Machine Perfusion Research Articles

Added Value of Histological Evaluation of Muscle Biopsies in Porcine Vascularized Composite Allografts.

Background: Machine perfusion (MP) offers extended preservation of vascularized complex allografts (VCA), but the diagnostic value of histology using hematoxylin and eosin (H&E) in detecting ischemia-reperfusion injury (IRI) in muscle cells remains unclear. This study aims to document the application of the Histology Injury Severity Score (HISS) and to assess whether additional staining for nicotinamide adenine dinucleotide (NADH) and membrane attack complex (MAC) improves IRI detection in a porcine limb replantation model. Methods: The forelimbs of 16 Dutch Landrace pigs were amputated and preserved for 24 h using hypothermic MP (n = 8) with Histidine-Tryptophan-Ketoglutarate (HTK) or for 4 h with SCS (n = 8) before heterotopic replantation and 7 days of follow-up. Muscle damage was assessed via biochemical markers and light microscopy using H&E, NADH, and MAC at baseline, post-intervention, and post-operative day (POD) 1, 3, and 7 timepoints, using the HISS and a self-developed NADH and MAC score. Results: H&E effectively identified damaged muscle fibers and contributed to IRI assessment in porcine limbs (p < 0.05). The highest HISS was measured on POD 3 between MP (4.9) and SCS (3.5) (p = 0.029). NADH scores of both preservation groups varied over the 7-day follow-up and were statistically insignificant compared with baseline measurements (p > 0.05). MAC revealed no to minimal necrotic tissue across the different timepoints. Conclusions: This study documents the application of the HISS with H&E to detect IRI in muscle fibers. NADH and MAC showed no significant added diagnostic utility. The 24 h MP showed similar muscle alterations using the HISS compared to that of the 4 h SCS after a 7-day follow up.

Current and Future Applications of Machine Perfusion and Other Dynamic Preservation Strategies in Liver Transplantation

Current and Future Applications of Machine Perfusion and Other Dynamic Preservation Strategies in Liver Transplantation

Impact of device variability and protocol differences on kidney function during normothermic machine perfusion: A comparative study using porcine and human kidneys.

A growing interest in renal normothermic machine perfusion (NMP) has resulted in more clinically available perfusion devices. While all perfusion systems have the same aim, there are significant differences in their circuits, pumps, sensors, and software. Therefore, our objective was to assess the impact of different perfusion protocols and devices on kidney function and perfusion parameters during NMP. Porcine kidneys were subjected to 30 min of warm ischemia, 24 h of static cold storage, and subsequently perfused for 6 h using (1) the Kidney Assist (KA) machine with a pressure of 75 mm Hg, (2) the KA device incorporating several adjustments and a pressure of 85 mm Hg (modified KA), or (3) the Perlife (PL) perfusion device (n = 4). Consecutively, discarded human kidneys were perfused using the KA or modified KA (n = 3) protocol. The PL group quickly reached the device's upper flow limit and consequently received a significantly lower pressure compared to the KA groups. The arterial pO2 was significantly lower in the PL group. Yet, hemoglobin concentration increased over time, and oxygen consumption was significantly higher compared to the KA groups. Fractional sodium excretion was significantly lower in the PL group. Tissue ATP levels, urine production, and creatinine clearance rates did not differ between groups. In human kidneys, the modified KA group showed significantly lower vascular resistance, higher oxygen delivery, and lower levels of lactate in the perfusate compared to the KA group. This study shows that perfusion characteristics and kidney function are significantly influenced by the perfusion protocol and the device and its settings during normothermic machine perfusion and therefore should be interpreted with caution.

Mitochondrial Injury assessed in Bile during Normothermic Machine Perfusion predicts Biliary Complications after Liver Transplantation

Mitochondrial Injury assessed in Bile during Normothermic Machine Perfusion predicts Biliary Complications after Liver Transplantation

Fueling success: Harnessing liver bioenergetics in clinical- and long-term machine perfusion

Fueling success: Harnessing liver bioenergetics in clinical- and long-term machine perfusion

Comparative Analysis of Digestion Methods for Bile Proteomics: The Key to Unlocking Biliary Biomarker Potential.

