 49 genes expressly represented on the array, we could not obtain meaningful data to explain the mechanisms by which CO potentiated the protective effects. Pretreatment with CO gas before organ procurement effectively protected cardiac grafts from ischemia reperfusion-induced injury in a rat heterotopic cardiac transplant model. A clinical report review indicated that CO-poisoned organ donors may be comparable to non-poisoned donors.

Successful Liver Retrieval After Acute Aortic Dissection Involving the Celiac Artery Origin

Successful Liver Retrieval After Acute Aortic Dissection Involving the Celiac Artery Origin

Soluble Thrombomodulin Ameliorates Ischemia-Reperfusion Injury of Liver Grafts by Modulating the Proinflammatory Role of High-Mobility Group Box 1

Transplantation using grafts obtained after cardiac death (CD) is considered a promising solution for graft shortages. However, no standard criteria for organ preservation have been established for CD donors. High-mobility group box 1 (HMGB1) is a DNA-binding protein that is released from dying hepatocytes as an early mediator of inflammation and organ tissue damage. HMGB1 stimulates immunocytes to produce inflammatory cytokines, thereby amplifying the inflammatory response. Thrombomodulin is an integral membrane protein that functions as an endothelial anticoagulant cofactor, and it binds HMGB1 through the extracellular domain. We investigated the effects of ART-123, recombinant human soluble thrombomodulin, on warm ischemia-reperfusion injury in liver grafts. Male Wistar rats were divided into four ex vivo groups: heart-beating (HB) group, in which livers were isolated from HB donors; CD group, in which livers were isolated from CD donors exposed to apnea-induced conditions and warm ischemic conditions for 30 min after cardiac arrest; and two CD groups pretreated with ART-123 (1 or 5 mg/kg). Each isolated liver was reperfused for 1 h after cold preservation for 6 h. The perfusate levels of HMGB1, LDH, TNF-α, and IL-6 were significantly lower in the CD group pretreated with ART-123 (5 mg/kg) than in the CD group. Bile production was significantly higher in the CD group pretreated with ART-123 (5 mg/kg) than in the CD group. The sinusoidal spaces were significantly narrower in the CD group than in the other groups. We propose that ART-123 maintains sinusoidal microcirculation by reducing endothelial cell damage during warm ischemia-reperfusion injury.

Bacteriological evaluation of bone grafts stored by cryogenic freezing at –24°C from a canine (Canis familiaris) bone bank

The scientific basis of bone transplantation was established during the mid-nineteenth century, when the osteogenic properties of the bone and periosteum, beneficial influence of cold preservation of bones, and first conventional attempt to store bones for elective use, were described. A bone bank has several advantages, such as the immediate availability of grafts in large quantities, and different shapes and sizes, and maintenance of osteoinductive activity in grafts. In addition, it reduces patient blood loss, surgical time, and quantity of anesthetics required. Clinical applications include the correction of comminuted fractures, treatment of non-union bones, and replacement of bone loss due to infections or malignancies. The success of these procedures depends on the preservation and integrity of the graft. This study aimed to evaluate the feasibility of maintaining a canine bone bank (Canis familiaris) by quantifying the bacterial contamination of bone grafts preserved at –24°C. The samples were evaluated through a monthly assessment of bacteriological cultures over a period of 6 months. The harvest method was efficient and sterile, reducing the risk of contamination. We conclude that the techniques chosen for the implementation of a bone bank were effective and feasible (use of a common freezer reaching a temperature below –24°C). The viability of the bones was attested during a 6-month period, and the samples demonstrated a 100% sterility rate.

Cold Preservation of Human Adult Hepatocytes for Liver Cell Therapy.

