Machine Perfusion Research Articles (Page 1)

Optimization of Oxygen Supply Conditions for Hypothermic Oxygenated Machine Perfusion.

Heart failure, a leading global health challenge, affects over 23 million people worldwide, with heart transplantation being the gold standard for end-stage disease. However, the scarcity of viable donor hearts presents a significant barrier, with only one-third of available grafts being used because of stringent selection criteria. Machine perfusion technologies, particularly normothermic machine perfusion (NMP), offer promise for improving graft preservation and assessment, yet their full potential for predicting transplantability remains underexplored. This study investigates 3 assessment methods to enhance human heart evaluation during NMP, focusing on mitochondrial function, left ventricular performance, and inflammatory markers. First, resonance Raman spectroscopy is used to assess mitochondrial redox state as a proxy for metabolic competency, offering a noninvasive and dynamic evaluation of mitochondrial function during ex vivo preservation. Second, left ventricular function is quantified using intraventricular balloons, providing critical insights into graft viability and performance. Third, inflammatory markers and endothelial activation are assessed from the perfusate to aid the prediction of posttransplant outcomes. These methods were successfully tested on human donor hearts that were declined for transplantation, preserved via static cold storage, and subsequently assessed with NMP in Langendorff mode. The results demonstrate that these parameters can be easily integrated into existing clinical perfusion workflows and hold potential for improving heart transplantation outcomes by enhancing graft selection and optimizing donor heart use. Future studies will further validate these biomarkers across different preservation techniques and evaluate their clinical applicability.

Initial Clinical Outcomes of Hypothermic Machine Perfusion in Deceased Donor Kidney Transplantation: A Pilot Comparative Feasibility Study from South Korea.

National utilization and outcomes of heart transplantation using ex situ machine perfusion.

Cellular Responses During Kidney Normothermic Machine Perfusion Reflect Graft Outcomes

Organoids derived from human pluripotent stem (hPS) cells hold promise for therapeutic purposes. However, technological advances to overcome their massive production while ensuring differentiation fidelity are still lacking. Here we report a procedure sustaining the derivation of kidney organoids from hPS cells (hPSC-kidney organoids) using a scalable, reproducible and affordable approach that allows hPSC-kidney organoid differentiation into different renal cell types. Using single-cell RNA sequencing, confocal image analysis, metabolic assays and CRISPR-Cas9 engineering for generation of fluorescent reporters, we show that hPSC-kidney organoids exhibit transcriptional variety and cellular composition following cell-to-cell contact. We infuse human kidney organoids into ex vivo porcine kidneys using normothermic machine perfusion, and demonstrate in vivo engraftment of hPSC-kidney organoids. We further evaluate the immune response, confirming the feasibility and viability of the procedure. We identify cells of human origin after normothermic machine perfusion and in vivo transplantation by means of in situ hybridization, immunohistochemistry, confocal microscopy, image analysis and quantification, in vivo imaging, and flow cytometry. This work provides a foundation for using hPSC-kidney organoids for ex vivo cell-based therapies in clinical trials.

Ischemia-reperfusion injury is a significant complication in kidney transplantation, often affecting the viability and function of organs. Normothermic machine perfusion is a technique used to improve the addition of organs prior to transplantation. In this study, we show that incorporating antioxidant poly(propylene sulfide) nanoparticles during cold-storage and normothermic machine perfusion significantly enhances its efficacy in reducing ischemia-reperfusion injury upon porcine kidney transplantation. We found that by scavenging reactive oxygen species, poly(propylene sulfide) nanoparticles reduced oxidative stress and inflammation that occur during ischemia-reperfusion with oxidized DNA reduced 5.3x and both TNF-α and complement activation approximately halved. Our studies show that this approach led to significantly improved hemodynamics, better renal function, and tissue health compared to normothermic machine perfusion alone. The results suggest that incorporating poly(propylene sulfide) nanoparticles into transplantation protocols may expand the pool of kidneys suitable for transplantation and enhance overall transplantation success rates. The broader impact of this work could extend to other organ transplants, suggesting a wider application of nanoantioxidant technologies in organ preservation.

Advanced perfusion techniques have been shown to improve liver transplantation (LT) outcomes in donation after determination of death by both circulatory (DCD) and neurological (DBD) criteria, but allocation strategies are still controversial. This study compared the outcomes of controlled DCD LT with normothermic regional perfusion and subsequent ex situ machine perfusion to those of DBD LT with static cold storage and extended criteria DBD (ECD) LT with dual hypothermic oxygenated perfusion (DHOPE), selected by propensity score matching. Three comparable cohorts were selected from transplants performed between January 2016 and June 2024: 61 DCD (DHOPE, n = 50; normothermic machine perfusion, n = 11), 122 DBD-static cold storage, and 122 ECD-DHOPE. Median functional warm ischemia time in DCD donors was 44 (39-48) min. Livers were assessed and accepted for LT based on normothermic regional perfusion parameters. All considered outcomes were comparable between groups and in line with benchmark values. One-year graft and patient survival exceeded 90% in all groups, whereas 3-y graft survival was 91.8%, 93.4%, and 88% in the DCD, DBD-static cold storage, and ECD-DHOPE groups, respectively. In the same groups, incidence of ischemic cholangiopathy was 3.3%, 4.9%, and 3.3%. Tailored application of advanced perfusion techniques allows achieving optimal outcomes in both DCD with prolonged warm ischemia time and ECD-DBD LT.