Bile's potential to reflect the health of the biliary system has led to increased attention, with proteomic analysis offering deeper understanding of biliary diseases and potential biomarkers. With the emergence of normothermic machine perfusion (NMP), bile can be easily collected and analyzed. However, the composition of bile can make the application of proteomics challenging. This study systematically evaluated various trypsin digestion methods to optimize proteomics of bile from human NMP livers. Bile was collected from 12 human donor livers that were accepted for transplantation after the NMP viability assessment. We performed tryptic digestion using six different methods: in-gel, in-solution, S-Trap, SMART, EasyPep, and filter-aided sample purification, with or without additional precipitation before digestion. Proteins were analyzed using untargeted proteomics. Methods were assessed for total protein IDs, variation, and protein characteristics to determine the most optimal method. Methods involving precipitation surpassed crude methods in protein identifications (4500 vs 3815) except for in-gel digestion. Filtered data (40%) resulted in 3192 versus 2469 for precipitated and crude methods, respectively. We found minimal differences in mass, cellular components, or hydrophobicity of proteins between methods. Intermethod variability was notably diverse, with in-gel, in-solution, and EasyPep outperforming others. Age-related biological comparisons revealed upregulation of metabolic-related processes in younger donors and immune response and cell cycle-related processes in older donors. Variability between methods emphasizes the importance of cross-validation across multiple analytical approaches to ensure robust analysis. We recommend the in-gel crude method for its simplicity and efficiency, avoiding additional precipitation steps. Sample processing speed, cost, cleanliness, and reproducibility should be considered when a digestion method is selected for bile proteomics.

Ex Vivo Simultaneous H215O Positron Emission Tomography and Magnetic Resonance Imaging of Porcine Kidneys-A Feasibility Study.

Pig kidneys (n = 7) were connected to a NMP circuit. PET/MRI was performed at two different pump flow levels: a blood-oxygenation-level-dependent (BOLD) MRI sequence performed simultaneously with a H215O PET sequence for determination of RBP. RBP was measured using H215O PET in all kidneys (flow 1: 0.42-0.76 mL/min/g, flow 2: 0.7-1.6 mL/min/g). We found a linear correlation between changes in delivered blood flow from the perfusion pump and changes in the measured RBP using PET imaging (r2 = 0.87). Our study demonstrated the feasibility of combined H215O PET/MRI during NMP of isolated porcine kidneys with tissue oxygenation being stable over time. The introduction of H215O PET/MRI in nephrological research could be highly relevant for future pre-transplant kidney evaluation and as a tool for studying renal physiology in healthy and diseased kidneys.

Providing red blood cells to facilitate organ transplant via normothermic perfusion techniques: A single-center experience.

Normothermic regional perfusion (NRP) and normothermic machine perfusion (NMP) are organ procurement and transport techniques that can improve organ quality, facilitate longer transport, and reduce postoperative complications, increasing organ availability and improving outcomes. NRP and NMP often require allogeneic red blood cells (RBCs). Our academic transfusion service began providing RBCs to support NRP and NMP for adult heart transplant (HT), orthotopic liver transplant (OLT), and multiorgan transplant (MOT) in August 2020. This single-center, retrospective study describes the implementation process and analyzes the characteristics of RBC support during the first 3 years of the perfusion programs. Timing and quantity of units issued and used, organ recipient demographics, and transplant outcomes were obtained from transfusion service and electronic medical records. From 2020 to 2023, the transfusion service received 233 requests to support NRP and NMP perfusion cases. Of these, 105 cases resulted in RBC use, and units were returned or discarded in 112 cases. A total of 131 patients received perfusion-facilitated transplants (92 HT, 27 OLT, and 12 MOT). The majority of perfusion-facilitated HTs utilized NRP (81/92, 88%), whereas most perfusion-facilitated OLTs utilized NMP (21/27, 78%). Across all 233 requests, a total of 381 RBC units were used to facilitate 131 transplants, averaging 2.91 units/transplant. Provision of RBCs for NRP and NMP techniques represents a novel method for transfusion services to support and facilitate life-saving organ transplants with only modest product use, about three RBC units per organ transplant in this single-center study.

