Mitochondrial Therapy During Machine Perfusion Restores the Function of DCD Livers: A Cross-Circulation Evaluation for Transplant Suitability

Mitochondrial Therapy During Machine Perfusion Restores the Function of DCD Livers: A Cross-Circulation Evaluation for Transplant Suitability | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Mitochondrial Therapy During Machine Perfusion Restores the Function of DCD Livers: A Cross-Circulation Evaluation for Transplant Suitability Sandra Lindstedt, Nicholas Bechet, Tibor Huzevka, Fanny Sveréus, and 14 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7871574/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Donation after circulatory death (DCD) has significantly increased the number of organs potentially available for transplantation. Livers are more vulnerable than most other solid organs to warm ischemia in the DCD process. The injury induced by warm ischemic time (WIT) is further exacerbated during cold ischemia and reperfusion, resulting in tissue damage caused by the production of reactive oxygen species. These are not only cytotoxic but also perpetuate mitochondrial dysfunction and cell death. To improve the utilization of livers from uncontrolled DCD (uDCD) or controlled DCD (cDCD) donors with prolonged WIT, new strategies to mitigate WIT must be developed. Ex vivo normothermic machine perfusion (NMP) has increased DCD organ utilization and improved the assessment of the viability of organs before transplantation. NMP could also serve as a platform for isolated treatment of organs prior to transplant. An innovative approach to mitigate organ injury is to treat damaged livers with mitochondrial transplantation (MTx). In this study, we tested the efficacy of xenogeneic mitochondrial administration to restore the function of porcine DCD livers with two hours of warm ischemia. DCD livers were explanted and connected to NMP, where mitochondrial transplantation was administered as a bolus dose in the portal vein and hepatic artery at the start of perfusion. After four hours of NMP, treated livers demonstrated a significant increase in bile production, improved bile quality, and restored cytoarchitecture. To further assess the transplantation suitability of these livers post-NMP, they were connected to cross-circulation (CC) with a recipient pig. After six hours of CC, untreated livers became oedematous, exhibited significant aspartate aminotransferase elevations in the bile, and showed cellular degeneration, including near-complete loss of Kupffer cells. In contrast, livers treated with MTx maintained function across CC and were comparable to healthy controls. These data validate the efficacy of MTx in restoring function in DCD livers exposed to prolonged ischaemic times, presenting a promising approach to expand the donor pool. Health sciences/Medical research/Translational research Biological sciences/Biological techniques/Biological models/Animal disease models Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Liver transplantation is a highly successful therapy for end-stage liver disease, but its availability is severely limited by disparities between organ supply and the need for transplantation, leading to many patients dying awaiting an organ 1 , 2 . While retrieval of organs from donation after brain death (DBD) donors remains the most common pathway in many countries, workflows and guidelines have been established to facilitate the retrieval and transplantation of organs from donation after circulatory death (DCD) donors 3 – 6 . Unlike kidneys or lungs, the liver is particularly vulnerable to the warm ischemic time (WIT) inherent to the DCD process. This presents a significant challenge even when livers are recovered with a super rapid retrieval approach, stored statically in ice, and subsequently transplanted 7 , 8 . Techniques such as normothermic regional perfusion (NRP), normothermic machine perfusion (NMP), and hypothermic oxygenated perfusion (HOPE) offer critical advantages for both organ quality and transplantation outcomes 9 , 10 . While the development of controlled DCD (cDCD) has substantially increased the donor pool, the greatest potential for expansion lies within uncontrolled DCD (uDCD) donors 11 , 12 . However, the success of using uDCD donors hinges on overcoming the challenges related to a prolonged WIT, which has been one of the main barriers to establishing successful clinical programs for these donors 13 , 14 . In cDCD liver transplantation, minimizing WIT remains a key consideration, as evidence consistently indicates that reducing ischemic time is essential to optimizing organ viability and function. A WIT ≥ 35 minutes was shown to result in a 1.8-fold higher graft loss rate than WIT ≤ 15 minutes 4 , 15 . The extended donor hepatectomy time, the interval between aortic cross-clamp and in situ cold perfusion, until the liver is removed from the body, incurs a negative impact on graft survival 4 , 16 . A recent analysis of the Eurotransplant registry, encompassing 12,974 recipients, revealed that the hepatectomy time is notably longer in DCD donors and is associated with a significantly higher risk of death-censored graft loss 16 . Ischemia-reperfusion injury (IRI), an unavoidable challenge in current transplantation practice, continues to impact outcomes significantly 17 – 19 . This injury occurs when the organ, subjected to ischemia during retrieval from the donor, is subsequently reperfused in the recipient, leading to inflammatory and oxidative stress responses 20 . Despite advancements in preservation and perfusion technologies, IRI remains a major barrier to optimal graft function and long-term survival, as it exacerbates tissue damage and influences both acute and chronic rejection risks 21 . Addressing IRI is essential for improving transplantation outcomes, highlighting the need for continued innovation in organ preservation and protective strategies. Although ex-situ MP has emerged as a valuable strategy to assess and treat marginal livers, these techniques shift the reperfusion process from the patient to the machine, and during this process, tissue injury may still occur 22 , 23 . While reperfusion injury in the liver after transplantation is thought to be mediated by transient portal hypertension, which causes hyperdynamic stress to the hepatic endothelial lining, more focus is now being placed on reperfusion-mediated oxidative stress and mitochondrial dysfunction 24 , 25 . It is postulated that IRI leads to the production of significant amounts of reactive oxygen species (ROS) by Kupffer cells and the intrahepatic neutrophils. ROS production is primarily triggered by mitochondrial respiratory dysfunction, which results in the accumulation of succinate and the reversal of the electron transition from complex I to complex II 25–27 . Reperfusion-induced oxidative stress typically peaks between 2–6 hours after reperfusion and results in sinusoidal endothelial cell, hepatocyte, and cholangiocyte death, mitochondrial swelling, and metabolic dysfunction 28 . This highlights a compelling link between mitochondrial dysfunction, WIT, and IRI, and as such, substantiates the use of mitochondrial therapies as a potential strategy to minimize hepatic IRI, expand the DCD liver pool, and in doing so, increase organ availability and improve graft survival 29 – 31 . We developed a novel approach for delivering respiration-competent, exogenously isolated mitochondria to neonatal, pediatric and adult, DCD hearts 32 , 33 . These transplanted mitochondria function as metabolic support for the endogenous mitochondrial pool, promoting normalization of transcriptomic and proteomic profiles to levels comparable to those observed in DBD hearts 34 . This normalization helps preserve cellular metabolism and attenuate the cascade of IRI. Although mitochondria transplantation (MTx) has been studied in rodent liver IRI models with relatively short WIT 35 , 36 , its’ application in ex vivo settings using machine perfusion, and a transplantation viability model has not been previously investigated. This study is the first to evaluate mitochondrial transplantation as a therapeutic strategy to mitigate long WIT-induced injury (two hours) in DCD livers using a clinically relevant porcine model in combination with normothermic machine perfusion (NMP). DCD porcine livers were subjected to NMP, with or without MTx, followed by cross-circulation with a recipient to assess transplant viability. RESULTS DCD livers exhibit signs of mitochondrial depletion To validate the rationale that DCD livers do indeed suffer mitochondrial dysfunction when exposed to a long WIT, biopsies from healthy livers and those exposed to a 2-hour WIT (2h DCD) were assessed with regards to ATP production (Fig. 1 a). The DCD samples exhibited a significant two-fold reduction in tissue ATP levels, pointing to a depletion of endogenous mitochondria (Fig. 1 b). Next, we sought to understand whether supplementation with exogenous mitochondria, isolated as previously described from the livers of C57BL/6 mice 37 (Fig. 1 c-d), renders any benefit to injured hepatocytes in in vitro conditions. When hepatocyte cultures derived from both healthy and IRI human liver tissue were supplemented with exogenous mitochondria, the IRI-derived hepatocytes exhibited an increase in ATP production compared to the healthy baseline (Fig. 1 e-f). This highlights that the addition of exogenous mitochondria into tissue appears to produce metabolic functional improvements in tissue that has suffered from mitochondrial dysfunction. This was further validated by live cell imaging after the addition of fluorescently labelled exogenous mitochondria, which revealed significantly greater levels of mitochondria uptake into IRI-hepatocytes (Fig. 1 g-h). Taken together, these data provide compelling evidence that the exogenous mitochondria are avidly taken up into DCD livers with extended WIT. Overview of the model, therapy, and assessment modalities To explore the benefit of MTx for DCD liver regeneration in a clinically relevant model, we established a workflow composed of three main parts: 1-generation of a porcine DCD model, 2-normothermic machine perfusion (NMP) of DCD livers, with or without MTx, 3-cross circulation of DCD livers with a recipient after NMP (Fig. 2 a). Ventricular fibrillation was induced in the donors by interference with the cardiac conduction system and livers were left in situ for two hours post circulatory arrest before retrieval (Fig. 2 a). Livers were then randomised (n = 6/group) into mitochondrial treatment and non-treated groups and placed on NMP for four hours. The surgeon, perfusionist, anesthesiologist, as well as the nurses and technicians performing the experiments, were blinded to the study arm. MTx or placebo were given as a bolus injection equally divided between the portal vein and the hepatic artery at the start of NMP. To further evaluate the transplant potential of the livers after NMP (n = 3/group), a cross-circulation model (CC) was established with a recipient pig for six hours. For comparison with a normal liver cohort, n = 9 porcine subjects were anesthetized, the abdomen was opened, and liver tissue samples were collected. Mitochondrial transplantation improves DCD liver function on normothermic machine perfusion Generation of DCD liver injury was confirmed through blinded scoring by a pathologist of tissue biopsies after two hours of warm ischemic time compared with healthy control liver tissue (Fig. 2 b-c), with DCD livers exhibiting consistent elevated cumulative injury scores (Fig. 2 d). In keeping with this, a comparative proteomic analysis of bile between healthy controls and DCD livers revealed that 580 proteins were significant differentially expressed, with the majority being upregulated (516 proteins) in contrast to the downregulated proteins (64 proteins) in the DCD group (Fig. 2 e). To explore the potential benefit of MTx in these organs, mitochondria were delivered serially in the first 10 minutes of NMP, and again after four hours. The regenerative impact of the mitochondria was already noticeable, with treated organs exhibiting a significant increase in bile production (Fig. 2 f), coupled with significant reduction in bile aspartate aminotransferase (AST) levels (Fig. 2 g). A more detailed bile composition analysis showed that both cohorts exhibited significant reductions in bilirubin concentration at four hours on NMP when compared to the start of NMP (Fig. 2 h). In both groups, there was a significant increase in bile glucose, though this difference was nearly 2.5-fold higher in non-treated livers, while treated livers only displayed a modest 30% increase (Fig. 2 i). No differences in pH were seen (Fig. 2 j), and while no significant differences in bile lactate were observed, it was evident that there was a trend for higher bile lactate levels in the non-treated livers after four hours of NMP (Fig. 2 k), indicating the maintenance of anaerobic metabolism in the absence of MTx. Gene set enrichment analysis (GSEA) was performed to visualize enriched biological processes found in bile in the treatment group after NMP, with the non-treated group as reference. Downregulated processes in the treatment group (blue) included mostly metabolic and catabolic processes at end of NMP while enriched processes (red) included response to stress, cell migration, cell adhesion and immune system processes, yielding a bile profile similar to one previously demonstrated in human livers deemed acceptable for transplantation (Fig. 2 l). Taken together these data infer a higher quality bile production in livers treated with MTx on NMP, which met the threshold for transplantation according to the bile composition 38 . Glucose concentrations in perfusate increased during NMP in the treated group and decreased in the non-treated group; however, this change was not statistically significant (Fig. 2 m). Differences in perfusate lactate and AST concentrations were also not significant but mirrored the trends seen in the bile samples after four hours of NMP (Fig. 2 n-o). A significant, but not clinically relevant difference was seen in portal vein flow after five minutes of perfusion, while after 4 hours of NMP a significant difference was seen between the cohorts, with the mitochondrial treatment group exhibiting higher portal flow (Fig. 2 p). Hepatic artery (HA) flow was equal after 5 min of NMP, while after 4 hours there was no significant difference between the groups (Fig. 2 q). However, the mean flow in HA in the control group declined between the start and 4h NMP, whilst flow increased in the treated group, indicating an overall improved perfusion and highlighting a trend towards a more rapid normalization of the graft haemodynamics post perfusion. First signs of tissue perturbation occur in non-treated livers during NMP To assess the degree of injury to the liver after treatment/placebo and NMP, a blinded pathologist scored hematoxylin and eosin-stained sections from each cohort to both healthy and DCD tissue (Fig. 3 a). After 4 hours of NMP both the MTx treated and non-treated samples exhibited similar cumulative injury scores, both of which were lower than in DCD injury, but moderately higher than healthy control levels, indicating a treatment independent improvement in gross tissue morphology after NMP (Fig. 3 b). Similar data were observed with regards to CK19 + cholangiocyte coverage, with no significant differences seen between groups at this timepoint, although the non-treated NMP group started to reveal a trend for reduced cholangiocyte coverage (Fig. 3 c-d). Interestingly, assessment of Kupffer cell coverage showed a trend for decreased coverage in DCD compared to healthy control tissue. There was a significant recovery