Acute rejection (AR) remains a major determinant of renal allograft outcomes, with the major histocompatibility complex (MHC) playing a pivotal role in its pathogenesis. Although immunosuppressive therapies have advanced, their reliance on high doses and lifelong administration increases the risks of infections, malignancies, and other serious complications. Normothermic machine perfusion (NMP) has emerged as a valuable tool in clinical transplantation, enabling organ preservation, functional assessment, and therapeutic intervention. Integrating NMP with genetic engineering approaches to modulate donor kidney MHC expression may offer a novel strategy for preventing AR. We synthesized cholesterol-modified small interfering RNA targeting B2m and Ciita (siB2m-Chol and siCiita-Chol) and set cholesterol-modified negative control small interfering RNA (siNC-Chol) as control. Interfering with MHC expression in transplanted kidneys using NMP combined with siB2m-Chol and siCiita-Chol pretreatment of donor kidneys to prevent AR of posttransplant allografts, we evaluated the efficacy of this approach by assessing postoperative survival, renal function, histological features, and inflammatory responses. NMP combined with siB2m-Chol and siCiita-Chol significantly reduced MHC expression on postoperative day 3, improved allograft function, and prolonged recipient survival. By postoperative day 7, pathological damage was reduced, and T cells, macrophages, B cells, donor-specific antibodies, and inflammatory cytokine production were markedly lower in treated grafts compared with the siNC-Chol control group. These findings demonstrate that ex vivo NMP effectively delivers cholesterol-modified small interfering RNA to renal grafts, substantially downregulating both MHC class I and II expression and consequently attenuating AR.

Recent advances in perfusion technology have positioned normothermic machine perfusion (NMP) as a promising alternative. However, the unavoidable occurrence of ischemia-reperfusion injury (IRI) during perfusion remains a critical barrier to enhancing postoperative outcomes and graft survival. This review systematically examines current pharmacological strategies to mitigate IRI in NMP, targeting key pathological mechanisms including oxidative stress, inflammatory cascades, apoptotic pathways, and endothelial dysfunction. We highlight promising therapeutic agents that have demonstrated efficacy in animal models, preclinical experiments and ex vivo human liver studies. In addition, we discuss the current challenges in clinical translation and future development directions.

Despite a record number of solid organs transplanted in 2023, ongoing issues with donor shortages leave thousands of people in the United States waiting. Increased utilization of donors after circulatory death (DCD) has been the primary driver of the increase in total transplants. Availability of newer technologies including normothermic machine perfusion (NMP), normothermic regional perfusion (NRP), and hypothermic machine perfusion (HMP) have been shown to attenuate ischemia-reperfusion injury and allow for graft viability assessment prior to implantation. Outcomes across all solid organs, especially in higher risk donors, have improved using machine perfusion to decrease rates of primary non-function and damage related to ischemia-reperfusion injury (IRI). Here, we present an overview of deceased donation practices across all solid organs.

BackgroundAssessment and treatment of severe ischemia-reperfusion-injury (IRI) remains an unmet challenge in kidney transplantation. Normothermic machine perfusion (NMP) recapitulates IRI ex situ, but there is limited understanding of the transcriptional pathways, and the associated cellular landscape, driving IRI during NMP and determining its severity. Such knowledge is essential for therapeutic targeting and organ resuscitation during machine perfusion.MethodsUsing tissue obtained at the time of NMP from kidneys subsequently transplanted as part of a randomized controlled trial, we undertook in-depth transcriptomic analyses comparing kidneys suffering severe IRI, (manifesting clinically as the development of delayed graft function (DGF)), to kidneys with mild IRI (defined by immediate graft function, IGF) post-transplantation.ResultsWe validated upregulation of previously described pro-inflammatory and immune transcriptomic pathways, including TNFa via NFkB signaling, Allograft Rejection and Inflammatory Response. Going further, we identified innate immune system driven processes at the core of the transcriptional signature in kidneys suffering severe IRI, such as recruitment and migration of myeloid leucocytes, macrophage activation, phagocytosis and inflammasome activation. Deconvolution using single-cell-RNAseq data showed kidneys with severe IRI and post-transplant DGF were enriched for pro-inflammatory mononuclear phagocytes, myofibroblasts and fibroblasts, but depleted of tubuloepithelial, cell signatures. These transcriptional findings were recapitulated in tissue biopsies obtained during NMP from an external cohort comparing kidneys with high acute tubular injury and severe IRI to kidneys with low acute tubular injury and mild IRI; these kidneys were histologically similar to the DGF/IGF kidneys, respectively.DiscussionTogether, our study characterizes the transcriptional signature of severe IRI during NMP, suggesting the role of pro-inflammatory innate/pro-fibrotic cells in this process. We describe a transcriptomic signature that may support future prospective therapeutic trials as a potential efficacy endpoint, and highlight potential cellular targets for therapeutic intervention during NMP in an era of precision medicine.

