Machine Perfusion and Bioengineering Strategies in Transplantation-Beyond the Emerging Concepts.

Solid organ transplantation has progressed rapidly over the decades from the first experimental procedures to its role in the modern era as an established treatment for end-stage organ disease. Solid organ transplantation including liver, kidney, pancreas, heart, and lung transplantation, is the definitive option for many patients, but despite the advances that have been made, there are still significant challenges in meeting the demand for viable donor grafts. Furthermore, post-operatively, the recipient faces several hurdles, including poor early outcomes like primary graft dysfunction and acute and chronic forms of graft rejection. In an effort to address these issues, innovations in organ engineering and treatment have been developed. This review covers efforts made to expand the donor pool including bioengineering techniques and the use of ex vivo graft perfusion. It also covers modifications and treatments that have been trialed, in addition to research efforts in both abdominal organs and thoracic organs. Overall, this article discusses recent innovations in machine perfusion and organ bioengineering with the aim of improving and increasing the quality of donor organs.

Isolation and Cryopreservation of Primary Macaque Hepatocytes.

Primary human hepatocytes (PHHs) are recognized as the "gold standard" for evaluating toxicity of various drugs or chemicals in vitro. However, due to their limited availability, primary hepatocytes isolated from rodents are more commonly used in various experimental studies than PHHs. However, bigger differences in drug metabolism were seen between humans and rats compared to those between human and non-human primates. Here, we describe a method to isolate primary hepatocytes from the liver of rhesus macaques (Macaca mulatta, a species of Old-World monkey) after in situ whole liver perfusion. Techniques for cryopreserving and recovering primary macaque hepatocytes (PMHs) are also described. Given the remarkable physiological and genetic similarity of non-human primates to humans, PMHs isolated using this protocol may serve as a reliable surrogate of PHHs in toxicological research and preclinical studies. Published 2024. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: In situ whole liver perfusion Basic Protocol 2: Primary macaque hepatocyte isolation and cell plating Basic Protocol 3: Cryopreservation and recovery of primary macaque hepatocytes.

Liver Graft Machine Perfusion: From History Perspective to Modern Approaches in Transplant Surgery.

The shortage of donor organs remains an unresolved issue in livertransplantation worldwide. Consequently, strategies for expanding the donor pool are currently being developed. Donors meeting extended criteria undergo thorough evaluation, as livers obtained from marginal donors yield poorer outcomes in recipients, including exacerbated reperfusion injury, acute kidney injury, early graft dysfunction, and primary nonfunctioning graft. However, the implementation of machine perfusion has shown excellent potential in preserving donor livers and improving their characteristics to achieve better outcomes for recipients. In this review, we analyzed the global experience of using machine perfusion in livertransplantation through the history ofthe development ofthis method to the latest trends and possibilities for increasing the number of liver transplants.

Normothermic Regional Perfusion in Controlled Donation After Circulatory Death Liver Transplantation: A Systematic Review and Meta-Analysis.

Liver grafts from controlled donation after circulatory death (cDCD) donors have lower utilization rates due to inferior graft and patient survival rates, largely attributable to the increased incidence of ischemic cholangiopathy, when compared with grafts from brain dead donors (DBD). Normothermic regional perfusion (NRP) may improve the quality of cDCD livers to allow for expansion of the donor pool, helping to alleviate the shortage of transplantable grafts. A systematic review and metanalysis was conducted comparing NRP cDCD livers with both non-NRP cDCD livers and DBD livers. In comparison to non-NRP cDCD outcomes, NRP cDCD grafts had lower rates of ischemic cholangiopathy [RR = 0.23, 95% CI (0.11, 0.49), p = 0.0002], primary non-function [RR = 0.51, 95% CI (0.27, 0.97), p = 0.04], and recipient death [HR = 0.5, 95% CI (0.36, 0.69), p < 0.0001]. There was no difference in outcomes between NRP cDCD donation compared to DBD liver donation. In conclusion, NRP improved the quality of cDCD livers compared to their non-NRP counterparts. NRP cDCD livers had similar outcomes to DBD grafts. This provides further evidence supporting the continued use of NRP in cDCD liver transplantation and offers weight to proposals for its more widespread adoption.

Surgical techniques for cardiac procurement, preparation and perfusion using the Organ Care System.