in coverage in treated livers on NMP, while non-treated tissue maintained the same reduced Kupffer cell coverage (Fig. 3 e-g). To assess the number of apoptotic cells in the tissue, a fluorescent TUNEL staining was used, revealing an increase in TUNEL + cells in DCD tissue, which declined in both treated and non-treated cohorts after NMP, again highlighting more treatment-independent outcomes due to NMP (Fig. 3 h-i). This was further validated through comparison of the bile protein landscape, which yielded only one significantly elevated protein in the treated group (Fig. 3 j). To assess the degree of tissue oedema, a wet-dry (W/D) ratio was carried out, yielding a non-significant increase in fluid in the non-treated group (Fig. 3 k). Cytokine analysis focused on interleukin-6 (IL-6) and interleukin-10 (IL-10) revealed non-significant increases in both cytokines in the untreated group (Fig. 3 l-m), pointing towards an immunomodulatory effect occurring in these livers. Taken together these data point to the origins of an immune-inflammatory cascade in both groups of organs, however, the non-treated group appears to be more greatly impacted, with a decline in Kupffer cell coverage, and greater apparent effort to immunomodulate the tissue environment. Cross circulation reveals a profound protective effect for mitochondrial transplantation To evaluate the transplant potential of the DCD livers treated with MTx or placebo after four hours of NMP, we undertook a six-hour cross circulation (CC) using recipient pigs. As with NMP, the total bile production remained significantly higher in the MTx-treated livers (Fig. 4 a), while bile AST levels remained significantly lower (Fig. 4 b), indicating the efficacy of the therapy at extended time points and a regeneration of treated livers. With regards to bile composition, further reductions in both bilirubin (Fig. 4 c) and lactate levels (Fig. 4 f) occurred in the treated livers on CC, also pointing to a restoration of normal liver function. The bile glucose levels manifested a significant reduction in the treated group on CC (Fig. 4 d) whilst the bile pH levels were normal in both treated and non-treated grafts at the end of CC (Fig. 4 e). Proteomic analysis of bile composition revealed that one protein, (UROC1) was significantly (p < 0.05) upregulated in the treatment group while 28 proteins were downregulated (Fig. 4 g). Gene set enrichment analysis from bile at end of cross circulation showed that the inflammatory response, humoral immune response, cell migration, response to wounding and regulation of coagulation were all upregulated in the treatment group (Fig. 4 h). Downregulated biological processes in the treatment group included metabolic processes, catabolic processes and translation. Perfusate analysis showed no significant differences in either glucose (Fig. 4 i) or lactate (Fig. 4 j), while AST levels yielded a comparable non-significant increase in the non-treated group as seen in the bile (Fig. 4 k) Treated grafts also exhibited a significant improvement in hepatic artery perfusion pressures (Fig. 4 m), while no perfusion differences were found in the portal vein (Fig. 4 l). These data point to an overall regeneration of liver function in the treated grafts as well as a substantial decline in those which did not receive mitochondrial treatment. These findings were recapitulated in the tissue sections, where the non-treated CC samples displayed apparent oedema and eosinophilia (Fig. 4 n). Blinded scoring revealed a significantly higher cumulative injury score in samples from non-treated livers, while those treated with mitochondria only exhibited a moderately non-significant elevation in injury score (Fig. 4 n). In line with the pathology observations, non-treated tissue yielded a 40-fold increase in apoptotic cells compared to healthy control tissue, while there were only 5 times more TUNEL + cells in those livers which received MTx (Fig. 4 o). The slight decline in Kupffer cells noted in the non-treated samples after 4 hours of NMP translated into a near complete abolishment of these cells after 6 hours on cross circulation, while in mitochondria treated livers, Kupffer cell coverage resembled that from healthy control tissue (Fig. 4 p). The same findings were noted with regards to CK19 + cholangiocyte coverage, indicating considerable damage to the biliary tree in the non-treated group (Fig. 4 q). In keeping with the widespread tissue damage and cell loss, the non-treated samples further experienced a significant increase in oedema as inferred by the significantly higher W/D ratio (Fig. 4 r). With regards to cytokine analyses, after six hours of cross circulation there were no differences between the levels of IL-6 and IL-10 expression (Fig. 4 s-t). Taken together, these data reaffirm the impairments in gross liver function seen in the non-treated group and further strengthen the efficacy of the MTx in mitigating liver degradation in DCD with a WIT of two hours. Proteomic cluster analysis and spatial proteomic profiling of MTx To analyze the proteomic landscape across all cohorts and treatment groups, an untargeted data-independent acquisition mass spectrometry method was performed. Across groups, the analysis identified 7276 unique proteins. After filtering for the proteins present in at least 75% of the samples per group, 1684 proteins remained for further downstream analyses. A heatmap analysis was performed to visualize the clustering of the bile samples according to significantly differentially expressed proteins. Unsupervised hierarchical clustering showed a clear distinction between the baseline and DCD groups, with downregulated proteins in the baseline group and upregulated proteins in the DCD group (Fig. 5 a). The mitochondria treated samples obtained at the end of cross circulation clustered closely to the baseline samples, while the treated samples at the end of 4h NMP clustered modestly to the baseline samples. To expand on the observed clustering, we sought to explore the differential expression of cell populations across cohorts, using a high-content imaging approach. This permitted the acquisition of 13 distinct markers (DAPI, CD3, CD4, PCNA, Ki67, vimentin, PCK, actin, CD163, LEA, HH3K4, IBA1, collagen-1) on the same sections at sub-cellular resolution (Fig. 5 b), totaling approximately 1.15 million cells. Several populations, including CD3+, Ki67+, vimentin+, and CD163 + cells, exhibited similar densities in the baseline and treated samples, both on NMP and CC, while non-treated samples showed an under- or over-expression of these cell populations (Fig. 5 c), corroborating the observations from the proteomics data. Both treated cohorts displayed significant elevations in the expression of HH3k4, an epigenetic marker associated with DNA repair and replication. More importantly, across all cell populations analyzed, it was clear that the non-treated CC samples suffered a massive loss of all cell types, highlighting the deterioration of these DCD livers in the absence of MTx. Moreover, those livers treated with MTx revealed an expression pattern similar to that observed in the baseline tissue, in keeping with the proteomics data, showing an improvement of the DCD livers over time after receiving the MTx. To explore spatial differences in a more hepatic-focused manner, an advanced segmentation map was generated to define 4 regions across a single functional hepatic unit (Fig. 6 a-b), starting from the collagen connective tissue then spanning liver zones 1–3, from the collagen border through the central vein, focusing on PCNA-, CD163-, Ki67-, CD3-, HH3-, LEA- and IBA-1-positive cell populations (Fig. 6 c-i). In keeping with the global data, it was evident that the zonal pattern of expression of the majority of markers was most comparable between the healthy control and treated cross-circulation datasets, indicating a profound cellular and morphological regeneration from the DCD state in mitochondrial treated livers. Taken together, these data indicate that four hours of NMP are not sufficient to fully inform on the state of the liver and that the most valuable insights come from longer cross-circulation time points, with improvement towards a healthy baseline organ, or complete deterioratation. DISCUSSION This study evaluates the efficacy of MTx in restoring the function of DCD livers subjected to an extended WIT of two hours. Livers were first placed on NMP, whereat we infused freshly isolated mitochondria to demonstrate that the addition of exogenous mitochondria to the organ mitigates IRI. We showed that this therapy improved liver function on NMP as validated by increasing bile production, reducing bile AST and lactate levels, and maintaining a normal cytoarchitecture. Next, to assess the liver’s viability in a transplant-like setting, we established a CC model with a recipient pig. While untreated livers went on to exhibit massive oedema, abolishment of both Kupffer cells and cholangiocytes, and a significant rise in bile AST levels, the livers treated with MTx maintained a normal liver function characterized by continuous bile production, low bile AST levels, low bile lactate levels, utilization of bile glucose and normal cytoarchitecture. These findings strongly support the efficacy of MTx in restoring DCD livers subjected to prolonged WIT. This approach for maintaining and regenerating DCD livers holds substantial potential to significantly expand the donor pool. The MTx was administered in two bolus doses, delivered simultaneously through both the portal vein and hepatic artery at the start of NMP. This dual-route approach was designed to optimize mitochondrial distribution across the entire liver, ensuring comprehensive exposure and uptake within the target tissues. The decision to administer mitochondria at the start of NMP was driven by the objective of achieving a high, targeted dose selectively within the liver parenchyma, at a point when endogenous mitochondria have not fully recovered to support a fully metabolically active organ. Administering mitochondria in vivo prior to donor death determination would result in uncertain dosing and unpredictable distribution within the body. While one might consider delivering the MTx as a bolus through the portal vein after opening the abdomen in DBD donors and before the cross-clamp, this approach would rely on the hope that the majority of mitochondria would be absorbed in the liver during the initial pass. However, in DCD—particularly uDCD—this approach would be impractical. Alternatively, administering mitochondria during CC was considered; however, potential dilution within the recipient’s circulation would create uncertainties regarding the effective dose reaching the liver, providing support. Moreover, from a clinical perspective, our study design aims to evaluate CC as a proxy for transplantation, aligning with realistic clinical protocols. In clinical practice, severely injured livers would not be transplanted into a recipient without prior evidence of functional restoration, to avoid exposing the recipient to undue transplantation risks. By administering MTx during NMP, we can maximize localized delivery and monitor early markers of liver recovery before considering exposure of the recipient, thus aligning with both therapeutic and safety objectives in clinical transplantation. Our CC model differs significantly from previously applied CC liver models 39 , 40 . In this model, after CC establishment, a clamp is placed over the liver hilum, completely obstructing the portal vein and hepatic artery. This approach was specifically chosen to simulate a clinical transplant scenario by effectively excluding the recipient’s native liver from circulation. In this way we have established an innovative way to test the functional capacity of the organ without the need for a transplant model. A challenge with MTx has been establishing a robust and reliable source of mitochondria to transplant, which were readily available, keeping in mind the unpredictable timing of transplant surgery. This is because not all tissues and individuals provide mitochondria of similar quality or yield, making the source highly important 37 . In addition, the mitochondrial isolation protocol is very sensitive with regards to tissue harvest time, as well as the time between isolation and delivery, which should not exceed 30 minutes 37 . Cryopreservation, which would potentially offer an off-shelf product, has been done but remains controversial due to concerns over organelle survival and function after the freeze-thaw process 41 . However, rodents, which are readily accessible across all research centres globally, may yet emerge as an ideal mitochondrial source to explore an advanced mitochondrial transplant therapy. In the present study, young mouse livers served as the source of mitochondria for therapeutic application, leveraging the liver’s naturally high mitochondrial concentration to yield a robust supply of high-quality, functionally potent mitochondria. Our previous work has demonstrated that mitochondria sourced from autologous, allogeneic, or xenogeneic origins are viable for therapeutic use, exhibiting compatibility without eliciting significant immune responses in recipients 37 . This finding broadens the potential for MTx by allowing flexibility in donor sources, which is especially valuable considering the unpredictable timing of transplantation and the limitations of mitochondrial isolation. The lack of immune rejection across these sources suggests that MTx may circumvent some of the immunogenic challenges that arise with other cellular therapies, such as xenogeneic mesenchymal stem cell (MSC)-based approaches or whole organ transplants 43 – 46 . Numerous studies have identified mitochondrial dysfunction as a central theme in ischemic tissue damage as well as IRI, highlighting mitochondria as a key target element in these pathophysiological cascades 47 – 49 . This was further reaffirmed in our study by the maintenance of elevated bile lactate levels in non-treated organs, which may relate both to impaired lactate clearance in the liver and maintenance of anaerobic metabolism in the absence of normally functioning mitochondria. Prior strategies to alleviate IRI have been focused either upstream or downstream of the mitochondria, whereas mitochondrial transplantation tackles this problem at the source. As the ROS released upon reperfusion invoke the release of damage-associated patterns (DAMPs) from resident hepatic cells 20 , 50 , the integration of cytokine filters into perfusion circuits has been explored. However, while these filters do remove cytokines from the perfusate, they are indiscriminate in removing both pro- and anti-inflammatory cytokines and have not yet been found to significantly alter end-outcomes when applied in NRP 51 – 55 . Downstream DAMP and cytokine inhibition has also been explored using caspase inhibitors and RNA interference, but further research is required to validate their effectiveness 51 , 56 , 57 . A more advanced approach is the use of CRISPR-Cas9 gene editing technology, which has been used to enhance the activity of immunomodulatory T-cells and dendritic cells 51 , 58 . An alternate approach was to increase the production of the anti-inflammatory cytokine IL-10, however, real-world application requires further investigation of delivery, transfection efficiency, and precisely where and how specific cells are targeted, especially in large mammalian organs 59 – 62 . Mesenchymal stem cells (MSC), which also possess strong immunomodulatory properties, have been delivered during machine perfusion to mitigate IRI, with several clinical trials underway exploring their effectiveness as an advanced therapy medicinal product (ATMP) 45 , 46 , 63 . An evolution from the use of MSCs focuses on the delivery of extracellular vesicles (EVs), which contain paracrine products of MSCs, are easier to produce at scale, and raise fewer of the immune rejection concerns when injecting cells 51 , 64 – 66 . In contrast, the upstream approaches focus on lessening mitochondrial damage and dysfunction to avoid the downstream DAMP and cytokine cascades. One such approach is the application of HOPE and controlled rewarming prior to NMP, with the initial use of HOPE thought to dampen the IRI-cascade when NMP is applied 9 , 10 , 67 , 68 . Priming with HOPE acted to reduce succinate metabolism upon reperfusion, which consequently attenuated the release of the damage-associated flavin-mononucleotide (FMN) 67 . Moreover, the authors concluded that one hour of HOPE treatment resulted in a mitochondrial reprogramming characterized by a more oxidized state 67 . One concern that still needs to be empirically addressed is the damage that mitochondria are known to sustain when exposed to cold temperatures, in balance with the protective effects 67 , 69 . An alternate focus has been on the modulation of the ischemia-induced accumulation of succinate, which manifests the profound ROS production upon reperfusion 70 . Application of the reversible complex II inhibitor, dimethyl malonate, was able to attenuate cardiac tissue damage after ischemia, however, its value in alleviating IRI is yet to be explored 70 . This work focused instead on replacing those mitochondria lost and damaged during both WIT and IRI, as a novel therapy to attempt to rescue DCD livers. This would further increase the viable liver pool for transplant by rescuing livers that would be discarded due to extended WIT. The data generated herein shows that DCD livers treated with MTx restored their function during NMP, and when further evaluated for their transplant potential in a CC model, treated livers were able to sustain a recipient, produced bile and displayed a well-preserved cyto-architecture, demonstrating the hallmarks of an organ potentially fit for transplant. This is further strengthened by the bile proteomic profile exhibited in treated livers, both on NMP and at the end of CC, which recapitulated a bile proteome previously validated in human livers deemed acceptable for transplantation 38 . Regarding therapeutic translation, recent work carried out in rodents found that treatment with MTx prevented ischemia-reperfusion induced hepatocellular injury 36 . Interestingly, this outcome was dependent on the survival of the Kupffer cell population, which were found to sequester the transplanted mitochondria 36 . In keeping with this, our data revealed that the absence of mitochondrial treatment in DCD results in an eventual complete loss of Kupffer cells, further highlighting them as a target for MTx. In conclusion, this study provides compelling evidence that xenogeneic MTx can effectively restore the function of DCD livers subjected to prolonged WIT, addressing a major barrier in liver transplantation. Administering mitochondria during NMP, enabled targeted, high-dose delivery to the liver parenchyma, optimizing cellular uptake and enhancing the therapeutic impact. The treated livers demonstrated improved bile production and composition, reduced tissue oedema, and preserved tissue integrity, with significant reductions in injury markers. Importantly, in a CC model using a recipient pig, treated livers maintained functional and structural integrity, underscoring the transplant and clinical potential of this therapy. By effectively mitigating WIT and IRI, MTx shows promise as an innovative strategy to expand the donor pool by rendering previously unsuitable DCD livers viable for transplantation. These findings support further exploration of mitochondrial transplantation as a powerful tool to increase the availability of viable organs, offering hope for patients in urgent need of liver transplants. METHODS Study Design 21 adult pigs ( Sus Scrofa Domesticus ), weighing approximately 50kg, were used in the study. Of these, 12 were used as a DCD liver source and subsequently placed on NMP, n = 6 (also used for NMP) were used for cross circulation, and n = 9 were used as non-treated controls. Pigs were housed in pens with a 12-hour light-dark cycle, ad libitum access to water prior to the experiment. All experimental procedures were performed according to ethical approval by the Ethical Committee on Animal Research (Dnr 5.2.18–8927/16) and conducted according to the directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes and Regulation (EU) 2019/1010 on the alignment of reporting obligation. Animals were randomised prior to the start of the study to either the treatment or the non-treatment group. For the experimental setup, circulatory arrest was achieved by inducing ventricular fibrillation by mechanical interference with the heart's conduction system. The donors were left untouched for two hours post arrest. The livers were then cold flushed in situ , explanted, and connected to NMP for four hours (n = 12), with half of the livers receiving mitochondria (n = 6) and the other half receiving a placebo (n = 6). Upon completion of NMP, three randomized livers from each group were connected to CC (n = 6). The DCD cohort was compared to a cohort of healthy control animals (n = 9), anesthetized with immediate biopsy of undisturbed liver tissue. Animal Preparation Twenty-one male and female adult farm-raised wild-type American Yorkshire pigs (Sus scrofa domesticus) with a mean weight of 50 kg were used in this study. Seraclone Anti-A (Bio-Rad, Medical Diagnostics GmbH, Dreieich, Germany) was used for determination of blood type that was then used to match the donor and recipient pairs along with weight. Prior to the start of the study, pairs were assigned randomly to either the donor treatment (n = 6) donor placebo (n = 6) group, recipient cross-circulation (n = 6) or control animals (n = 9). All were premedicated with xylazine (Rompun® vet. 20 mg/mL; Bayer AG, Leverkusen, Germany; 2 mg/kg) and ketamine (Ketaminol® vet. 100 mg/mL; Farmaceutici Gellini S.p.A., Aprilia, Italy; 20 mg/kg). A peripheral intravenous (IV) line was inserted in the earlobe, and a urinary catheter was inserted in the bladder. General anesthesia was accomplished with ketamine (Ketaminol® vet), midazolam (Midazolam Panpharma®, Oslo, Norway) and fentanyl (Leptanal®, Lilly, France) infusions. Mechanical ventilation was established using a Siemens-Elema ventilator (Servo 900C, Siemens, Solna, Sweden). The animals were intubated with a 7.5 size endotracheal tube. The ventilator was set to volume-controlled ventilation (VCV) with the flow pattern switch in “constant flow” which lowers the peak pressures according to the manufacturer’s instructions. Inspiration time is set to 25% with pause time 10% to give an I:E ratio of 1:2. Ventilation was adjusted to maintain carbon dioxide levels (PaCO 2 ) between 33–41 mmHg. Tidal volume (Vt) was kept at 6–8 mL/kg. Dynamic compliance was calculated by the equation \(\:{\text{C}}_{\text{d}\text{y}\text{n}}=\:\frac{{\text{V}}_{\text{T}}}{(\text{p}\text{e}\text{a}\text{k}\:\text{p}\text{r}\text{e}\text{s}\text{s}\text{u}\text{r}\text{e}-\text{P}\text{E}\text{E}\text{P})}\) . A pulmonary artery catheter (Swan-Ganz CCOmbo V and Introflex, Edwards Lifesciences Services GmbH, Unterschleissheim, Germany) was inserted in the right internal jugular vein, and an arterial line (Secalon-T™, Merit Medical Ireland Ltd, Galway, Ireland) was placed in the right common carotid artery. Figure 2 shows the overview of the experimental setup. Dihydrostreptomycinsulfate (0.1 mL/kg) (Boehringer Ingelheim Animal Health Nordics A/S, Copenhagen, Denmark) was given subcutaneously before initiation of surgery in all animals. Additionally, hemodynamic measurements were recorded at sixty minutes with thermodilution through a Swan-Ganz catheter and an arterial line. The following parameters were recorded: Heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP), central venous pressure (CVP), cardiac output (CO), systolic pulmonary pressure (SPP), diastolic pulmonary pressure (DPP), mean pulmonary pressure (MPP), pulmonary artery wedge pressure (PAWP), systemic vascular resistance (SVR), and pulmonary vascular resistance (PVR). For healthy control animals, a midline sternotomy and laparotomy were done, and tissue biopsies were taken randomly according to a previous randomization chart from the different liver lobes. For DCD animals, ventricular fibrillation was induced by mechanical interference with the heart's conduction system. 10,000 U of heparin was administered prior to ventricular fibrillation. After circulatory arrest, the donors were left untouched for two hours. After a midline sternotomy and laparotomy, the infra-renal inferior vena cava (IVC) and abdominal aorta were dissected free and prepared for cannulation. Cannulas were inserted in the IVC and abdominal aorta patc The liver was flushed with 2000 mL of cold (4–6°C) Institute George Lopez 1 preservation solution (Institute George Lopez, Lissieu, France) via the aorta and 2000 mL via the PV. The bile duct, portal vein (PV), and hepatic artery (HA) were dissected free. The liver was then completely mobilized and procured by sectioning the supra- and infra-hepatic IVC, the bile duct, the PV, and the coeliac axis with a patch of aorta. After hepatectomy, the liver was kept on ice slush and prepared for perfusion. Normothermic machine perfusion The coeliac axis artery (HA) was cannulated using a Medtronic DLP 8Fr arterial cannula (Medtronic, Minneapolis, MN, USA). The portal vein (PV) was cannulated using a Medtronic DLP 24Fr venous cannula (Medtronic, Minneapolis, MN, USA). After flushing of the liver and de-airing of the cannulas with 500 mL of gelofusine (Braun, Melsungen, Germany). the liver was then connected to the NMP. The inferior vena cava (IVC) was left open for free outflow of perfusate to keep the venous pressure gradient < 10 mmHg. The inflow of the PV was maintained to 0.5–0.7 L/min using a biomedical pump (Bio-Pump® Centrifugal pump, Medtronic, Minneapolis, MN, USA) and inflow to the HA was maintained to 0.15–0.3 L/min using a roller-pump (Jostra, Maquet HL20, Getinge, Sweden) not exceeding pressure > 150mmHg. Inflow and outflow pressures were monitored across machine perfusion. The common bile duct was cannulated with an 8 Fr cannula (Bio-Medicus, Medtronic, MN, USA), and bile was collected. Circuit temperature was maintained at 37°C using a water heater (Gaymar Stryker TP700 T/pump, Stryker Corporation, Kalamazoo, Michigan, USA) and oxygenator water jacket (Medos Hilite 7000). Sweep flow was approximately 1.5 L/min, Fi 60% with 93% N/7% CO 2 mixed gas. The system had been primed with Steen™ Solution (XVIVO perfusion, Gothenburg, Sweden), with 10,000 U of heparin, and maintained a hematocrit level of 20% using red blood cells from the donor animal taken before induced ventricular fibrillation. Mitochondrial Isolation and Function Mitochondria that were used for MTx were isolated from fresh liver tissue of C57BL/6 mice. To further evaluate mitochondrial function under control and IRI conditions, an additional set of mitochondria was isolated from porcine liver tissue for ATP quantification. Briefly, harvested tissue underwent homogenization in a 5 mL respiration buffer solution (250 mmol/ sucrose, 20 mmol/ K+-HEPES (4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid, pH 7.2), followed by a 10-minute Subtilisin, enzymatic digestion process, conducted on ice. Subsequently, the digested tissue underwent a series of filtration steps, and the mitochondria were precipitated via centrifugation at 9,500 rpm for 5 minutes at 4°C, then resuspended for subsequent delivery, as previously detailed 71 . Multisizer 4e Coulter Counter (Beckman, Miami, FL, USA) was employed to assess the size and quantity of the isolated mitochondria, while viability was determined through the ATPlite assay (Revvity Inc, Sweden) 72 – 75 . Mitochondrial dose and delivery Following the start of machine perfusion, livers were randomly assigned to receive either 1x10 10 xenogeneic mitochondria (in 10 mL of isolation buffer), or a placebo (10 mL of isolation buffer) as a control. In previous studies, we have shown that mitochondrial dose concentrations for efficacy vary with the organ being treated. For the heart and kidney, it has been shown that 2×10 5 to 2×10 6 mitochondria per gram of wet weight is safe and efficacious 29 , 31 . Mitochondria were isolated in suspension, which was divided into equal volumes and delivered as an antegrade bolus divided between the hepatic artery and portal vein through separate ports in each line over 5 seconds. The liver was then perfused for four hours on NMP. Isolation and cryopreservation of primary human hepatocytes. Primary human hepatocytes were isolated by a three-step perfusion technique as previously described 76 , 77 . Briefly, the liver was perfused with buffer (Hank’s balanced salt solution, Sigma-Aldrich, St. Louis, MO) containing 0.5 mM EGTA (Sigma-Aldrich, St. Louis, MO) followed by a washout and perfusion with 250 mg/L collagenase (Collagenase XI, Sigma-Aldrich, St. Louis, MO or CIzyme, Vitacyte, Indianapolis, IN). The degraded tissue was cut to release cells and cells were washed by centrifugation to remove non-parenchymal cells. Cells were cryopreserved at a concentration of 7 million cells/mL and with 10% DMSO (Sigma-Aldrich, St. Louis, MO) in IGL-1 preservation solution. The solution with the cells was left on ice for 15 minutes followed by 2 hours at -80°C. They were then transferred and stored in liquid nitrogen. Cell Culture For culture, primary human hepatocytes were thawed using Hepatocyte Thaw Medium (Gibco, Thermo Fisher Scientific, Waltham MA, USA) and seeded in Williams' E Medium (no phenol red; Gibco) supplemented with Primary Hepatocyte Maintenance Supplements (Gibco). Plates were pre-coated with collagen (50 µg/mL in 0.02 M acetic acid). Cells were seeded at 5 × 10 3 cells/well in 96 well plate and incubated overnight at 37°C in 5% CO₂. The following day, cells were washed with warm DPBS and incubated with 50 µL of media containing freshly isolated mitochondria (1 × 10⁵/well) for four hours. Mitochondrial function was assessed using the ATPlite assay (Revvity Inc., Waltham, MA, USA), and luminescence was measured with a GloMax® plate reader (Promega, Madison, WI, USA). Live Cell Imaging Human hepatocytes were seeded at a density of 3 × 10 4 cells per well in collagen-coated 8-well slides (ibidi GmbH, Gräfelfing, Germany) and incubated overnight at 37°C in a 5% CO₂ atmosphere. Host cells were stained with CellMask™ (C10045, Invitrogen, Thermo Fisher Scientific) for 30 minutes and nuclei were stained with Hoechst 33342. Isolated mitochondria were labeled with MitoTracker Green FM (Invitrogen). Fluorescence and confocal microscopy were used to visualize mitochondrial uptake and localization. Cross circulation Recipient pigs (n = 6) underwent sedation and general anaesthesia in a similar fashion to donor pigs. A right neck cut-down exposed the right internal jugular vein, and a right groin cut-down exposed the femoral artery. A 15,000 U heparin bolus was administered, and cannulation with 15F catheters (Life Support Medtronic) in the right internal jugular vein and a 12F catheter (DLP Medtronic) in the femoral artery was performed using the Seldinger technique. Immediately before the initiation of cross-circulation, recipient pigs were intravenously administered one gram of methylprednisolone (APP Pharmaceuticals) and 500 mg of calcium chloride (Hospira). Donor blood used to prime the circuit during NMP was not removed. The tubing was spliced, deaired, and connected the