Colorectal cancer liver metastasis (CRLM) is associated with poor survival, primarily due to acquired therapy resistance. While novel therapies arise, translation is limited by the lack of tumor models accurately representing dynamic microenvironmental interplay. Here, we show that ex vivo normothermic machine perfusion (NMP) offers a novel preclinical framework to study the intratumoral dynamics of CRLM biology. Six resected metastatic human livers were preserved for two days and subjected to multi-omic profiling of serially sampled adjacent liver and metastatic tissue using single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (ST). Tissue integrity was assessed and cross-validated by immunofluorescence (IF), high-resolution respirometry (HRR) and flow-cytometry. NMP was successfuly applied to metastatic livers with minimal surgical adaptations, preserving both intrinsic hepatic properties and tissue viability over an extended duration. Single-cell and spatial mapping confirmed preservation of CRLM phenotypic properties and demonstrated high clinical translatability by applicability of the intrinsic epithelial consensus molecular subtypes to metastasis. Spatially deconvoluted pathway activities reflected functional tissue-microenvironments. Transcriptomic profiles - including those of tumor-associated myeloid cells - were preserved during NMP. Finally, we demonstrate tumor-associated myeloid cell persistence as a driver of disease progression and poor survival in colorectal cancer. Our findings represent the basis for future innovative applications adopting NMP in the context of CRLM, providing a new preclinical tumor model avenue.

The global shortage of donor livers has led to an increased reliance on extended criteria donor livers, including from donors after circulatory death. Utilizing marginal livers necessitates distinguishing between injured but recoverable livers and those that are irreversibly damaged. Bile duct injury, prevalent in these grafts, poses significant risks, as ischemic injuries can be challenging to assess and may lead to recipient bile duct strictures and ultimately graft failure. This study aimed to identify objective measurements that enhance existing viability criteria and facilitate the diagnosis of ischemic injuries. We employed ex situ machine perfusion (MP) to monitor porcine livers subjected to standardized biliary injury (BileINJ), standardized global liver injury (GlobalINJ), and no injury (CTRL) (n=23). Using microdialysis catheters and pCO2 sensors, we analyzed liver tissue, hilar plate, and bile duct, in addition to standard viability assessments, collecting data on 77 variables. Principal component analysis indicated distinct clustering of the GlobalINJ and partially the BileINJ group away from the CTRL group during MP. Feature extraction models highlighted microdialysate lactate, lactate/pyruvate ratio, glycerol, tissue pCO2, and blood gas hematocrit as critical indicators for classifying liver state. Histopathological evaluations confirmed group-specific liver and bile injuries. We identified unique metabolic patterns that differentiate ischemic injured from non-injured porcine livers and were able to distinguish liver and biliary injury. Real-time monitoring of livers using microdialysis and tissue pCO2 during MP is feasible and clinically available. Measurements taken in the hilar plate show the ability to identify bile duct damage while not introducing measurement devices to the vulnerable bile duct itself. Our findings enable more objective and timely selection of transplantable livers and warrant further investigation in clinical studies.

Utilization rates of potential deceased donors for liver transplantation has declined following the implementation of the acuity circles system in 2020. Since then, ex situ machine perfusion (es-MP) and normothermic regional perfusion (NRP) have been introduced in the United States. Liver graft utilization rates were analyzed using the US national registry data from 2022 to 2024. The associations of es-MP and NRP practices with donation after circulatory death (DCD) utilization rates among organ procurement organizations (OPOs) and transplant center volume were evaluated. DCD donor utilization significantly increased from 21.5% in 2022 to 42.5% in 2024 (P < 0.001). Utilization of extended criteria donors (ECDs), including DCD donors aged ≥60 y or with a body mass index ≥40 kg/m2, also rose substantially, from 7.1% in 2022 to 33.2% in 2024 (P < 0.001). At the transplant center level, a significant correlation was found between the increase in transplant volume and both the es-MP use (r = 0.29; P = 0.01) and ECD utilization (r = 0.26; P = 0.02). At the OPO level, a significant association was observed between the increase in DCD utilization rate and NRP use (r = 0.29; P = 0.03). DCD liver utilization has significantly increased, with a notable rise in the utilization of ECDs, which may be driven by new technologies such as es-MP and NRP. While es-MP at the center level use may increase transplant volume, NRP use at the OPO level appears to significantly improve DCD liver utilization.