Hepatocyte transplantation is a promising alternative therapy for the treatment of hepatic failure, hepatocellular deficiency, and genetic metabolic disorders. Hypothermic preservation of isolated human hepatocytes is potentially a simple and convenient strategy to provide on-demand hepatocytes in sufficient quantity and of the quality required for biotherapy. In this study, first we assessed how cold storage in three clinically safe preservative solutions (UW, HTS-FRS, and IGL-1) affects the viability and in vitro functionality of human hepatocytes. Then we evaluated whether such cold-preserved human hepatocytes could engraft and repopulate damaged livers in a mouse model of liver failure. Human hepatocytes showed comparable viabilities after cold preservation in the three solutions. The ability of fresh and cold-stored hepatocytes to attach to a collagen substratum and to synthesize and secrete albumin, coagulation factor VII, and urea in the medium after 3 days in culture was also equally preserved. Cold-stored hepatocytes were then transplanted in the spleen of immunodeficient mice previously infected with adenoviruses containing a thymidine kinase construct and treated with a single dose of ganciclovir to induce liver injury. Engraftment and liver repopulation were monitored over time by measuring the blood level of human albumin and by assessing the expression of specific human hepatic mRNAs and proteins in the recipient livers by RT-PCR and immunohistochemistry, respectively. Our findings show that cold-stored human hepatocytes in IGL-1 and HTS-FRS preservative solutions can survive, engraft, and proliferate in a damaged mouse liver. These results demonstrate the usefulness of human hepatocyte hypothermic preservation for cell transplantation.

Ex Vivo Perfusion With Adenosine A2A Receptor Agonist Enhances Rehabilitation of Murine Donor Lungs After Circulatory Death.

Ex vivo lung perfusion (EVLP) enables assessment and rehabilitation of marginal donor lungs before transplantation. We previously demonstrated that adenosine A2A receptor (A2AR) agonism attenuates lung ischemia-reperfusion injury. The current study utilizes a novel murine EVLP model to test the hypothesis that A2AR agonist enhances EVLP-mediated rehabilitation of donation after circulatory death (DCD) lungs. Mice underwent euthanasia and 60 minutes warm ischemia, and lungs were flushed with Perfadex and underwent cold static preservation (CSP, 60 minutes). Three groups were studied: no EVLP (CSP), EVLP with Steen solution for 60 minutes (EVLP), and EVLP with Steen solution supplemented with ATL1223, a selective A2AR agonist (EVLP + ATL1223). Lung function, wet/dry weight, cytokines and neutrophil numbers were measured. Microarrays were performed using the Affymetrix GeneChip Mouse Genome 430A 2.0 Array. Ex vivo lung perfusion significantly improved lung function versus CSP, which was further, significantly improved by EVLP + ATL1223. Lung edema, cytokines, and neutrophil counts were reduced after EVLP and further, significantly reduced after EVLP + ATL1223. Gene array analysis revealed differential expression of 1594 genes after EVLP, which comprise canonical pathways involved in inflammation and innate immunity including IL-1, IL-8, IL-6, and IL-17 signaling. Several pathways were uniquely regulated by EVLP + ATL1223 including the downregulation of genes involved in IL-1 signaling, such as ADCY9, ECSIT, IRAK1, MAPK12, and TOLLIP. Ex vivo lung perfusion modulates proinflammatory genes and reduces pulmonary dysfunction, edema, and inflammation in DCD lungs, which are further reduced by A2AR agonism. This murine EVLP model provides a novel platform to study rehabilitative mechanisms of DCD lungs.

Oxygenated Static Preservation of Donation after Cardiac Death Liver Grafts Improves Hepatocyte Viability and Function

Background: Cell therapy, such as hepatocyte transplantation (HTx), is promising for the treatment of metabolic liver diseases or as a bridge to orthotopic liver transplantation in patients with fulminant liver failure. However, one of the limitations of this therapy is the shortage of donors. The present study aims to investigate whether the two-layer method (TLM) of cold preservation with oxygenation improves the viability and activity of hepatocytes from rat donation after cardiac death (DCD) donors compared with results obtained with the University of Wisconsin (UW) solution. Moreover, we evaluated the hepatocyte function after culture or transplantation into the spleen. Materials and Methods: We used male Sprague-Dawley rats for this study. The DCD model was induced by phrenotomy after injecting heparin. We assigned rats based on warm ischemia times of 15 and 30 min to groups S and L, respectively. Each group (n = 5) was then subdivided as follows: (1) group S: not preserved (S/N), preserved by TLM for 3 h (S/TLM3) and 12 h (S/TLM12), and in the UW solution for 3 h (S/UW3) and 12 h (S/UW12), and (2) group L: not preserved (L/N), preserved by TLM for 3 h (L/TLM3) and 12 h (L/TLM12), and in the UW solution for 3 h (L/UW3) and 12 h (L/UW12). The cell viability and function of isolated DCD hepatocytes were analyzed for culture or HTx into the spleen. Results: The viability and ATP levels of DCD hepatocytes significantly improved after TLM compared with the values after preservation in cold UW solution in group S/N (p < 0.059). The levels of albumin production and urea synthesis by hepatocytes after culture were significantly higher in groups S/TLM3 and S/TLM12 than in groups S/UW3 and S/UW12 (p < 0.05), respectively. Further, serum albumin levels after HTx were also markedly higher in groups S/TLM3 and S/TLM12 than in groups S/UW3 and S/UW12. The morphological features revealed that cultured and transplanted hepatocytes remained clearly viable and maintained an expression for specific hepatic function, such as the production of albumin and glycogen. Conclusion: This novel method of oxygenated cold preservation of DCD livers can expand the hepatocyte donor pool for HTx and establish a wider application of this developing technique.