recipient pig to the NMP circuit, marking the start of cross-circulation (CC). AV-AV cross circulation circuit was secured, and flow was maintained using a roller-pump (Jostra HL-20 pump console; Maquet). Circuit flows were titrated to 0.15-0.3L/min to the HA, 0.65-0.8L/min to the PV, and 1L/min of arterial return to the recipient for veno-arterial circulatory support. Pressure (PA and PV), flow (PA and PV), and temperature data were continuously monitored using System M® by Spectrum Medical, USA. Circuit temperature was maintained at 37°C using a water heater (Gaymar Stryker TP700 T/pump) and oxygenator water jacket (Medos Hilite 7000). Throughout the duration of cross-circulation, the recipient was maintained on a continuous heparin infusion (initial rate of 25 U kg − 1 h − 1 ). Activated clotting time was measured using a HemoChron whole blood microcoagulation system (Accriva Diagnostics), and the heparin drip was adjusted to maintain a target value of 250–350 s. Physiological parameters of the recipient, including heart rate, electrocardiogram, blood pressure (cuff and arterial A-line pressure), mean arterial pressure (MAP), oxygen saturation (SpO 2 ), end-tidal CO 2 , temperature, and respiratory rate, were continuously monitored and recorded using a multi-parameter MX750 monitor (Philips Healthcare, Lund, Sweden). Upon establishment of CC, a laparotomy was done, the portal vein (PV), and hepatic artery (HA) were dissected free. The PV and the HA of the recipient liver were clamped such that the liver on NMP was the only one filtering the blood of the recipient. Cross circulation was maintained for six hours, after which the liver and recipient perfusions were discontinued. Blood and bile collection Blood samples and bile samples were collected every hour during the experimental time course. Blood gases and bile samples from the donor liver were drawn and analyzed every hour during the experimental time course, on an ABL 90 FLEX blood gas analyzer (Radiometer Medical ApS, Brønshøj, Denmark). Tissue Sampling/Processing Biopsies were taken randomly within the same liver lobe for all biological replicates repeatedly during the experimental timeline. Samples were fixed in 4% paraformaldehyde for 48 hours before transfer to 0.01% sodium azide in PBS. For hematoxylin and eosin (H&E) and fluorescent staining samples were embedded in paraffin wax after dehydration in ethanol series. Hematoxylin and eosin staining (Merck Millipore, Germany) was performed on 4 µm sections. Sections (cut using a microtome) were de-paraffinized, starting with xylene (100%), followed by immersion in a graded ethanol series (from 99.99% to 70%) and ending with rinses in distilled water. After H&E staining (Histolab, Askim, Sweden), slides were dehydrated in ethanol, 70%, 96% and 99.99%, and finally with xylene (100%). Sections were mounted in Pertex (Histolab, Gothenburg, Sweden). Fluorescent Staining Staining was carried out directly on glass slides using a PAP pen to create a hydrophobic barrier. After permeabilization and blocking slices were incubated with primary antibody (IBA1, 1:750; CK19, 1:250) overnight at 4 degrees Celsius. Following washes samples were incubated with secondary antibody (goat anti-rabbit 568, 1:1000) for 90 minutes at 4 degrees Celsius. Finally, samples were incubated with DAPI (1:1000) and tomato lectin (1:500) for 30 minutes at room temperature before final washes and mounting with fluoromount-G. All imaging was carried out using a Nikon confocal A1RHD. Image Acquisition For H&E staining, imaging was carried out on an Olympus light microscope. Representative images of each sample were captured using a 4x, 10x, and 20x objective. For fluorescent staining, imaging was carried out on a Nikon A1RHD confocal. All images were captured at a resolution of 2765 2 pixels (total field of view (FOV) = 2.8mm 2 ), at a resolution of 0.61µm/pixel, using a 20x objective, NA-0.75, RI-1.0. Four random FOV’s were imaged per sample. Laser and gain settings were kept constant across samples. Nikon files were exported as 8-bit TIFF images, one per channel, for analysis. Confocal Image Analysis For fluorescent staining, images were analyzed using Fiji 78 . Macros were written for all analytics carried out in this manuscript. Briefly, both IBA1 and CK19 coverage were analyzed by thresholding the image and analyzing the signal coverage area as a % of the whole FOV. Histopathological Scoring H&E images, at 4x, 10x and 20x magnifications, from all timepoints (healthy control, NMP-end and CC-end) were compiled into a blinded slide deck. The compiled images were sent to a pathologist for scoring where the tissue was assessed according to the BANFF criteria 79 , accounting for portal inflammation, bile ductal inflammation/damage and venous endothelial inflammation, with additional scoring for degree of edema and hemorrhage. Cytokine Evaluation Cytokine evaluation was carried out on tissue homogenate at the end of machine perfusion, and end of cross circulation. A porcine kit for IL-6 and IL-10 was used (EPX090-60829-901, Thermo Fisher Scientific) according to the manufacturer’s instructions. The kit was run using a Bioplex-200system (BioRad, Hercules, CA, USA). TUNEL Staining, Imaging & Quantification TUNEL Assay Kit BrdU Red was used (Abcam, ab66110). 10 µm FFPE liver sections were de-paraffinised and rehydrated according to standard protocols. Rehydrated samples were permeabilised for 10 min at room temperature in 3% triton-x-100 after which staining was carried out according to suppliers instructions. Before mounting samples were counterstained with tomato lectin-488. Imaging was carried out on Nikon ti2 epifluorescence microscope. Samples were imaged blinded and 4 random FOVs were acquired from each biopsy. Analysis was carried out in Fiji and TUNEL positive cells were automatically detected using a pre-developed macro. Wet-dry Ratio Hepatic oedema was assessed by measuring the wet weight to dry weight ratio in liver tissue from the distal liver segments from four hours of NMP and after completed CC. Liver tissue pieces were weighed, lyophilised for 24 hours, and then weighed again. The ratio between the wet and dry weight was then calculated. Mass spectrometry analysis Proteins from bile and liver tissue were extracted and digested with trypsin (Promega, Madison, WI, USA) using a S-Trap digestion protocol (ProTifi, Fairport, New York, USA). Mass spectra were acquired through the data-independent acquisition (DIA) method on a timsTOF HT instrument coupled to an EVOSEP one and analyzed using DIA-NN v1.8.1 80 . Downstream analyses were performed in RStudio v2024.09.01, using R v4.4.2. Differentially expressed proteins were determined through thresholds q-value < 0.05 (FDR-corrected p-value) and log2-fold change estimated from bootstrapping methods 81 . Ultrahigh-Content Imaging Imaging was carried out on 10µm FFPE sections using the automated cyclic staining platform, MACSima™ (Miltenyi Biotec, Bergisch Gladbach, Germany) with MACSwell 4 frames. Prior to imaging, sections underwent antigen retrieval by boiling in TEC buffer for 30 minutes. Before samples were loaded into the MACSima prestaining with DAPI (1:10) was done for 10 minutes followed by 3 washes in running buffer. Finally, each well was filled with 250µL running buffer before loading into MACSima™. Antibodies used from this readout were a combination of those produced and pre-validated by Miltenyi Biotec for ultrahigh-content imaging, as well as antibodies from external sources. All antibodies were directly conjugated with either FITC, PE, APC, Alexa-fluor-488 or Alexa-fluor-635. Staining conditions which include antibody dilution, incubation time, staining cycle sequence, photobleaching, and exposure time were kept constant across all runs. Image acquisition took place with a 20x long working distance objective (NA-0.45). MACSima Image Analysis Image pre-processing and analysis was carried out using MACS-iQ View software (Miltenyi Biotech), which includes stitching, spatial alignment and registration, and subtraction of residual intensities from autofluorescence. Single cell segmentation was carried out using the Advanced Morphology for Tissue algorithm based on DAPI as a reference channel with a Donut method to segment the cytoplasm. Cell classification was achieved using a histogram-based gating strategy for each marker. Statistics Data collection was conducted with Microsoft Excel. Data were analyzed using GraphPadPrism10. Normality was difficult to assess due to the small sample sizes, however based on histogram assessment all data were considered normal. Data were analyzed using either a one-way ANOVA with multiple comparisons using Tukey’s test, when comparing more than two groups, or a t-test when comparing two groups. Significance was defined as p < 0.05(*), p < 0.01(**), p < 0.001(***), p < 0.0001(****). Declarations Ethics: All experimental procedures were performed according to ethical approval by the ethical Committee on Animal Research (Dnr 5.2.18–8927/16) and conducted according to the CODEX guidelines by the Swedish Research Council, Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes and Regulation (EU) 2019/1010 on the alignment of reporting obligations and complies with ARRIVE guidelines. Competing interests: The authors declare that they have no competing interests. FUNDING Grants awarded by the Marianne and Marcus Wallenberg Foundation, the Knut and Alice Wallenberg Foundation, and the ALF Foundation were used for this research. 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MS-DAP platform for downstream data analysis of label-free proteomics uncovers optimal workflows in benchmark data sets and increased sensitivity in analysis of Alzheimer’s biomarker data. J Proteome Res 22, 374–386 (2022). Additional Declarations There is NO Competing Interest. Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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1","display":"","copyAsset":false,"role":"figure","size":343838,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eDCD livers suffer mitochondrial depletion but respond to MTx in vitro\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003eOverview of tissue sampling from baseline and DCD livers. \u003cstrong\u003eb \u003c/strong\u003eQuantification of tissue ATP levels (µM) from baseline and DCD liver samples. \u003cstrong\u003ec-d\u003c/strong\u003e Representative transmission electron microscopy (\u003cstrong\u003ec\u003c/strong\u003e) and confocal (\u003cstrong\u003ed\u003c/strong\u003e) images of mitochondria isolated from mouse liver. \u003cstrong\u003ee \u003c/strong\u003eSchematic showing approach for adding exogenous mitochondria to hepatocyte cell cultures derived from a control and IRI human livers. \u003cstrong\u003ef \u003c/strong\u003eQuantification of ATP levels after MTx measured as % change from baseline (100%). \u003cstrong\u003eg \u003c/strong\u003eRepresentative confocal images of single cells in control in IRI cultures showing mitochondrial uptake. \u003cstrong\u003eh \u003c/strong\u003eQuantification of average Mitotracker signal (average fluorescence units) within cells from control and IRI cell cultures. T-tests *p \u0026lt; 0.05, **p \u0026lt; 0.01, **p \u0026lt; 0.001, ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"OnlineFig12.png","url":"https://assets-eu.researchsquare.com/files/rs-7871574/v1/93cbf36a1c7b6a053e987ab2.png"},{"id":96244515,"identity":"fa653085-3db5-4700-b299-52e2e2b40565","added_by":"auto","created_at":"2025-11-19 07:18:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":413268,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eMTx maintains liver function on NMP after extended DWIT\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. a \u003c/strong\u003eTo produce a DCD model, ventricular fibrillation was induced in the donor through interference with the conduction system. Warm ischemic time (WIT) was 2 hours after which livers were retrieved and then perfused using normothermic machine perfusion (NMP) for 4 hours, receiving either MTx or placebo. Finally, livers were connected via NMP to a living recipient to establish a cross-circulation (CC) approach, which was maintained for 6 hours. Baseline liver biopsies were obtained from n=9 healthy control pigs. \u003cstrong\u003eb-c \u003c/strong\u003eRepresentative H\u0026amp;E images from healthy control and DCD livers. \u003cstrong\u003ec \u003c/strong\u003eQuantification of cumulative injury score between healthy control and DCD liver samples. \u003cstrong\u003ee \u003c/strong\u003eVolcano plot showing significantly differentially expressed proteins in bile with upregulated proteins highlighted in red and downregulated proteins highlighted in blue in the DCD group in reference to the baseline group. \u003cstrong\u003ef \u003c/strong\u003eQuantification of total bile production (mL) across NMP between non-treated and treated livers. \u003cstrong\u003eg \u003c/strong\u003eQuantification of bile aspartate aminotransferase (AST, m(/mL) levels between non-treated and treated livers. \u0026nbsp;\u003cstrong\u003eh-k \u003c/strong\u003eQuantification of bile bilirubin (\u003cstrong\u003eh, \u003c/strong\u003eµmol/L), glucose (\u003cstrong\u003eI, \u003c/strong\u003emmol/L), pH (\u003cstrong\u003ej\u003c/strong\u003e), and lactate levels (\u003cstrong\u003ek, \u003c/strong\u003emmol/L) between non-treated and treated livers, at 5 minutes and 4 hours on NMP. \u003cstrong\u003el \u003c/strong\u003eGene set enrichment analysis (GSEA) was performed to display significantly differentially expressed biological processes in bile at end of NMP. Gene set enrichment analysis (GSEA) was performed to identify significantly altered biological processes in bile at the end of NMP. Upregulated processes (shown in red) and downregulated processes (shown in blue) in the mitochondrial treatment group are presented relative to the no-treatment group. NES = normalized enrichment score\u003cem\u003e. \u003c/em\u003e\u003cstrong\u003em-o \u003c/strong\u003eQuantification of plasma glucose (\u003cstrong\u003em\u003c/strong\u003e), lactate(\u003cstrong\u003en\u003c/strong\u003e) and AST (\u003cstrong\u003eo\u003c/strong\u003e) levels between non-treated and treated livers, at 5 min and 4h on NMP. \u003cstrong\u003ep\u003c/strong\u003e Quantification of portal vein flow between non-treated and treated livers, at 5 min and 4h on NMP. \u003cstrong\u003eq\u003c/strong\u003e Quantification of hepatic artery flow between non-treated and treated livers, at 5 min and 4h on NMP. n=6/group. t-test or two-way ANOVA. *p \u0026lt; 0.05, **p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"OnlineFig2.png","url":"https://assets-eu.researchsquare.com/files/rs-7871574/v1/5bcbe525e97f5bcda63c2673.png"},{"id":95945312,"identity":"bf620e76-129e-4609-b29a-407d023c22cd","added_by":"auto","created_at":"2025-11-14 17:29:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1122743,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eInception of tissue perturbation and Kupffer cell decline occurs in untreated livers on NMP\u003c/strong\u003e\u003c/em\u003e. \u003cstrong\u003ea\u003c/strong\u003e Representative H\u0026amp;E images from healthy control, DCD 2h, treated NMP and non-treated NMP samples. \u003cstrong\u003eb\u003c/strong\u003e Quantification of cumulative injury score between healthy control, DCD 2h, treated NMP and non-treated NMP samples. \u003cstrong\u003ec\u003c/strong\u003e Representative confocal images stained for Lycopersicon Esculentum (LEA) lectin and CK19 (cholangiocytes) in healthy control, DCD 2h, treated NMP and non-treated NMP samples. \u003cstrong\u003ed \u003c/strong\u003eQuantification of CK19+ cholangiocyte coverage between healthy control, DCD 2h, treated NMP and non-treated NMP samples. \u003cstrong\u003ee \u003c/strong\u003eRepresentative confocal images stained for Lycopersicon Esculentum (LEA) lectin and IBA1 (Kupffer cells) in healthy control, DCD 2h, treated NMP and non-treated NMP samples. \u003cstrong\u003ef \u003c/strong\u003eQuantification of Kupffer cell coverage between healthy control, DCD 2h and treated NMP samples. \u003cstrong\u003eg \u003c/strong\u003eQuantification of Kupffer cell coverage between healthy control, DCD 2h and non-treated NMP samples. \u003cstrong\u003eh \u003c/strong\u003eRepresentative images showing TUNEL staining in healthy control, DCD 2h, treated NMP and non-treated NMP samples. \u003cstrong\u003ei\u003c/strong\u003e Quantification of TUNEL+ cell count/FOV between healthy control, DCD 2h, treated NMP and non-treated NMP samples. \u003cstrong\u003ej \u003c/strong\u003eVolcano plot showing significantly differentially expressed proteins in bile with upregulated proteins highlighted in red and downregulated proteins highlighted in blue in the treatment group in comparison to the no treatment group at end of NMP. Threshold for significant log2 foldchange was set through bootstrapping in the MS-DAP package in R and significant q-value (-log10 FDR adjusted p-value) was set to \u0026lt;0.05.