We provide an audio-visual step-by-step guide to the preparation of a donor heart for the application of normothermic, ex situ cardiac perfusion on the TransMedics Organ Care System using a heart donated after brain death. The use of the Organ Care System increases heart transplantation activity by enabling the utilization of hearts donated after circulatory death, the use of extended criteria grafts and the extension of out-of-body time, which can help overcome geographic or surgical barriers. Ex situ cardiac perfusion is a new technique and is therefore not yet routinely performed in many centres. However, it can be assumed that this technique will become more established and widespread in the future. Our video tutorial, which summarizes all important steps, can therefore be of benefit to surgical teams for planning, training or as a refresher.

Perfusate Exchange Does Not Improve Outcomes in 24-hour Ex Situ Lung Perfusion.

BACKGROUND: Reliable 24-hour preservation is required to optimize the rehabilitation potential of Ex Situ Lung Perfusion (ESLP). Other ESLP protocols include fresh perfusate replacement to counteract an accumulation of deleterious by-products. We describe the results of our reliable 24-hour negative pressure ventilation (NPV)-ESLP protocol with satisfactory acute post-transplant outcomes and investigate perfusate exchange (PE) as a modification to enhance prolonged ESLP. METHODS: Twelve pig lungs underwent 24 hours of NPV-ESLP using 1.5L of cellular perfusate (500 mL packed red blood cells and 1 L buffered perfusate). The Control (n = 6) had no PE; the PE (n = 6) had 500 mL replaced after 12 hours of NPV-ESLP with 1000 mL fresh perfusate. Three left lungs per group were transplanted. RESULTS: Results are reported as Control vs PE (mean ± SEM). Both groups demonstrated stable and acceptable oxygenation during 24 hours of ESLP with final PF ratios of 527.5 ± 42.19 and 488.4 ± 35.38 (P = .25). Final compliance measurements were 20.52 ± 3.59 and 18.55 ± 2.91 (P = .34). There were no significant differences in pulmonary artery pressure after 24 hours of ESLP (10.02 ± 2.69 vs 14.34 ± 1.64, P = .10), and pulmonary vascular resistance only differed significantly at T12 (417.6 ± 53.06 vs 685.4 ± 81.19, P = .02). Percentage weight gain between groups was similar (24.32 ± 8.4 and 45.33 ± 7.76, P = .07). Post-transplant left lung oxygenation was excellent (327.3 ± 14.62 and 313.3 ± 15.38, P = .28). There was no significant difference in % weight gain of lungs post-transplant (22.20 ± 7.22 vs 14.36 ± 9.96, P = .28). CONCLUSION: Acceptable lung function was maintained during 24-hour NPV-ESLP and post-transplant regardless of PE.

Moderate-Flow Perfusion is Superior to Low-Flow Perfusion in Ex Situ Lung Perfusion.

BACKGROUND: Full-flow perfusion during prolonged ex situ lung perfusion (ESLP) results in unacceptable pulmonary edema formation. Clinical ESLP at 30% to 50% predicted cardiac output (CO) supports acceptable physiologic outcomes; however, progressive pulmonary edema still develops. Lower flow rates may provide equivalent physiologic preservation with less edema formation due to reduced hydrostatic pressures. We report our results of moderate-flow (MF; 30% CO) vs low-flow (LF; 10% CO) negative pressure ventilation (NPV)-ESLP with transplantation. METHODS: Twelve pig lungs underwent 12-hours of NPV-ESLP with 30% or 10% CO (n = 6/group). Three left lungs per group were transplanted post-ESLP and assessed in vivo over 4 hours. Lung function was assessed by physiologic parameters, weight-gain, and pro-inflammatory cytokine profiles. RESULTS: Results are MF vs LF (mean ± SEM). All lungs demonstrated acceptable oxygenation post-ESLP (454.2 ± 40.85 vs 422.7 ± 31.68, P = .28); however, after transplantation, the MF lungs demonstrated significantly better oxygenation (300.7 ± 52.26 vs 141.9 ± 36.75, P = .03). There was no significant difference in compliance after ESLP (21.38 ± 2.28 vs 16.48 ± 2.34, P = .08); however, pulmonary artery pressure (PAP; 10.89 ± 2.28 vs 21.11 ± 0.93, P = .06) and pulmonary vascular resistance (PVR; 438.60 ± 97.97 vs 782.20 ± 162.20, P = .05) were significantly higher in the LF group. Weight gain (%) post-ESLP and post-transplant was similar between groups (29.42 ± 5.72 vs 24.17 ± 4.42, P = .24; and 29.63 ± 7.23 vs 57.04 ± 15.78, P = .09). TNF-α and IL-6 were significantly greater throughout LF ESLP. CONCLUSIONS: The MF NPV-ESLP results in superior lung function with less inflammation compared to LF NPV-ESLP.