First Comparison of Hypothermic Oxygenated PErfusion Versus Static Cold Storage of Human Donation After Cardiac Death Liver Transplants: An International-matched Case Analysis.

Exposure of donor liver grafts to prolonged periods of warm ischemia before procurement causes injuries including intrahepatic cholangiopathy, which may lead to graft loss. Due to unavoidable prolonged ischemic time before procurement in donation after cardiac death (DCD) donation in 1 participating center, each liver graft of this center was pretreated with the new machine perfusion "Hypothermic Oxygenated PErfusion" (HOPE) in an attempt to improve graft quality before implantation. HOPE-treated DCD livers (n = 25) were matched and compared with normally preserved (static cold preservation) DCD liver grafts (n = 50) from 2 well-established European programs. Criteria for matching included duration of warm ischemia and key confounders summarized in the balance of risk score. In a second step, perfused and unperfused DCD livers were compared with liver grafts from standard brain dead donors (n = 50), also matched to the balance of risk score, serving as baseline controls. HOPE treatment of DCD livers significantly decreased graft injury compared with matched cold-stored DCD livers regarding peak alanine-aminotransferase (1239 vs 2065 U/L, P = 0.02), intrahepatic cholangiopathy (0% vs 22%, P = 0.015), biliary complications (20% vs 46%, P = 0.042), and 1-year graft survival (90% vs 69%, P = 0.035). No graft failure due to intrahepatic cholangiopathy or nonfunction occurred in HOPE-treated livers, whereas 18% of unperfused DCD livers needed retransplantation. In addition, HOPE-perfused DCD livers achieved similar results as control donation after brain death livers in all investigated endpoints. HOPE seems to offer important benefits in preserving higher-risk DCD liver grafts.

Extracorporeal Hypothermic Perfusion Device for Intestinal Graft Preservation to Decrease Ischemic Injury During Transportation

IntroductionThe small intestine is one of the most ischemia-sensitive organs used in transplantation. To better preserve the intestinal graft viability and decrease ischemia-reperfusion injury, a device for extracorporeal perfusion was developed. We present the results for the first series of perfused human intestine with an intestinal perfusion unit (IPU). MethodsFive human intestines were procured for the protocol. (1) An experimental segment was perfused by the IPU delivering cold preservation solution to the vascular and luminal side continually at 4 ºC for 8 h. (2) Control (jejunum and ileum) segments were preserved in static cold preservation. Tissue samples were obtained for histopathologic grading according to the Park/Chiu scoring system (0 = normal, 8 = transmural infarction). ResultsJejunal experimental segments scored 2.2 with the Park/Chiu system compared to the control segments, which averaged 3.2. Overall scoring for ileum experimental and control segments was equal with 1.6. ConclusionThis data presents proof of concept that extracorporeal intestinal perfusion is feasible. The evidence shows that the IPU can preserve the viability of human intestine, and histopathologic evaluation of perfused intestine is favorable. Our early results can eventually lead to expanding the possibilities of intestinal preservation.

Effects of Inhibitors of Necroptosis and Autophagy on Morphofunctional Characteristics of the Myocardium during Static Cold Storage of Donor Rat Heart.

Cardioprotective effects of necrostatin-1 (necroptosis inhibitor) and 3-methyladenine (autophagy inhibitor) were studied on the model of long-term cold preservation of rat heart. Addition of necrostatin-1 (490 nmol/liter) or 3-methyladenine (4.5 mmol/liter) to custodiol preserving solution reduced myocardial infarction size and improved left-ventricular function during reperfusion after 8-h preservation at 4°C. Inhibition of necroptosis and autophagy contributed to the cardioprotective effect under conditions of cold preservation of the donor heart.