\u003cem\u003e \u003c/em\u003e\u003cstrong\u003e\u0026nbsp;k\u003c/strong\u003e Wet/dry ratio quantification to assess oedema between treated NMP and non-treated NMP samples. \u003cstrong\u003el-m\u003c/strong\u003e Quantification of interleukin-6 and interleukin-10 levels between treated NMP and non-treated NMP samples (pg mL\u003csup\u003e-1\u003c/sup\u003e). n=6/group. t-test or ANOVA. *p \u0026lt; 0.05, **p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"OnlineFig3.png","url":"https://assets-eu.researchsquare.com/files/rs-7871574/v1/ae214dd7d62566f428da656c.png"},{"id":96245149,"identity":"f406c9d6-ff26-49d4-8bc0-475e1886eddf","added_by":"auto","created_at":"2025-11-19 07:19:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":664581,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eMTx regenerates livers on cross circulation\u003c/strong\u003e\u003c/em\u003e. \u003cstrong\u003ea\u003c/strong\u003e Quantification of total bile production (mL) across CC between non-treated and treated livers. \u003cstrong\u003eb\u003c/strong\u003e Quantification of bile aspartate aminotransferase (AST) levels (mU/mL) between non-treated and treated livers. \u003cstrong\u003ec-f\u003c/strong\u003e Quantification of bile bilirubin (\u003cstrong\u003ec, \u003c/strong\u003eµmol/mL), glucose (\u003cstrong\u003ed, \u003c/strong\u003emmol/L), pH (\u003cstrong\u003ee\u003c/strong\u003e) and lactate (\u003cstrong\u003ef, \u003c/strong\u003emmol/L) levels between non-treated and treated livers on CC. \u003cstrong\u003eg \u003c/strong\u003eVolcano plot showing significantly differentially expressed proteins in bile with upregulated proteins highlighted in red and downregulated proteins highlighted in blue in the treatment group in comparison to the non-treated group at the end of cross circulation. The threshold for significant log2 foldchange was set through bootstrapping in the MS-DAP package in R, and significant q-value (-log10 FDR adjusted p-value) was set to \u0026lt;0.05\u003cem\u003e. \u003c/em\u003e\u003cstrong\u003eh \u003c/strong\u003eGSEA of biological processes at the end of cross circulation displayed as q-values. Upregulated processes (red) and downregulated processes (blue) in the treatment group in reference to the non-treated group. NES = normalized enrichment score.\u003cstrong\u003e \u0026nbsp;i-k\u003c/strong\u003e Quantification of plasma glucose (\u003cstrong\u003ei\u003c/strong\u003e, mmol/L), lactate (\u003cstrong\u003ej\u003c/strong\u003e, mmol/L), and AST levels (\u003cstrong\u003ek\u003c/strong\u003e, mU/mL) between non-treated and treated livers on CC.\u003cstrong\u003e l\u003c/strong\u003e Quantification of portal vein flow (L/min) between non-treated and treated livers, at 5 min and 4h on NMP.\u003cstrong\u003e m\u003c/strong\u003e Quantification of hepatic artery flow (L/min) between non-treated and treated livers, at 5 min and 4h on NMP. \u003cstrong\u003en \u003c/strong\u003eRepresentative H\u0026amp;E images and quantification of cumulative injury score from healthy control, treated CC, and non-treated CC samples. \u003cstrong\u003eo \u003c/strong\u003eRepresentative TUNEL staining images and quantification of TUNEL+ cells from healthy control, treated CC, and non-treated CC samples. \u003cstrong\u003ep \u003c/strong\u003eRepresentative confocal images stained for Lycopersicon Esculentum (LEA) lectin and IBA1 (Kupffer cells) and Kupffer cell coverage quantification in healthy control, treated CC, and non-treated CC samples. \u003cstrong\u003eq \u003c/strong\u003eRepresentative confocal images stained for Lycopersicon Esculentum (LEA) lectin and CK19 and CK19+ cell coverage quantification in healthy control, treated CC, and non-treated CC samples. \u003cstrong\u003er \u003c/strong\u003eWet/dry ratio quantification to assess oedema between treated CC, and non-treated CC samples. \u003cstrong\u003es-t\u003c/strong\u003e Quantification of interleukin-6 and interleukin-10 levels between treated CC and non-treated CC samples (pg mL\u003csup\u003e-1\u003c/sup\u003e). n=3/group. t-test. *p \u0026lt; 0.05. \u0026nbsp;\u003c/p\u003e","description":"","filename":"OnlineFig4.png","url":"https://assets-eu.researchsquare.com/files/rs-7871574/v1/dc97967a613a56d393610e99.png"},{"id":96244748,"identity":"80144122-c029-43a8-83d2-a2d05454c827","added_by":"auto","created_at":"2025-11-19 07:19:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1275286,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eMTx generates proteomic and cell population profiles comparable to healthy tissue.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003eHeat map of hierarchical clustering of significantly differentially expressed proteins in bile. Columns represent individual samples from the different groups and rows represent individual protein, where blue color represents under-expressed proteins and red color represents overexpressed proteins. \u003cstrong\u003eb \u003c/strong\u003eRepresentative images from baseline, DCD 2h, non-treated NMP, treated NMP, non-treated CC, and treated CC acquired using MACSima, stained for DAPI, CD3, CD4, PCNA, Ki67, vimentin, actin, CD163, LEA, HH3K4, IBA1, collagen-1. \u003cstrong\u003ec \u003c/strong\u003eCell population qualifications between cohorts for CD3+, CD4+, PCNA+, Ki67+, vimentin+, CD163+, PCK+, LEA+, HH3K4+, and IBA1+ cells. ANOVA. *p \u0026lt; 0.05, **p \u0026lt; 0.01, **p \u0026lt; 0.001, ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"OnlineFig5.png","url":"https://assets-eu.researchsquare.com/files/rs-7871574/v1/8010aa6e674ed0ffee8de68b.png"},{"id":96244812,"identity":"820d3034-afd3-4f70-9eb0-d2fbf6be2f75","added_by":"auto","created_at":"2025-11-19 07:19:18","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":798269,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eHepatic\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ezonal analysis shows distinct patterns of cell expression and highlight a regenerative effect for MTx.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea-b \u003c/strong\u003eRepresentative images of the hepatic functional unit with and without segmentation. Segmentation includes collagen border (orange), zone-1 (pink), zone-2 (green), zone-3 (yellow). \u003cstrong\u003ec-i \u003c/strong\u003eSpatial\u003cstrong\u003e \u003c/strong\u003ecell population qualifications by zone between cohorts for PCNA+, CD163+, Ki67+, CD3+, HH3+, IBA-1+ cells and LEA+. Two-way ANOVA. *p \u0026lt; 0.05, **p \u0026lt; 0.01, **p \u0026lt; 0.001, ****p \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"OnlineFig6.png","url":"https://assets-eu.researchsquare.com/files/rs-7871574/v1/e4a03e451a5d8873e05f6bc5.png"},{"id":96255595,"identity":"95ccff0f-e00e-45de-a72d-fe2e2f9c4db8","added_by":"auto","created_at":"2025-11-19 07:48:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7183401,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7871574/v1/1639c368-7309-44f1-a427-5bf892676482.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Mitochondrial Therapy During Machine Perfusion Restores the Function of DCD Livers: A Cross-Circulation Evaluation for Transplant Suitability","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eLiver transplantation is a highly successful therapy for end-stage liver disease, but its availability is severely limited by disparities between organ supply and the need for transplantation, leading to many patients dying awaiting an organ\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. While retrieval of organs from donation after brain death (DBD) donors remains the most common pathway in many countries, workflows and guidelines have been established to facilitate the retrieval and transplantation of organs from donation after circulatory death (DCD) donors\u003csup\u003e\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eUnlike kidneys or lungs, the liver is particularly vulnerable to the warm ischemic time (WIT) inherent to the DCD process. This presents a significant challenge even when livers are recovered with a super rapid retrieval approach, stored statically in ice, and subsequently transplanted\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Techniques such as normothermic regional perfusion (NRP), normothermic machine perfusion (NMP), and hypothermic oxygenated perfusion (HOPE) offer critical advantages for both organ quality and transplantation outcomes\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. While the development of controlled DCD (cDCD) has substantially increased the donor pool, the greatest potential for expansion lies within uncontrolled DCD (uDCD) donors\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. However, the success of using uDCD donors hinges on overcoming the challenges related to a prolonged WIT, which has been one of the main barriers to establishing successful clinical programs for these donors\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn cDCD liver transplantation, minimizing WIT remains a key consideration, as evidence consistently indicates that reducing ischemic time is essential to optimizing organ viability and function. A WIT\u0026thinsp;\u0026ge;\u0026thinsp;35 minutes was shown to result in a 1.8-fold higher graft loss rate than WIT\u0026thinsp;\u0026le;\u0026thinsp;15 minutes\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. The extended donor hepatectomy time, the interval between aortic cross-clamp and in situ cold perfusion, until the liver is removed from the body, incurs a negative impact on graft survival\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. A recent analysis of the \u003cem\u003eEurotransplant\u003c/em\u003e registry, encompassing 12,974 recipients, revealed that the hepatectomy time is notably longer in DCD donors and is associated with a significantly higher risk of death-censored graft loss\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Ischemia-reperfusion injury (IRI), an unavoidable challenge in current transplantation practice, continues to impact outcomes significantly\u003csup\u003e\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. This injury occurs when the organ, subjected to ischemia during retrieval from the donor, is subsequently reperfused in the recipient, leading to inflammatory and oxidative stress responses\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Despite advancements in preservation and perfusion technologies, IRI remains a major barrier to optimal graft function and long-term survival, as it exacerbates tissue damage and influences both acute and chronic rejection risks\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Addressing IRI is essential for improving transplantation outcomes, highlighting the need for continued innovation in organ preservation and protective strategies. Although ex-situ MP has emerged as a valuable strategy to assess and treat marginal livers, these techniques shift the reperfusion process from the patient to the machine, and during this process, tissue injury may still occur\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. While reperfusion injury in the liver after transplantation is thought to be mediated by transient portal hypertension, which causes hyperdynamic stress to the hepatic endothelial lining, more focus is now being placed on reperfusion-mediated oxidative stress and mitochondrial dysfunction\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. It is postulated that IRI leads to the production of significant amounts of reactive oxygen species (ROS) by Kupffer cells and the intrahepatic neutrophils. ROS production is primarily triggered by mitochondrial respiratory dysfunction, which results in the accumulation of succinate and the reversal of the electron transition from complex I to complex II\u003csup\u003e25\u0026ndash;27\u003c/sup\u003e. Reperfusion-induced oxidative stress typically peaks between 2\u0026ndash;6 hours after reperfusion and results in sinusoidal endothelial cell, hepatocyte, and cholangiocyte death, mitochondrial swelling, and metabolic dysfunction\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. This highlights a compelling link between mitochondrial dysfunction, WIT, and IRI, and as such, substantiates the use of mitochondrial therapies as a potential strategy to minimize hepatic IRI, expand the DCD liver pool, and in doing so, increase organ availability and improve graft survival\u003csup\u003e\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eWe developed a novel approach for delivering respiration-competent, exogenously isolated mitochondria to neonatal, pediatric and adult, DCD hearts\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. These transplanted mitochondria function as metabolic support for the endogenous mitochondrial pool, promoting normalization of transcriptomic and proteomic profiles to levels comparable to those observed in DBD hearts\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. This normalization helps preserve cellular metabolism and attenuate the cascade of IRI. Although mitochondria transplantation (MTx) has been studied in rodent liver IRI models with relatively short WIT\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, its\u0026rsquo; application in ex vivo settings using machine perfusion, and a transplantation viability model has not been previously investigated. This study is the first to evaluate mitochondrial transplantation as a therapeutic strategy to mitigate long WIT-induced injury (two hours) in DCD livers using a clinically relevant porcine model in combination with normothermic machine perfusion (NMP). DCD porcine livers were subjected to NMP, with or without MTx, followed by cross-circulation with a recipient to assess transplant viability.