Ischemia-Free Liver Transplant: Technical Aspects and Considerations After First Case in Western Countries.

The use of marginal donor livers, particularly steatotic livers, could help to resolve the problem of organ shortage and wait list mortality. Ischemia-free liver transplant with the potential to avoid ischemiareperfusion injury and related complications, particularly early allograft dysfunction, could positively encourage the use of marginal donorlivers and extend the donor pool. Here, we describe the first case in a Western country of ischemia-free liver transplant of a marginal donor liver. To date, a research team in China is the only group to have described and used this technique. The technical and setup aspects are illustrated, and present controversies are discussed. A 58-year-old female patient received a transplant of a >60% steatotic donor liver. The transplant was accomplished with the ischemia-free liver transplant technique, and the donor liver was procured and transplanted under continuous normothermic machine perfusion. The donor liver functional parameters under normothermic machine perfusion were reassuring, and recipient recovery was uneventful. Although ischemia-free liver transplant is a technically and organizationally demanding procedure, our case demonstrates the feasibility of the ischemia-free liver transplant technique and encourages the development and expansion of its use.

The Effects of Oxygen-Derived Free-Radical Scavengers During Normothermic Ex-Situ Heart Perfusion.

Oxidative stress occurs during ex-situ heart perfusion (ESHP) and may negatively affect functional preservation of the heart. We sought to assess the status of key antioxidant enzymes during ESHP, and the effects of augmenting these antioxidants on the attenuation of oxidative stress and improvement of myocardial and endothelial preservation in ESHP. Porcine hearts were perfused for 6 hours with oxygen-derived free-radical scavengers polyethylene glycol (PEG)-catalase or PEG-superoxide dismutase (SOD) or with naive perfusate (control). The oxidative stress-related modifications were determined in the myocardium and coronary vasculature, and contractile function, injury, and endothelial integrity were compared between the groups. The activity of key antioxidant enzymes decreased and adding catalase and SOD restored the enzyme activity. Cardiac function and endothelial integrity were preserved better with restored catalase activity. Catalase and SOD both decreased myocardial injury and catalase reduced ROS production and oxidative modification of proteins in the myocardium and coronary vasculature. The activity of antioxidant enzymes decrease in ESHP. Catalase may improve the preservation of cardiac function and endothelial integrity during ESHP. While catalase and SOD may both exert cardioprotective effects, unbalanced SOD and catalase activity may paradoxically increase the production of reactive species during ESHP.

Normothermic ex vivo kidney perfusion preserves mitochondrial and graft function after warm ischemia and is further enhanced by AP39.

We previously reported that normothermic ex vivo kidney  perfusion (NEVKP) is superior in terms of organ protection compared to static cold storage (SCS), which is still the standard method of organ preservation, but the mechanisms are incompletely understood. We used a large animal kidney autotransplant model to evaluate mitochondrial function during organ preservation and after kidney transplantation, utilizing live cells extracted from fresh kidney tissue. Male porcine kidneys stored under normothermic perfusion showed preserved mitochondrial function and higher ATP levels compared to kidneys stored at 4 °C (SCS). Mitochondrial respiration and ATP levels were further enhanced when AP39, a mitochondria-targeted hydrogen sulfide donor, was administered during warm perfusion. Correspondingly, the combination of NEVKP and AP39 was associated with decreased oxidative stress and inflammation, and with improved graft function after transplantation. In conclusion, our findings suggest that the organ-protective effects of normothermic perfusion are mediated by maintenance of mitochondrial function and enhanced by AP39 administration. Activation of mitochondrial function through the combination of AP39 and normothermic perfusion could represent a new therapeutic strategy for long-term renal preservation.

Optimization of an in situ liver perfusion method to evaluate hepatic function of juvenile American alligators (Alligator mississippiensis).

American alligators (Alligator mississippiensis) are a sentinel species whose health is representative of environmental quality. However, their susceptibility to various natural or anthropogenic stressors is yet to be comprehensively studied. Understanding hepatic function in such assessments is essential as the liver is the central organ in the metabolic physiology of an organism, and therefore influences its adaptive capability. In this study, a novel liver perfusion system was developed to study the hepatic physiology of juvenile alligators. First, a cannulation procedure was developed for an in situ liver perfusion preparation. Second, an optimal flow rate of 0.5 ml/min/g liver was determined based on the oxygen content in the effluent perfusate. Third, the efficacy of the liver preparation was tested by perfusing the liver with normoxic or hypoxic Tyrode's buffer while various biomarkers of hepatic function were monitored in the effluent perfusate. Our results showed that in the normoxic perfusion, the aspartate transferase (AST) and lactate/pyruvate ratio in the perfusate remained stable and within an acceptable physiological range for 6 h. In contrast, hypoxia exposure significantly increased the lactate/pyruvate ratio in the perfusate after 2 h, indicating an induction of anaerobic metabolism. These results suggest that the perfused liver remained viable during the perfusion period and exhibited the expected physiological response under hypoxia exposure. The liver perfusion system developed in this study provides an experimental framework with which to study the basic hepatic physiology of alligators and elucidate the effects of environmental or anthropogenic stressors on the metabolic physiology of this sentinel species.