Sub-zero non-freezing of vascularized composite allografts in a rodent partial hindlimb model

Ischemia is a major limiting factor in Vascularized Composite Allotransplantation (VCA) as irreversible muscular injury can occur after as early as 4–6 h of static cold storage (SCS). Organ preservation technologies have led to the development of storage protocols extending rat liver ex vivo preservation up to 4 days. Development of such a protocol for VCAs has the added challenge of inherent ice nucleating factors of the graft, therefore, this study focused on developing a robust protocol for VCA supercooling. Rodent partial hindlimbs underwent subnormothermic machine perfusion (SNMP) with several loading solutions, followed by a storage solution with cryoprotective agents (CPA) developed for VCAs. Storage occurred in suspended animation for 24h and VCAs were recovered using SNMP with modified Steen. This study shows a robust VCA supercooling preservation protocol in a rodent model. Further optimization is expected to allow for its application in a transplantation model, which would be a breakthrough in the field of VCA preservation.

Hypothermic oxygenated machine perfusion influences the immunogenicity of donor livers in humans.

Hypothermic oxygenated machine perfusion (HOPE) is an organ preservation strategy shown to reduce ischemia-reperfusion-injury (IRI)-related complications following liver transplantation (LT). In animal models HOPE can also decrease alloimmune responses post-transplantation, but this remains to be evaluated in humans. Our study, involving 27 LT patients enrolled in 2 randomised controlled trials comparing static cold storage (SCS) with HOPE (14 HOPE- and 13 SCS-treated), delves into the impact of HOPE on the molecular profile of liver allografts and on the immune responses elicited post-transplantation. Following HOPE treatment, fewer intra-hepatic immune cells were observed in liver perfusates compared to SCS. Analysis of liver tissue transcriptome at reperfusion revealed an effect of HOPE on the reactive oxygen species pathway. Two weeks post-transplantation, HOPE recipients exhibited increased circulating CD4+FOXP3+CD127lo regulatory T cells (Tregs) (p<0.01), which corresponded to a higher frequency of donor specific Tregs (p<0.01) and was followed by reduced alloreactivity index of CD8+ T cells 3 months post-transplant. Our study provides novel mechanistic insight into the capacity of HOPE to influence liver IRI and to modulate effector and regulatory donor-specific T cell responses post-transplantation. These findings, which confirm observations made in animal models, help explain the decreased rejection rates reported in patients receiving HOPE-treated allografts.

Prolongation of the Time Window From Traumatic Limb Amputation to Replantation From 6 to 33 Hours Using Ex Vivo Limb Perfusion.

Continuous extracorporeal perfusion (ECP), or machine perfusion, holds promise for prolonged skeletal muscle preservation in limb ischemia-reperfusion injury. This study aimed to extend the amputation-to-replantation time window from currently 6 hours to 33 hours using a 24-hour ECP approach. Six large white pigs underwent surgical forelimb amputation under general anesthesia. After amputation, limbs were kept for 9 hours at room temperature and then perfused by 24-hour ECP with a modified histidine-tryptophan-ketoglutarate (HTK) solution. After ECP, limbs were orthotopically replanted and perfused in vivo for 12 hours. Clinical data, blood, and tissue samples were collected and analyzed. All 6 forelimbs could be successfully replanted and in vivo reperfused for 12 hours after 9 hours of room temperature ischemia followed by 24 hours ECP. Adequate limb perfusion was observed after replantation as shown by thermography and laser Doppler imaging. All pigs survived without severe organ failure, and no significant increase in inflammatory cytokines was found. Macroscopy and histology showed marked interstitial muscular edema of the limbs, whereas myofiber necrosis was not evident, implying the preservation of muscular integrity. The use of a 24-hour ECP has successfully extended limb preservation to 33 hours. The modified histidine-tryptophan-ketoglutarate perfusate demonstrated its ability for muscle protection. This innovative approach not only facilitates limb replantation after combat injuries, surmounting geographical barriers, but also broadens the prospects for well-matched limb allotransplants across countries and continents.