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eDCD livers exhibit signs of mitochondrial depletion\u003c/h2\u003e\u003cp\u003eTo validate the rationale that DCD livers do indeed suffer mitochondrial dysfunction when exposed to a long WIT, biopsies from healthy livers and those exposed to a 2-hour WIT (2h DCD) were assessed with regards to ATP production (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). The DCD samples exhibited a significant two-fold reduction in tissue ATP levels, pointing to a depletion of endogenous mitochondria (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Next, we sought to understand whether supplementation with exogenous mitochondria, isolated as previously described from the livers of C57BL/6 mice\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec-d), renders any benefit to injured hepatocytes in \u003cem\u003ein vitro\u003c/em\u003e conditions. When hepatocyte cultures derived from both healthy and IRI human liver tissue were supplemented with exogenous mitochondria, the IRI-derived hepatocytes exhibited an increase in ATP production compared to the healthy baseline (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee-f). This highlights that the addition of exogenous mitochondria into tissue appears to produce metabolic functional improvements in tissue that has suffered from mitochondrial dysfunction. This was further validated by live cell imaging after the addition of fluorescently labelled exogenous mitochondria, which revealed significantly greater levels of mitochondria uptake into IRI-hepatocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg-h). Taken together, these data provide compelling evidence that the exogenous mitochondria are avidly taken up into DCD livers with extended WIT.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eOverview of the model, therapy, and assessment modalities\u003c/h3\u003e\n\u003cp\u003eTo explore the benefit of MTx for DCD liver regeneration in a clinically relevant model, we established a workflow composed of three main parts: 1-generation of a porcine DCD model, 2-normothermic machine perfusion (NMP) of DCD livers, with or without MTx, 3-cross circulation of DCD livers with a recipient after NMP (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Ventricular fibrillation was induced in the donors by interference with the cardiac conduction system and livers were left \u003cem\u003ein situ\u003c/em\u003e for two hours post circulatory arrest before retrieval (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Livers were then randomised (n\u0026thinsp;=\u0026thinsp;6/group) into mitochondrial treatment and non-treated groups and placed on NMP for four hours. The surgeon, perfusionist, anesthesiologist, as well as the nurses and technicians performing the experiments, were blinded to the study arm. MTx or placebo were given as a bolus injection equally divided between the portal vein and the hepatic artery at the start of NMP. To further evaluate the transplant potential of the livers after NMP (n\u0026thinsp;=\u0026thinsp;3/group), a cross-circulation model (CC) was established with a recipient pig for six hours. For comparison with a normal liver cohort, n\u0026thinsp;=\u0026thinsp;9 porcine subjects were anesthetized, the abdomen was opened, and liver tissue samples were collected.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eMitochondrial transplantation improves DCD liver function on normothermic machine perfusion\u003c/h3\u003e\n\u003cp\u003eGeneration of DCD liver injury was confirmed through blinded scoring by a pathologist of tissue biopsies after two hours of warm ischemic time compared with healthy control liver tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb-c), with DCD livers exhibiting consistent elevated cumulative injury scores (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). In keeping with this, a comparative proteomic analysis of bile between healthy controls and DCD livers revealed that 580 proteins were significant differentially expressed, with the majority being upregulated (516 proteins) in contrast to the downregulated proteins (64 proteins) in the DCD group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). To explore the potential benefit of MTx in these organs, mitochondria were delivered serially in the first 10 minutes of NMP, and again after four hours. The regenerative impact of the mitochondria was already noticeable, with treated organs exhibiting a significant increase in bile production (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef), coupled with significant reduction in bile aspartate aminotransferase (AST) levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg). A more detailed bile composition analysis showed that both cohorts exhibited significant reductions in bilirubin concentration at four hours on NMP when compared to the start of NMP (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh). In both groups, there was a significant increase in bile glucose, though this difference was nearly 2.5-fold higher in non-treated livers, while treated livers only displayed a modest 30% increase (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ei). No differences in pH were seen (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ej), and while no significant differences in bile lactate were observed, it was evident that there was a trend for higher bile lactate levels in the non-treated livers after four hours of NMP (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ek), indicating the maintenance of anaerobic metabolism in the absence of MTx. Gene set enrichment analysis (GSEA) was performed to visualize enriched biological processes found in bile in the treatment group after NMP, with the non-treated group as reference. Downregulated processes in the treatment group (blue) included mostly metabolic and catabolic processes at end of NMP while enriched processes (red) included response to stress, cell migration, cell adhesion and immune system processes, yielding a bile profile similar to one previously demonstrated in human livers deemed acceptable for transplantation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003el). Taken together these data infer a higher quality bile production in livers treated with MTx on NMP, which met the threshold for transplantation according to the bile composition\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eGlucose concentrations in perfusate increased during NMP in the treated group and decreased in the non-treated group; however, this change was not statistically significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003em). Differences in perfusate lactate and AST concentrations were also not significant but mirrored the trends seen in the bile samples after four hours of NMP (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003en-o). A significant, but not clinically relevant difference was seen in portal vein flow after five minutes of perfusion, while after 4 hours of NMP a significant difference was seen between the cohorts, with the mitochondrial treatment group exhibiting higher portal flow (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ep). Hepatic artery (HA) flow was equal after 5 min of NMP, while after 4 hours there was no significant difference between the groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eq). However, the mean flow in HA in the control group declined between the start and 4h NMP, whilst flow increased in the treated group, indicating an overall improved perfusion and highlighting a trend towards a more rapid normalization of the graft haemodynamics post perfusion.\u003c/p\u003e\n\u003ch3\u003eFirst signs of tissue perturbation occur in non-treated livers during NMP\u003c/h3\u003e\n\u003cp\u003eTo assess the degree of injury to the liver after treatment/placebo and NMP, a blinded pathologist scored hematoxylin and eosin-stained sections from each cohort to both healthy and DCD tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). After 4 hours of NMP both the MTx treated and non-treated samples exhibited similar cumulative injury scores, both of which were lower than in DCD injury, but moderately higher than healthy control levels, indicating a treatment independent improvement in gross tissue morphology after NMP (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Similar data were observed with regards to CK19\u0026thinsp;+\u0026thinsp;cholangiocyte coverage, with no significant differences seen between groups at this timepoint, although the non-treated NMP group started to reveal a trend for reduced cholangiocyte coverage (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec-d). Interestingly, assessment of Kupffer cell coverage showed a trend for decreased coverage in DCD compared to healthy control tissue. There was a significant recovery in coverage in treated livers on NMP, while non-treated tissue maintained the same reduced Kupffer cell coverage (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee-g). To assess the number of apoptotic cells in the tissue, a fluorescent TUNEL staining was used, revealing an increase in TUNEL\u0026thinsp;+\u0026thinsp;cells in DCD tissue, which declined in both treated and non-treated cohorts after NMP, again highlighting more treatment-independent outcomes due to NMP (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh-i). This was further validated through comparison of the bile protein landscape, which yielded only one significantly elevated protein in the treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ej). To assess the degree of tissue oedema, a wet-dry (W/D) ratio was carried out, yielding a non-significant increase in fluid in the non-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ek). Cytokine analysis focused on interleukin-6 (IL-6) and interleukin-10 (IL-10) revealed non-significant increases in both cytokines in the untreated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003el-m), pointing towards an immunomodulatory effect occurring in these livers. Taken together these data point to the origins of an immune-inflammatory cascade in both groups of organs, however, the non-treated group appears to be more greatly impacted, with a decline in Kupffer cell coverage, and greater apparent effort to immunomodulate the tissue environment.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eCross circulation reveals a profound protective effect for mitochondrial transplantation\u003c/h3\u003e\n\u003cp\u003eTo evaluate the transplant potential of the DCD livers treated with MTx or placebo after four hours of NMP, we undertook a six-hour cross circulation (CC) using recipient pigs. As with NMP, the total bile production remained significantly higher in the MTx-treated livers (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), while bile AST levels remained significantly lower (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb), indicating the efficacy of the therapy at extended time points and a regeneration of treated livers. With regards to bile composition, further reductions in both bilirubin (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec) and lactate levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef) occurred in the treated livers on CC, also pointing to a restoration of normal liver function. The bile glucose levels manifested a significant reduction in the treated group on CC (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed) whilst the bile pH levels were normal in both treated and non-treated grafts at the end of CC (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). Proteomic analysis of bile composition revealed that one protein, (UROC1) was significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) upregulated in the treatment group while 28 proteins were downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eg). Gene set enrichment analysis from bile at end of cross circulation showed that the inflammatory response, humoral immune response, cell migration, response to wounding and regulation of coagulation were all upregulated in the treatment group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eh). Downregulated biological processes in the treatment group included metabolic processes, catabolic processes and translation. Perfusate analysis showed no significant differences in either glucose (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ei) or lactate (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ej), while AST levels yielded a comparable non-significant increase in the non-treated group as seen in the bile (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ek) Treated grafts also exhibited a significant improvement in hepatic artery perfusion pressures (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003em), while no perfusion differences were found in the portal vein (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003el). These data point to an overall regeneration of liver function in the treated grafts as well as a substantial decline in those which did not receive mitochondrial treatment.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThese findings were recapitulated in the tissue sections, where the non-treated CC samples displayed apparent oedema and eosinophilia (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003en). Blinded scoring revealed a significantly higher cumulative injury score in samples from non-treated livers, while those treated with mitochondria only exhibited a moderately non-significant elevation in injury score (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003en). In line with the pathology observations, non-treated tissue yielded a 40-fold increase in apoptotic cells compared to healthy control tissue, while there were only 5 times more TUNEL\u0026thinsp;+\u0026thinsp;cells in those livers which received MTx (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eo). The slight decline in Kupffer cells noted in the non-treated samples after 4 hours of NMP translated into a near complete abolishment of these cells after 6 hours on cross circulation, while in mitochondria treated livers, Kupffer cell coverage resembled that from healthy control tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ep). The same findings were noted with regards to CK19\u0026thinsp;+\u0026thinsp;cholangiocyte coverage, indicating considerable damage to the biliary tree in the non-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eq). In keeping with the widespread tissue damage and cell loss, the non-treated samples further experienced a significant increase in oedema as inferred by the significantly higher W/D ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003er). With regards to cytokine analyses, after six hours of cross circulation there were no differences between the levels of IL-6 and IL-10 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003es-t). Taken together, these data reaffirm the impairments in gross liver function seen in the non-treated group and further strengthen the efficacy of the MTx in mitigating liver degradation in DCD with a WIT of two hours.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eProteomic cluster analysis and spatial proteomic profiling of MTx\u003c/h2\u003e\u003cp\u003eTo analyze the proteomic landscape across all cohorts and treatment groups, an untargeted data-independent acquisition mass spectrometry method was performed. Across groups, the analysis identified 7276 unique proteins. After filtering for the proteins present in at least 75% of the samples per group, 1684 proteins remained for further downstream analyses. A heatmap analysis was performed to visualize the clustering of the bile samples according to significantly differentially expressed proteins. Unsupervised hierarchical clustering showed a clear distinction between the baseline and DCD groups, with downregulated proteins in the baseline group and upregulated proteins in the DCD group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). The mitochondria treated samples obtained at the end of cross circulation clustered closely to the baseline samples, while the treated samples at the end of 4h NMP clustered modestly to the baseline samples.