Ex Situ Limb Perfusion System to Extend Vascularized Composite Tissue Allograft Survival in Swine.

Organ perfusion systems have successfully been applied in solid organ transplantations. Their use in limb transplantation and replantation has not been widely investigated. In this study, we tested the potential for ex situ perfusion system to prolong limb allograft viability in a swine forelimb amputation/replantation model. Fourteen swine were used. In group 1 (n = 4), we perfused 4 amputated limbs for 12 hours using warm (27 °C-32 °C) autologous blood. Group 2 (n = 3) served as a cold preservation control group, preserving limbs for 6 hours at 4 °C. All limbs were transplanted into healthy swine (n = 7) and observed for another 12 hours. Hemodynamic variables of circulation, as well as perfusate gases and electrolytes (pH, pCO2, pO2, O2 saturation, Na(+), K(+), Cl(-), Ca(2+), HCO3(-), glucose, lactate) were measured. Muscle samples were used to measure single-muscle fiber contractility. In the control group, no microcirculation was observed after 6 hours of cold storage. In the pump perfusion group, all limbs displayed a gradual increase in lactate levels (P < 0.05) during ex situ perfusion that returned to normal after transplantation and reperfusion (P = 0.05). The pH and potassium remained stable throughout the experiment. Single-muscle fiber contractility testing showed near normal contractility at the end of the reperfusion period (P > 0.05). Limb weight did not increase significantly between the end of pump perfusion and reperfusion (P > 0.05). We demonstrated the potential to preserve limb allograft using ex vivo circulation. This approach promises to extend the narrow time frame for revascularization of procured extremities in limb transplantation.

Elevated Cardiac Troponin I in Preservation Solution Is Associated With Primary Graft Dysfunction

BackgroundAlthough primary graft dysfunction (PGD) is a leading cause of mortality and morbidity early post-heart transplant, relatively little is known regarding mechanisms involved in PGD development. Methods and ResultsWe examined the relationship between cardiac troponin I (cTnI) concentrations in the preservation solution from 43 heart transplant procedures and the development of PGD. Donor hearts were flushed with cold preservation solution (University of Wisconsin [UW] or Custodiol) and stored in the same solution. cTnI concentrations were measured utilizing the i-STAT System and normalized to left ventricular mass. Recipient medical records were reviewed to determine PGD according to the 2014 ISHLT consensus conference. Nineteen patients developed PGD following cardiac transplantation. For both UW and Custodiol, normalized cTnI levels were significantly increased (P = .031 and .034, respectively) for those cases that developed PGD versus no PGD. cTnI levels correlated with duration of ischemic time in the UW group, but not for the Custodiol group. Donor age and donor cTnI (obtained prior to organ procurement) did not correlate with preservation cTnI levels in either UW or Custodiol. ConclusionsIncreased preservation solution cTnI is associated with the development of PGD suggesting preservation injury may be a dominant mechanism for the development of PGD.

The Effects of Direct Oxygen Supply During Static Cold Preservation of Rat Livers: An Experimental Study.

We aimed to determine the biochemical and histopathologic effects of direct oxygen supply to the preservation fluid of static cold storage system with a simple method on rat livers. Sixteen rats were randomly divided into 2 groups: the control group, which contained Ringer's lactate as preservation fluid; and the oxygen group, which contained oxygen and Ringer's lactate for preservation. Each liver was placed in a bag containing 50 mL Ringer's lactate and placed in ice-filled storage containers. One hundred percent oxygen supplies were given via a simple, inexpensive system created in our laboratory, to the livers in oxygen group. We obtained samples for histopathologic evaluation in the 12th hour. In addition, 3 mL of preservation fluid was subjected to biochemical analysis at 0, sixth, and twelfth hours. Aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, and pH levels were measured from the preservation fluid. In oxygen-supplemented group, the acceleration speed of increase in alanine aminotransferase and lactate dehydrogenase levels at sixth hour and lactate dehydrogenase, alanine aminotransferase, and lactate dehydrogenase levels at 12th hour were statistically significantly reduced. In histopathologic examination, all parameters except ballooning were statistically significantly better in the oxygen-supplemented group. This simple system for oxygenation of liver tissues during static cold storage was shown to be effective with good results in biochemical and histopathologic assessments. Because this is a simple, inexpensive, and easily available method, larger studies are warranted to evaluate its effects (especially in humans).