Improving normothermic machine perfusion and blood transfusion through biocompatible blood silicification

The growing world population and increasing life expectancy are driving the need to improve the quality of blood transfusion, organ transplantation, and preservation. Here, to improve the ability of red blood cells (RBCs) for normothermic machine perfusion, a biocompatible blood silicification approach termed "shielding-augmenting RBC-in-nanoscale amorphous silica (SARNAS)" has been developed. The key to RBC surface engineering and structure augmentation is the precise control of the hydrolysis form of silicic acid to realize stabilization of RBC within conformal nanoscale silica-based exoskeletons. The formed silicified RBCs (Si-RBCs) maintain membrane/structural integrity, normal cellular functions (e.g., metabolism, oxygen-carrying capability), and enhance resistance to external stressors as well as tunable mechanical properties, resulting in nearly 100% RBC cryoprotection. In vivo experiments confirm their excellent biocompatibility. By shielding RBC surface antigens, the Si-RBCs provide universal blood compatibility, the ability for allogeneic mechanical perfusion, and more importantly, the possibility for cross-species transfusion. Being simple, reliable, and easily scalable, the SARNAS strategy holds great promise to revolutionize the use of engineered blood for future clinical applications.

A meta-analysis of perfusion parameters affecting weight gain in exvivo perfusion.

Ex vivo machine perfusion (EVMP) has been established to extend viability of donor organs. However, EVMP protocols are inconsistent. We hypothesize that there is a significant relationship between specific parameters during EVMP and perfusion outcomes. A meta-analysis of literature was conducted in accordance with the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) Statement. The search encompassed articles published before July 25, 2023. PubMed, Embase, and CENTRAL databases were screened using search terms "ex-vivo," "ex-situ," "machine," and "perfusion." Weight gain, an indicator of organ viability, was chosen to compare outcomes. Extracted variables included perfused organ, warm and cold ischemia time before perfusion, perfusion duration, perfusate flow, pressure, temperature, perfusate composition (presence of cellular or acellular oxygen carrier, colloids, and other supplements) and percent weight change. Data were analyzed using SPSS statistical software. Overall, 44 articles were included. Red blood cell-based perfusates resulted in significantly lower weight gain compared to acellular perfusates without oxygen carriers (11.3% vs. 27.0%, p < 0.001). Hemoglobin-based oxygen carriers resulted in significantly lower weight gain compared to acellular perfusates (16.5% vs. 27%, p = 0.006). Normothermic perfusion led to the least weight gain (14.6%), significantly different from hypothermic (24.3%) and subnormothermic (25.0%) conditions (p < 0.001), with no significant difference between hypothermic and subnormothermic groups (24.3% vs. 25.0%, p = 0.952). There was a positive correlation between flow rate and weight gain (ß = 13.1, R = 0.390, p < 0.001). Oxygen carriers, low flow rates, and normothermic perfusate temperature appear to improve outcomes in EVMP. These findings offer opportunities for improving organ transplantation outcomes.

Innovations in transplant techniques for complex anomalies.

With advances in the field of congenital cardiac surgery and in the management of congenital heart defects in early life, the population of adult congenital heart disease (ACHD) patients is increasing. End-stage heart failure is currently the main cause of cardiovascular mortality and is expected to increase in the coming years. This review summarizes recent innovations in transplant techniques, with special attention to what is known in the population of ACHD recipients. The use of machine perfusion for heart preservation enables longer preservation times. Normothermic (organ care system - OCS) and hypothermic (hypothermic oxygenated perfusion - HOPE) machine perfusion will alleviate the time pressure associated with heart transplantation in the ACHD population, may allow for expansion of the geographical range in which donors can be matched and may improve graft quality. Donation after circulatory death (DCD) heart transplantation, either through direct procurement-machine perfusion (DP-MP) or thoraco-abdominal normothermic regional perfusion (TA-NRP) is a viable strategy to further expand the donor pool. The use of machine perfusion and DCD donors in ACHD is feasible and shows promise. Time pressure and shortage of donors is even more critical in ACHD than in other patient populations, making these innovations particularly relevant. Further clinical experience and research is needed to elucidate their impact.