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo expand on the observed clustering, we sought to explore the differential expression of cell populations across cohorts, using a high-content imaging approach. This permitted the acquisition of 13 distinct markers (DAPI, CD3, CD4, PCNA, Ki67, vimentin, PCK, actin, CD163, LEA, HH3K4, IBA1, collagen-1) on the same sections at sub-cellular resolution (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb), totaling approximately 1.15\u0026nbsp;million cells. Several populations, including CD3+, Ki67+, vimentin+, and CD163\u0026thinsp;+\u0026thinsp;cells, exhibited similar densities in the baseline and treated samples, both on NMP and CC, while non-treated samples showed an under- or over-expression of these cell populations (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec), corroborating the observations from the proteomics data. Both treated cohorts displayed significant elevations in the expression of HH3k4, an epigenetic marker associated with DNA repair and replication. More importantly, across all cell populations analyzed, it was clear that the non-treated CC samples suffered a massive loss of all cell types, highlighting the deterioration of these DCD livers in the absence of MTx. Moreover, those livers treated with MTx revealed an expression pattern similar to that observed in the baseline tissue, in keeping with the proteomics data, showing an improvement of the DCD livers over time after receiving the MTx.\u003c/p\u003e\u003cp\u003eTo explore spatial differences in a more hepatic-focused manner, an advanced segmentation map was generated to define 4 regions across a single functional hepatic unit (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea-b), starting from the collagen connective tissue then spanning liver zones 1\u0026ndash;3, from the collagen border through the central vein, focusing on PCNA-, CD163-, Ki67-, CD3-, HH3-, LEA- and IBA-1-positive cell populations (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec-i). In keeping with the global data, it was evident that the zonal pattern of expression of the majority of markers was most comparable between the healthy control and treated cross-circulation datasets, indicating a profound cellular and morphological regeneration from the DCD state in mitochondrial treated livers. Taken together, these data indicate that four hours of NMP are not sufficient to fully inform on the state of the liver and that the most valuable insights come from longer cross-circulation time points, with improvement towards a healthy baseline organ, or complete deterioratation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis study evaluates the efficacy of MTx in restoring the function of DCD livers subjected to an extended WIT of two hours. Livers were first placed on NMP, whereat we infused freshly isolated mitochondria to demonstrate that the addition of exogenous mitochondria to the organ mitigates IRI. We showed that this therapy improved liver function on NMP as validated by increasing bile production, reducing bile AST and lactate levels, and maintaining a normal cytoarchitecture. Next, to assess the liver\u0026rsquo;s viability in a transplant-like setting, we established a CC model with a recipient pig. While untreated livers went on to exhibit massive oedema, abolishment of both Kupffer cells and cholangiocytes, and a significant rise in bile AST levels, the livers treated with MTx maintained a normal liver function characterized by continuous bile production, low bile AST levels, low bile lactate levels, utilization of bile glucose and normal cytoarchitecture. These findings strongly support the efficacy of MTx in restoring DCD livers subjected to prolonged WIT. This approach for maintaining and regenerating DCD livers holds substantial potential to significantly expand the donor pool.\u003c/p\u003e\u003cp\u003eThe MTx was administered in two bolus doses, delivered simultaneously through both the portal vein and hepatic artery at the start of NMP. This dual-route approach was designed to optimize mitochondrial distribution across the entire liver, ensuring comprehensive exposure and uptake within the target tissues. The decision to administer mitochondria at the start of NMP was driven by the objective of achieving a high, targeted dose selectively within the liver parenchyma, at a point when endogenous mitochondria have not fully recovered to support a fully metabolically active organ. Administering mitochondria \u003cem\u003ein vivo\u003c/em\u003e prior to donor death determination would result in uncertain dosing and unpredictable distribution within the body. While one might consider delivering the MTx as a bolus through the portal vein after opening the abdomen in DBD donors and before the cross-clamp, this approach would rely on the hope that the majority of mitochondria would be absorbed in the liver during the initial pass. However, in DCD\u0026mdash;particularly uDCD\u0026mdash;this approach would be impractical.\u003c/p\u003e\u003cp\u003eAlternatively, administering mitochondria during CC was considered; however, potential dilution within the recipient\u0026rsquo;s circulation would create uncertainties regarding the effective dose reaching the liver, providing support. Moreover, from a clinical perspective, our study design aims to evaluate CC as a proxy for transplantation, aligning with realistic clinical protocols. In clinical practice, severely injured livers would not be transplanted into a recipient without prior evidence of functional restoration, to avoid exposing the recipient to undue transplantation risks. By administering MTx during NMP, we can maximize localized delivery and monitor early markers of liver recovery before considering exposure of the recipient, thus aligning with both therapeutic and safety objectives in clinical transplantation. Our CC model differs significantly from previously applied CC liver models\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. In this model, after CC establishment, a clamp is placed over the liver hilum, completely obstructing the portal vein and hepatic artery. This approach was specifically chosen to simulate a clinical transplant scenario by effectively excluding the recipient\u0026rsquo;s native liver from circulation. In this way we have established an innovative way to test the functional capacity of the organ without the need for a transplant model.\u003c/p\u003e\u003cp\u003eA challenge with MTx has been establishing a robust and reliable source of mitochondria to transplant, which were readily available, keeping in mind the unpredictable timing of transplant surgery. This is because not all tissues and individuals provide mitochondria of similar quality or yield, making the source highly important\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. In addition, the mitochondrial isolation protocol is very sensitive with regards to tissue harvest time, as well as the time between isolation and delivery, which should not exceed 30 minutes\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Cryopreservation, which would potentially offer an off-shelf product, has been done but remains controversial due to concerns over organelle survival and function after the freeze-thaw process\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. However, rodents, which are readily accessible across all research centres globally, may yet emerge as an ideal mitochondrial source to explore an advanced mitochondrial transplant therapy. In the present study, young mouse livers served as the source of mitochondria for therapeutic application, leveraging the liver\u0026rsquo;s naturally high mitochondrial concentration to yield a robust supply of high-quality, functionally potent mitochondria. Our previous work has demonstrated that mitochondria sourced from autologous, allogeneic, or xenogeneic origins are viable for therapeutic use, exhibiting compatibility without eliciting significant immune responses in recipients\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. This finding broadens the potential for MTx by allowing flexibility in donor sources, which is especially valuable considering the unpredictable timing of transplantation and the limitations of mitochondrial isolation. The lack of immune rejection across these sources suggests that MTx may circumvent some of the immunogenic challenges that arise with other cellular therapies, such as xenogeneic mesenchymal stem cell (MSC)-based approaches or whole organ transplants\u003csup\u003e\u003cspan additionalcitationids=\"CR44 CR45\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eNumerous studies have identified mitochondrial dysfunction as a central theme in ischemic tissue damage as well as IRI, highlighting mitochondria as a key target element in these pathophysiological cascades\u003csup\u003e\u003cspan additionalcitationids=\"CR48\" citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. This was further reaffirmed in our study by the maintenance of elevated bile lactate levels in non-treated organs, which may relate both to impaired lactate clearance in the liver and maintenance of anaerobic metabolism in the absence of normally functioning mitochondria. Prior strategies to alleviate IRI have been focused either upstream or downstream of the mitochondria, whereas mitochondrial transplantation tackles this problem at the source. As the ROS released upon reperfusion invoke the release of damage-associated patterns (DAMPs) from resident hepatic cells\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e, the integration of cytokine filters into perfusion circuits has been explored. However, while these filters do remove cytokines from the perfusate, they are indiscriminate in removing both pro- and anti-inflammatory cytokines and have not yet been found to significantly alter end-outcomes when applied in NRP\u003csup\u003e\u003cspan additionalcitationids=\"CR52 CR53 CR54\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Downstream DAMP and cytokine inhibition has also been explored using caspase inhibitors and RNA interference, but further research is required to validate their effectiveness\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. A more advanced approach is the use of CRISPR-Cas9 gene editing technology, which has been used to enhance the activity of immunomodulatory T-cells and dendritic cells\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. An alternate approach was to increase the production of the anti-inflammatory cytokine IL-10, however, real-world application requires further investigation of delivery, transfection efficiency, and precisely where and how specific cells are targeted, especially in large mammalian organs\u003csup\u003e\u003cspan additionalcitationids=\"CR60 CR61\" citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. Mesenchymal stem cells (MSC), which also possess strong immunomodulatory properties, have been delivered during machine perfusion to mitigate IRI, with several clinical trials underway exploring their effectiveness as an advanced therapy medicinal product (ATMP)\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e,\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. An evolution from the use of MSCs focuses on the delivery of extracellular vesicles (EVs), which contain paracrine products of MSCs, are easier to produce at scale, and raise fewer of the immune rejection concerns when injecting cells\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan additionalcitationids=\"CR65\" citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn contrast, the upstream approaches focus on lessening mitochondrial damage and dysfunction to avoid the downstream DAMP and cytokine cascades. One such approach is the application of HOPE and controlled rewarming prior to NMP, with the initial use of HOPE thought to dampen the IRI-cascade when NMP is applied\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e,\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. Priming with HOPE acted to reduce succinate metabolism upon reperfusion, which consequently attenuated the release of the damage-associated flavin-mononucleotide (FMN)\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. Moreover, the authors concluded that one hour of HOPE treatment resulted in a mitochondrial reprogramming characterized by a more oxidized state\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. One concern that still needs to be empirically addressed is the damage that mitochondria are known to sustain when exposed to cold temperatures, in balance with the protective effects\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e,\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e. An alternate focus has been on the modulation of the ischemia-induced accumulation of succinate, which manifests the profound ROS production upon reperfusion\u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. Application of the reversible complex II inhibitor, dimethyl malonate, was able to attenuate cardiac tissue damage after ischemia, however, its value in alleviating IRI is yet to be explored\u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThis work focused instead on replacing those mitochondria lost and damaged during both WIT and IRI, as a novel therapy to attempt to rescue DCD livers. This would further increase the viable liver pool for transplant by rescuing livers that would be discarded due to extended WIT. The data generated herein shows that DCD livers treated with MTx restored their function during NMP, and when further evaluated for their transplant potential in a CC model, treated livers were able to sustain a recipient, produced bile and displayed a well-preserved cyto-architecture, demonstrating the hallmarks of an organ potentially fit for transplant. This is further strengthened by the bile proteomic profile exhibited in treated livers, both on NMP and at the end of CC, which recapitulated a bile proteome previously validated in human livers deemed acceptable for transplantation\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Regarding therapeutic translation, recent work carried out in rodents found that treatment with MTx prevented ischemia-reperfusion induced hepatocellular injury\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Interestingly, this outcome was dependent on the survival of the Kupffer cell population, which were found to sequester the transplanted mitochondria\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. In keeping with this, our data revealed that the absence of mitochondrial treatment in DCD results in an eventual complete loss of Kupffer cells, further highlighting them as a target for MTx.\u003c/p\u003e\u003cp\u003eIn conclusion, this study provides compelling evidence that xenogeneic MTx can effectively restore the function of DCD livers subjected to prolonged WIT, addressing a major barrier in liver transplantation. Administering mitochondria during NMP, enabled targeted, high-dose delivery to the liver parenchyma, optimizing cellular uptake and enhancing the therapeutic impact. The treated livers demonstrated improved bile production and composition, reduced tissue oedema, and preserved tissue integrity, with significant reductions in injury markers. Importantly, in a CC model using a recipient pig, treated livers maintained functional and structural integrity, underscoring the transplant and clinical potential of this therapy. By effectively mitigating WIT and IRI, MTx shows promise as an innovative strategy to expand the donor pool by rendering previously unsuitable DCD livers viable for transplantation. These findings support further exploration of mitochondrial transplantation as a powerful tool to increase the availability of viable organs, offering hope for patients in urgent need of liver transplants.