Defining the Spatial Resolution of Analyte Recovery during Microperfusion-Based Sampling of Brain Parenchyma.

The unique architecture of the brain and the blood-brain barrier imposes challenges for the measurement of parenchyma-derived biomarkers that prevent sufficient understanding of transient neuropathogenic processes. One solution to this challenge is direct sampling of brain interstitial fluid via implanted microperfusion probes. Seeking to understand spatial limitations to microperfusion in the brain, we employed computational fluid dynamics modeling and empirical recovery of fluorescently labeled dextrans in an animal model. We found that dextrans were successfully recovered via microperfusion over a 6 h sampling period, especially at probes implanted 2 mm from the dextran infusion point relative to probes implanted 5 mm from the injection site. Experimental recovery was consistently around 1% of simulated, suggesting that this parameter can be used to set practical limits on the maximal tissue concentration of proteins measured in microperfusates and on the spatial domain sampled by our multimodal microperfusion probe.

Using An In Vitro Tissue Perfusion System to Detect the Functional Activities of Isolated Intestinal Tubes in Real Time.

Gastrointestinal diseases, which have a high incidence, pose considerable challenges for humans. The small intestine is integral to food and drug digestion and absorption and plays a crucial role in treating these diseases. The intestinal tube movement experiment, a common and essential in vitro method, is utilized to study gastrointestinal dynamics. This includes the preparation of the isolated intestinal tube, as well as the suspension of the prepared intestinal tube in the bath and its connection to a signal detector. This is followed by the recording and analysis of a series of parameters, such as tension, which can be used to assess intestinal motor function, as well as considerations for keeping the intestinal tube active in vitro. The standardized program from sampling to data collection greatly improves the repeatability of the experimental data and ensures the authenticity of the recording of intestinal tension after physiological, pathological, and drug intervention. Here we present the key problems in experimental operation and a valuable reference experimental protocol for studying drugs that regulate gastrointestinal motility.

Impact of Hemoglobin Level in Ex Vivo Heart Perfusion on Donation After Circulatory Death Hearts: A Juvenile Porcine Experimental Model.

BACKGROUND: Ex vivo heart perfusion (EVHP) of donation after circulatory death (DCD) hearts has become an effective strategy in adults; however, the small circulating volume in pediatrics poses the challenge of a low-hemoglobin (Hb) perfusate. We aimed to determine the impact of perfusate Hb levels during EVHP on DCD hearts using a juvenile porcine model. METHODS: Sixteen DCD piglet hearts (11-14 kg) were reperfused for 4 h in unloaded mode followed by working mode. Metabolism, cardiac function, and cell damage were compared between the low-Hb (Hb, 5.0-5.9 g/dL; n = 8) and control (Hb, 7.5-8.4 g/dL; n = 8) groups. Between-group differences were evaluated using 2-sample t -tests or Fisher's Exact tests. RESULTS: During unloaded mode, the low-Hb group showed lower myocardial oxygen consumption ( P < 0.001), a higher arterial lactate level ( P = 0.001), and worse systolic ventricular function ( P < 0.001). During working mode, the low-Hb group had a lower cardiac output (mean, 71% versus 106% of normal cardiac output, P = 0.010) and a higher arterial lactate level ( P = 0.031). Adjusted cardiac troponin-I ( P = 0.112) did not differ between the groups. Morphological myocyte injury in the left ventricle was more severe in the low-Hb group ( P = 0.028). CONCLUSIONS: Low-Hb perfusate with inadequate oxygen delivery induced anaerobic metabolism, resulting in suboptimal DCD heart recovery and declined cardiac function. Arranging an optimal perfusate is crucial to organ protection, and further endeavors to refine the priming volume of EVHP or the transfusion strategy are required.

Two-day Static Cold Preservation of α1,3-Galactosyltransferase Knockout Kidney Grafts Before Simulated Xenotransplantation.