Assessment of hemodynamic and blood parameters that may reflect macroscopic quality of porcine kidneys during normothermic machine perfusion using whole blood

PurposeUsing ex vivo normothermic machine perfusion (NMP) with whole blood we assessed marginal porcine kidneys under reperfusion. The aim was to link measureable machine and clinical blood parameters with the currently used visual assessment. This could serve as a baseline for a standardized evaluation score to identify potentially transplantable kidneys in the future.MethodsKidneys and autologous whole blood were procured from slaughterhouse pigs (n = 33) and were perfused for 4 h using NMP. The hemodynamic parameters arterial pressure (AP), renal blood flow (RBF) and intrarenal resistance (IRR) were measured. Activity of aspartate transaminase (AST), gamma-glutamyltransferase (GGT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH) and lactate were assessed in blood at 0/1/2/4 h. Kidneys were grouped into “potentially transplantable” (PT) or “not transplantable” (NT) based on their overall macroscopic appearance after NMP by an experienced physician.ResultsPT-kidneys (n = 20) had a significantly lower IRR and higher RBF than NT-kidneys (n = 13). GGT, ALP and LDH did not differ significantly, but at 4 h, AST was significantly higher in PT-kidneys compared to NT-kidneys. Lactate levels kept increasing during NMP in NT-kidneys and were significantly higher at 1/2/4 h than in PT-kidneys.ConclusionThe immediately assessed macroscopic aspects of examined kidneys correlated with hemodynamic parameters, increased lactate and lower AST in this study. In the future, NMP with whole blood could be a useful tool to extend the donor pool by allowing the assessment of otherwise unknown characteristics of marginal kidneys before transplantation.

Protective effects of mesenchymal stem cells-derived extracellular vesicles against ischemia-reperfusion injury of hearts donated after circulatory death: Preliminary study in a pig model

IntroductionInsufficient supply of cardiac grafts represents a severe obstacle in heart transplantation. Donation after Circulatory Death (DCD), in addition to conventional donation after brain death, is one promising option to overcome the organ shortage. However, DCD organs undergo an inevitable more extended period of warm unprotected ischemia between circulatory arrest and graft procurement. Mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) have shown remarkable protective effects against ischemia-reperfusion injury. Thus, we aimed to enhance grafts preservation from DCD donors, through treatment with MSC-EVs. MethodsFemale pigs were euthanized by barbiturate overdose and after 20 min of a flat EKG, the chest was opened, the heart harvested and subsequently connected to an extracorporeal perfusion machine. MSC-EVs, isolated by ion exchange chromatography, were added to the perfusion solution (1×1011 particles) and the heart was perfused for 2 h. Then, heart tissue biopsies were taken to assess histological changes, mitochondrial morphology, antioxidant enzyme activity and inflammation mediators’ expression. Biochemical parameters of myocardial viability were assessed in the perfusate. ResultsThe treatment with MSC-EVs significantly prevented mitochondria swelling, mitochondrial cristae loss and oxidative stress in cardiac tissue. The protective effect of MSC-EVs was confirmed by the delayed increase of the cardiac-specific enzymes CK and TnC in the perfusate and the reduction of caspase-3+ cells in tissue sections. ConclusionMSC-EVs improve graft quality by preserving the mitochondrial ultrastructure protecting the myocardium against oxidative stress, reducing apoptosis of cardiac cells and preventing the increase of pro-inflammatory cytokines.

Mitochondrial Function After Normothermic Regional Perfusion or Direct Procurement Followed by Hypothermic Oxygenated Machine Perfusion in Heart Transplantation After Circulatory Death.