Utilization and Transplantation of Unused Kidneys After Assessment Using Normothermic Machine Perfusion Technology.

Genetically engineered organs for early reporting of transplant rejection.

Normothermic machine perfusion (NMP) could serve as a platform to assess deceased-donor kidney viability before transplantation, yet it remains unclear which parameters indicate renal viability. As vascular integrity is important for adequate renal function after transplantation, this study aimed to investigate the influence of warm ischemic injury on vascular smooth muscle cell (VSMC) responsiveness to vasoactive drugs during NMP. Fourteen porcine kidneys (n = 7 per group) were exposed to either 30 or 60 min of warm ischemia (WI), followed by 3.5 h of cold machine perfusion. After cold perfusion, kidneys underwent 4 h of NMP (37°C). During NMP, vasoactive drugs were sequentially infused into the renal artery at 30-min intervals, starting with epoprostenol (10 μg), followed by dopamine (1 mg), sodium nitroprusside (2 mg), acetylcholine (1 mg), norepinephrine (10 μg), and finally verapamil (2.5 mg). Renal blood flow during NMP changed significantly in both groups after administration of dopamine, acetylcholine, norepinephrine, and verapamil, but not following epoprostenol and sodium nitroprusside infusion. In kidneys subjected to 30 min of WI, the response to dopamine and norepinephrine was more pronounced, and oxygen consumption and blood pH were higher compared to kidneys that sustained 60 min of WI. This study indicates that prolonged WI damage diminishes the contractility of VSMCs through the α-adrenergic receptors. Our findings suggest that the renal vascular responses to dopamine and norepinephrine, as well as decreased oxygen consumption and blood pH, could serve as objective indicators to quantify warm ischemic injury during renal NMP.

Normothermic machine perfusion (NMP) of donor kidneys provides an opportunity not only for organ preservation but also for therapeutic intervention to reduce ischemia-reperfusion injury (IRI) and support tissue repair. Sevoflurane, a volatile anesthetic known to protect against IRI in other organ systems, has not previously been explored in the context of kidney NMP. This study aimed to establish a stable and reproducible porcine kidney NMP model incorporating sevoflurane delivery. Different administration techniques and oxygenator setups were evaluated to identify the most effective method of sevoflurane application. Administering sevoflurane directly as a liquid into the perfusate resulted in excessive gas formation and unstable drug concentrations. In contrast, using a vaporizer connected to an Inspire 8F M oxygenator allowed for stable and reproducible sevoflurane levels over a 90-min perfusion period, while maintaining sufficient oxygenation. This method proved to be a reliable approach for sevoflurane delivery in kidney NMP. However, oxygenators exposed to sevoflurane should not be reused due to the risk of membrane damage. These findings support the feasibility of using sevoflurane in kidney NMP and provide a platform for further investigation into its potential to improve renal graft outcomes.

Ex situ machine perfusion (EVMP) leads to the accumulation of metabolites and electrolytes in the perfusate. This review aimed to examine the impact of dialysis on biochemical and functional parameters during EVMP. A systematic review was conducted per PRISMA guidelines. Studies integrating dialysis in lungs, liver, pancreas, and heart EVMP, either in large animals or humans, were included. Biochemical and metabolic parameters, organ weight, inflammatory markers, and organ-specific outcomes were extracted. Meta-analyses and longitudinal regression were conducted. Nineteen studies were included. Dialysis maintained sodium (p = 0.17), potassium (p = 0.75), lactate concentrations (p = 0.3), and pH (p = 0.1). A significant decrease was seen in chloride concentration (p < 0.01), while calcium levels increased significantly over time in the dialysis group (p < 0.001). Interleukin 6 was elevated in dialysis relative to the controls during porcine lung perfusion (p < 0.05) and trended toward significance in humans (p = 0.08). During heart perfusion, the dialysis group exhibited higher cardiac contractility, expressed as a percentage of baseline (71.2% ± 7.7 vs. 55.3% ± 8.2, p < 0.05). During liver perfusion, bile production increased significantly from baseline (β = +1.85 IU/L/h, p < 0.001). Lung compliance was stable in the dialysis (β = -2.34 mL/H2O/h, p = 0.36) and control groups (β = +0.12 mL/H2O/h, p = 0.27). Dialysis maintained biochemical homeostasis during EVMP. Significant functional benefits were seen in cardiac EVMP and should be investigated in other organs, including appropriate control groups.