Transplantation remains the preferred treatment for end-stage kidney disease but is critically limited by the number of available organs. Xenografts from genetically modified pigs have become a promising solution to the loss of life while waiting for transplantation. However, the current clinical model for xenotransplantation will require off-site procurement, leading to a period of ischemia during transportation. As of today, there is limited understanding regarding the preservation of these organs, including the duration of viability, and the associated molecular changes. Thus, our aim was to evaluate the effects of static cold storage (SCS) on α1,3-galactosyltransferase knockout (GGTA1 KO) kidney. After SCS, viability was further assessed using acellular sub-normothermic ex vivo perfusion and simulated transplantation with human blood. Compared to baseline, tubular and glomerular interstitium was preserved after 2 days of SCS in both WT and GGTA1 KO kidneys. Bulk RNA-sequencing demonstrated that only eight genes were differentially expressed after SCS in GGTA1 KO kidneys. During sub-normothermic perfusion, kidney function, reflected by oxygen consumption, urine output, and lactate production was adequate in GGTA1 KO grafts. During a simulated transplant with human blood, macroscopic and histological assessment revealed minimal kidney injury. However, GGTA1 KO kidneys exhibited higher arterial resistance, increased lactate production, and reduced oxygen consumption during the simulated transplant. In summary, our study suggests that SCS is feasible for the preservation of porcine GGTA1 KO kidneys. However, alternative preservation methods should be evaluated for extended preservation of porcine grafts.

Hypothermic oxygenated perfusion of the donor heart in heart transplantation: the short-term outcome from a randomised, controlled, open-label, multicentre clinical trial.

BACKGROUND: Static cold storage (SCS) remains the gold standard for preserving donor hearts before transplantation but is associated with ischaemia, anaerobic metabolism, and organ injuries, leading to patient morbidity and mortality. We aimed to evaluate whether continuous, hypothermic oxygenated machine perfusion (HOPE) of the donor heart is safe and superior compared with SCS. METHODS: We performed a multinational, multicentre, randomised, controlled, open-label clinical trial with a superiority design at 15 transplant centres across eight European countries. Adult candidates for heart transplantation were eligible and randomly assigned in a 1:1 ratio. Donor inclusion criteria were age 18-70 years with no previous sternotomy and donation after brain death. In the treatment group, the preservation protocol involved the use of a portable machine perfusion system ensuring HOPE of the resting donor heart. The donor hearts in the control group underwent ischaemic SCS according to standard practices. The primary outcome was time to first event of a composite of either cardiac-related death, moderate or severe primary graft dysfunction (PGD) of the left ventricle, PGD of the right ventricle, acute cellular rejection at least grade 2R, or graft failure (with use of mechanical circulatory support or re-transplantation) within 30 days after transplantation. We included all patients who were randomly assigned, fulfilled inclusion and exclusion criteria, and received a transplant in the primary analysis and all patients who were randomly assigned and received a transplant in the safety analyses. This trial was registered with ClicalTrials.gov (NCT03991923) and is ongoing. FINDINGS: A total of 229 patients were enrolled between Nov 25, 2020, and May 19, 2023. The primary analysis population included 204 patients who received a transplant. There were no patients who received a transplant lost to follow-up. All 100 donor hearts preserved with HOPE were transplantable after perfusion. The primary endpoint was registered in 19 (19%) of 101 patients in the HOPE group and 31 (30%) of 103 patients in the SCS group, corresponding to a risk reduction of 44% (hazard ratio 0·56; 95% CI 0·32-0·99; log-rank test p=0·059). PGD was the primary outcome event in 11 (11%) patients in the HOPE group and 29 (28%) in the SCS group (risk ratio 0·39; 95% CI 0·20-0·73). In the HOPE group, 63 (65%) patients had a reported serious adverse event (158 events) versus 87 (70%; 222 events) in the SCS group. Major adverse cardiac transplant events were reported in 18 (18%) and 33 (32%) patients in the HOPE and SCS group (risk ratio 0·56; 95% CI 0·34-0·92). INTERPRETATION: Although there was not a significant difference in the primary endpoint, the 44% risk reduction associated with HOPE was suggested to be a clinically meaningful benefit. Post-transplant complications, measured as major adverse cardiac transplant events, were reduced. Analysis of secondary outcomes suggested that HOPE was beneficial in reducing primary graft dysfunction. HOPE in donor heart preservation addresses the existing challenges associated with graft preservation and the increasing complexity of donors and heart transplantation recipients. Future investigation will help to further elucidate the benefit of HOPE. FUNDING: XVIVO Perfusion.

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).

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.