Strategies to minimize ischemic damage during heart transplantation (HTX) by donation after circulatory death (DCD) are warranted because the inevitable ischemic injury linked to DCD HTX deteriorates mitochondrial respiratory capacity and ultimately graft quality. This study aimed to examine the myocardial mitochondrial function during DCD HTX with hypothermic oxygenated machine perfusion (HOPE) and compare the effect of normothermic regional perfusion (NRP) with that of direct procurement and perfusion (DPP). A porcine DCD HTX model was used with hearts subjected to either DPP (n = 6) or NRP (n = 7) followed by HOPE and orthotopic HTX. Mitochondrial respiratory function was analyzed by high-resolution respirometry in left ventricle biopsies at baseline, after 180 min of HOPE, and after 60 min of reperfusion post-HTX. Mitochondrial oxidative phosphorylation (P = 0.0008), respiratory control ratio (P = 0.04), and coupling efficiency (P = 0.04) declined during DCD HTX. Fatty acid oxidation was preserved after 3 h of HOPE with a modest, statistically nonsignificant decline after reperfusion (P = 0.2). Oxidative phosphorylation was inversely correlated with troponin-T levels (r = -0.70, P = 0.0004). No statistically significant difference in mitochondrial respiratory capacity was observed between participants exposed to NRP and DPP. Mitochondrial respiratory capacity declined gradually throughout the course of DCD HTX and correlated with the degree of myocardial damage. Following HOPE, the extent of mitochondrial deterioration was comparable between NRP and DPP.

Machine Perfusion of Donor Hearts – The Recovery and Transplantation of Previously Unrecoverable Hearts

Heart transplantation remains the gold standard treatment for end-stage heart failure. With an increasing population and improving medical therapy there is an increasing number of patients who would benefit from heart transplantation but are unable to receive one due to a shortage of suitable donor organs. This ongoing need, and significant limitations with traditional cold static storage (CSS), have driven the development of machine perfusion technology that have expanded the donor pool. Machine perfusion is divided into hypothermic and normothermic, with both technologies perfusing the heart with oxygenated solution to reduce the degree of ischaemia-reperfusion injury that occurs. Both technologies have allowed for the safe retrieval and transplantation of hearts with prolonged ischaemic times or following donation after circulatory determination of death and have significantly increased the potential donor pool and number of transplants able to be safely performed. The aim of this review is to summarise the current options available to clinicians for donor heart organ preservation, with a focus on how the development of machine perfusion technology has allowed for the recovery of hearts previously considered unrecoverable using CSS.

Revolutionizing deceased donor transplantation: How new approaches to machine perfusion broadens the horizon for organ donation

Solid organ transplantation is lifesaving for persons with end-stage organ disease. Thanks to advancements in organ preservation, surgeons are now able to successfully transplant organs that were previously considered high risk for poor graft function. Innovations in perfusion machine types, preservation solutions and additives to preservation solutions have significantly improved the ability to utilize organs previously thought unusable.Newer organ preservation techniques are offering a promising outlook for extending graft longevity and improving transplant outcomes. This review explores the impact of deceased donor type on graft quality and highlights emerging strategies designed to improve the function and viability of deceased donor organs.

Cardiac Loading using Passive Left Atrial Pressurization and Passive Afterload for Graft Assessment.

Ex vivo machine perfusion or normothermic machine perfusion is a preservation method that has gained great importance in the transplantation field. Despite the immense opportunity for assessment due to the beating state of the heart, current clinical practice depends on limited metabolic trends for graft evaluation. Hemodynamic measurements obtained from left ventricular loading have garnered significant attention within the field due to their potential as objective assessment parameters. In effect, this protocol provides an easy and effective manner of incorporating loading capabilities to established Langendorff perfusion systems through the simple addition of an extra reservoir. Furthermore, it demonstrates the feasibility of employing passive left atrial pressurization for loading, an approach that, to our knowledge, has not been previously demonstrated. This approach is complemented by a passive Windkessel base afterload, which acts as a compliance chamber to maximize myocardial perfusion during diastole. Lastly, it highlights the capability of capturing functional metrics during cardiac loading, including left ventricular pulse pressure, contractility, and relaxation, to uncover deficiencies in cardiac graft function after extended periods of preservation times (˃6 h).