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eStudy Design\u003c/h2\u003e\u003cp\u003e21 adult pigs (\u003cem\u003eSus Scrofa Domesticus\u003c/em\u003e), weighing approximately 50kg, were used in the study. Of these, 12 were used as a DCD liver source and subsequently placed on NMP, n\u0026thinsp;=\u0026thinsp;6 (also used for NMP) were used for cross circulation, and n\u0026thinsp;=\u0026thinsp;9 were used as non-treated controls. Pigs were housed in pens with a 12-hour light-dark cycle, \u003cem\u003ead libitum\u003c/em\u003e access to water prior to the experiment. All experimental procedures were performed according to ethical approval by the Ethical Committee on Animal Research (Dnr 5.2.18\u0026ndash;8927/16) and conducted according to the directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes and Regulation (EU) 2019/1010 on the alignment of reporting obligation. Animals were randomised prior to the start of the study to either the treatment or the non-treatment group.\u003c/p\u003e\u003cp\u003eFor the experimental setup, circulatory arrest was achieved by inducing ventricular fibrillation by mechanical interference with the heart's conduction system. The donors were left untouched for two hours post arrest. The livers were then cold flushed \u003cem\u003ein situ\u003c/em\u003e, explanted, and connected to NMP for four hours (n\u0026thinsp;=\u0026thinsp;12), with half of the livers receiving mitochondria (n\u0026thinsp;=\u0026thinsp;6) and the other half receiving a placebo (n\u0026thinsp;=\u0026thinsp;6). Upon completion of NMP, three randomized livers from each group were connected to CC (n\u0026thinsp;=\u0026thinsp;6). The DCD cohort was compared to a cohort of healthy control animals (n\u0026thinsp;=\u0026thinsp;9), anesthetized with immediate biopsy of undisturbed liver tissue.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eAnimal Preparation\u003c/h2\u003e\u003cp\u003eTwenty-one male and female adult farm-raised wild-type American Yorkshire pigs (Sus scrofa domesticus) with a mean weight of 50 kg were used in this study. Seraclone Anti-A (Bio-Rad, Medical Diagnostics GmbH, Dreieich, Germany) was used for determination of blood type that was then used to match the donor and recipient pairs along with weight. Prior to the start of the study, pairs were assigned randomly to either the donor treatment (n\u0026thinsp;=\u0026thinsp;6) donor placebo (n\u0026thinsp;=\u0026thinsp;6) group, recipient cross-circulation (n\u0026thinsp;=\u0026thinsp;6) or control animals (n\u0026thinsp;=\u0026thinsp;9). All were premedicated with xylazine (Rompun\u0026reg; vet. 20 mg/mL; Bayer AG, Leverkusen, Germany; 2 mg/kg) and ketamine (Ketaminol\u0026reg; vet. 100 mg/mL; Farmaceutici Gellini S.p.A., Aprilia, Italy; 20 mg/kg). A peripheral intravenous (IV) line was inserted in the earlobe, and a urinary catheter was inserted in the bladder. General anesthesia was accomplished with ketamine (Ketaminol\u0026reg; vet), midazolam (Midazolam Panpharma\u0026reg;, Oslo, Norway) and fentanyl (Leptanal\u0026reg;, Lilly, France) infusions. Mechanical ventilation was established using a Siemens-Elema ventilator (Servo 900C, Siemens, Solna, Sweden). The animals were intubated with a 7.5 size endotracheal tube. The ventilator was set to volume-controlled ventilation (VCV) with the flow pattern switch in \u0026ldquo;constant flow\u0026rdquo; which lowers the peak pressures according to the manufacturer\u0026rsquo;s instructions. Inspiration time is set to 25% with pause time 10% to give an I:E ratio of 1:2. Ventilation was adjusted to maintain carbon dioxide levels (PaCO\u003csub\u003e2\u003c/sub\u003e) between 33\u0026ndash;41 mmHg. Tidal volume (Vt) was kept at 6\u0026ndash;8 mL/kg. Dynamic compliance was calculated by the equation \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\text{C}}_{\\text{d}\\text{y}\\text{n}}=\\:\\frac{{\\text{V}}_{\\text{T}}}{(\\text{p}\\text{e}\\text{a}\\text{k}\\:\\text{p}\\text{r}\\text{e}\\text{s}\\text{s}\\text{u}\\text{r}\\text{e}-\\text{P}\\text{E}\\text{E}\\text{P})}\\)\u003c/span\u003e\u003c/span\u003e. A pulmonary artery catheter (Swan-Ganz CCOmbo V and Introflex, Edwards Lifesciences Services GmbH, Unterschleissheim, Germany) was inserted in the right internal jugular vein, and an arterial line (Secalon-T\u0026trade;, Merit Medical Ireland Ltd, Galway, Ireland) was placed in the right common carotid artery. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the overview of the experimental setup. Dihydrostreptomycinsulfate (0.1 mL/kg) (Boehringer Ingelheim Animal Health Nordics A/S, Copenhagen, Denmark) was given subcutaneously before initiation of surgery in all animals. Additionally, hemodynamic measurements were recorded at sixty minutes with thermodilution through a Swan-Ganz catheter and an arterial line. The following parameters were recorded: Heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP), central venous pressure (CVP), cardiac output (CO), systolic pulmonary pressure (SPP), diastolic pulmonary pressure (DPP), mean pulmonary pressure (MPP), pulmonary artery wedge pressure (PAWP), systemic vascular resistance (SVR), and pulmonary vascular resistance (PVR).\u003c/p\u003e\u003cp\u003eFor healthy control animals, a midline sternotomy and laparotomy were done, and tissue biopsies were taken randomly according to a previous randomization chart from the different liver lobes.\u003c/p\u003e\u003cp\u003eFor DCD animals, ventricular fibrillation was induced by mechanical interference with the heart's conduction system. 10,000 U of heparin was administered prior to ventricular fibrillation. After circulatory arrest, the donors were left untouched for two hours. After a midline sternotomy and laparotomy, the infra-renal inferior vena cava (IVC) and abdominal aorta were dissected free and prepared for cannulation. Cannulas were inserted in the IVC and abdominal aorta patc The liver was flushed with 2000 mL of cold (4\u0026ndash;6\u0026deg;C) Institute George Lopez 1 preservation solution (Institute George Lopez, Lissieu, France) via the aorta and 2000 mL via the PV. The bile duct, portal vein (PV), and hepatic artery (HA) were dissected free. The liver was then completely mobilized and procured by sectioning the supra- and infra-hepatic IVC, the bile duct, the PV, and the coeliac axis with a patch of aorta. After hepatectomy, the liver was kept on ice slush and prepared for perfusion.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eNormothermic machine perfusion\u003c/h2\u003e\u003cp\u003eThe coeliac axis artery (HA) was cannulated using a Medtronic DLP 8Fr arterial cannula (Medtronic, Minneapolis, MN, USA). The portal vein (PV) was cannulated using a Medtronic DLP 24Fr venous cannula (Medtronic, Minneapolis, MN, USA). After flushing of the liver and de-airing of the cannulas with 500 mL of gelofusine (Braun, Melsungen, Germany). the liver was then connected to the NMP. The inferior vena cava (IVC) was left open for free outflow of perfusate to keep the venous pressure gradient\u0026thinsp;\u0026lt;\u0026thinsp;10 mmHg. The inflow of the PV was maintained to 0.5\u0026ndash;0.7 L/min using a biomedical pump (Bio-Pump\u0026reg; Centrifugal pump, Medtronic, Minneapolis, MN, USA) and inflow to the HA was maintained to 0.15\u0026ndash;0.3 L/min using a roller-pump (Jostra, Maquet HL20, Getinge, Sweden) not exceeding pressure\u0026thinsp;\u0026gt;\u0026thinsp;150mmHg. Inflow and outflow pressures were monitored across machine perfusion. The common bile duct was cannulated with an 8 Fr cannula (Bio-Medicus, Medtronic, MN, USA), and bile was collected. Circuit temperature was maintained at 37\u0026deg;C using a water heater (Gaymar Stryker TP700 T/pump, Stryker Corporation, Kalamazoo, Michigan, USA) and oxygenator water jacket (Medos Hilite 7000). Sweep flow was approximately 1.5 L/min, Fi 60% with 93% N/7% CO\u003csub\u003e2\u003c/sub\u003e mixed gas. The system had been primed with Steen\u0026trade; Solution (XVIVO perfusion, Gothenburg, Sweden), with 10,000 U of heparin, and maintained a hematocrit level of 20% using red blood cells from the donor animal taken before induced ventricular fibrillation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eMitochondrial Isolation and Function\u003c/h2\u003e\u003cp\u003eMitochondria that were used for MTx were isolated from fresh liver tissue of C57BL/6 mice. To further evaluate mitochondrial function under control and IRI conditions, an additional set of mitochondria was isolated from porcine liver tissue for ATP quantification. Briefly, harvested tissue underwent homogenization in a 5 mL respiration buffer solution (250 mmol/ sucrose, 20 mmol/ K+-HEPES (4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid, pH 7.2), followed by a 10-minute Subtilisin, enzymatic digestion process, conducted on ice. Subsequently, the digested tissue underwent a series of filtration steps, and the mitochondria were precipitated via centrifugation at 9,500 rpm for 5 minutes at 4\u0026deg;C, then resuspended for subsequent delivery, as previously detailed\u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e. Multisizer 4e Coulter Counter (Beckman, Miami, FL, USA) was employed to assess the size and quantity of the isolated mitochondria, while viability was determined through the ATPlite assay (Revvity Inc, Sweden) \u003csup\u003e\u003cspan additionalcitationids=\"CR73 CR74\" citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eMitochondrial dose and delivery\u003c/h2\u003e\u003cp\u003eFollowing the start of machine perfusion, livers were randomly assigned to receive either 1x10\u003csup\u003e10\u003c/sup\u003e xenogeneic mitochondria (in 10 mL of isolation buffer), or a placebo (10 mL of isolation buffer) as a control. In previous studies, we have shown that mitochondrial dose concentrations for efficacy vary with the organ being treated. For the heart and kidney, it has been shown that 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e to 2\u0026times;10\u003csup\u003e6\u003c/sup\u003e mitochondria per gram of wet weight is safe and efficacious\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Mitochondria were isolated in suspension, which was divided into equal volumes and delivered as an antegrade bolus divided between the hepatic artery and portal vein through separate ports in each line over 5 seconds. The liver was then perfused for four hours on NMP.\u003c/p\u003e\u003cp\u003e\u003cem\u003eIsolation and cryopreservation of primary human hepatocytes.\u003c/em\u003e\u003c/p\u003e\u003cp\u003ePrimary human hepatocytes were isolated by a three-step perfusion technique as previously described\u003csup\u003e\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e,\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u003c/sup\u003e. Briefly, the liver was perfused with buffer (Hank\u0026rsquo;s balanced salt solution, Sigma-Aldrich, St. Louis, MO) containing 0.5 mM EGTA (Sigma-Aldrich, St. Louis, MO) followed by a washout and perfusion with 250 mg/L collagenase (Collagenase XI, Sigma-Aldrich, St. Louis, MO or CIzyme, Vitacyte, Indianapolis, IN). The degraded tissue was cut to release cells and cells were washed by centrifugation to remove non-parenchymal cells.\u003c/p\u003e\u003cp\u003eCells were cryopreserved at a concentration of 7\u0026nbsp;million cells/mL and with 10% DMSO (Sigma-Aldrich, St. Louis, MO) in IGL-1 preservation solution. The solution with the cells was left on ice for 15 minutes followed by 2 hours at -80\u0026deg;C. They were then transferred and stored in liquid nitrogen.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eCell Culture\u003c/h2\u003e\u003cp\u003eFor culture, primary human hepatocytes were thawed using Hepatocyte Thaw Medium (Gibco, Thermo Fisher Scientific, Waltham MA, USA) and seeded in Williams' E Medium (no phenol red; Gibco) supplemented with Primary Hepatocyte Maintenance Supplements (Gibco). Plates were pre-coated with collagen (50 \u0026micro;g/mL in 0.02 M acetic acid). Cells were seeded at 5 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells/well in 96 well plate and incubated overnight at 37\u0026deg;C in 5% CO₂. The following day, cells were washed with warm DPBS and incubated with 50 \u0026micro;L of media containing freshly isolated mitochondria (1 \u0026times; 10⁵/well) for four hours. Mitochondrial function was assessed using the ATPlite assay (Revvity Inc., Waltham, MA, USA), and luminescence was measured with a GloMax\u0026reg; plate reader (Promega, Madison, WI, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eLive Cell Imaging\u003c/h2\u003e\u003cp\u003eHuman hepatocytes were seeded at a density of 3 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well in collagen-coated 8-well slides (ibidi GmbH, Gr\u0026auml;felfing, Germany) and incubated overnight at 37\u0026deg;C in a 5% CO₂ atmosphere. Host cells were stained with CellMask\u0026trade; (C10045, Invitrogen, Thermo Fisher Scientific) for 30 minutes and nuclei were stained with Hoechst 33342. Isolated mitochondria were labeled with MitoTracker Green FM (Invitrogen). Fluorescence and confocal microscopy were used to visualize mitochondrial uptake and localization.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eCross circulation\u003c/h2\u003e\u003cp\u003eRecipient pigs (n\u0026thinsp;=\u0026thinsp;6) underwent sedation and general anaesthesia in a similar fashion to donor pigs. A right neck cut-down exposed the right internal jugular vein, and a right groin cut-down exposed the femoral artery. A 15,000 U heparin bolus was administered, and cannulation with 15F catheters (Life Support Medtronic) in the right internal jugular vein and a 12F catheter (DLP Medtronic) in the femoral artery was performed using the Seldinger technique. Immediately before the initiation of cross-circulation, recipient pigs were intravenously administered one gram of methylprednisolone (APP Pharmaceuticals) and 500 mg of calcium chloride (Hospira). Donor blood used to prime the circuit during NMP was not removed. The tubing was spliced, deaired, and connected the recipient pig to the NMP circuit, marking the start of cross-circulation (CC). AV-AV cross circulation circuit was secured, and flow was maintained using a roller-pump (Jostra HL-20 pump console; Maquet). Circuit flows were titrated to 0.15-0.3L/min to the HA, 0.65-0.8L/min to the PV, and 1L/min of arterial return to the recipient for veno-arterial circulatory support. Pressure (PA and PV), flow (PA and PV), and temperature data were continuously monitored using System M\u0026reg; by Spectrum Medical, USA. Circuit temperature was maintained at 37\u0026deg;C using a water heater (Gaymar Stryker TP700 T/pump) and oxygenator water jacket (Medos Hilite 7000). Throughout the duration of cross-circulation, the recipient was maintained on a continuous heparin infusion (initial rate of 25 U kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Activated clotting time was measured using a HemoChron whole blood microcoagulation system (Accriva Diagnostics), and the heparin drip was adjusted to maintain a target value of 250\u0026ndash;350 s. Physiological parameters of the recipient, including heart rate, electrocardiogram, blood pressure (cuff and arterial A-line pressure), mean arterial pressure (MAP), oxygen saturation (SpO\u003csub\u003e2\u003c/sub\u003e), end-tidal CO\u003csub\u003e2\u003c/sub\u003e, temperature, and respiratory rate, were continuously monitored and recorded using a multi-parameter MX750 monitor (Philips Healthcare, Lund, Sweden). Upon establishment of CC, a laparotomy was done, the portal vein (PV), and hepatic artery (HA) were dissected free. The PV and the HA of the recipient liver were clamped such that the liver on NMP was the only one filtering the blood of the recipient. Cross circulation was maintained for six hours, after which the liver and recipient perfusions were discontinued.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eBlood and bile collection\u003c/h2\u003e\u003cp\u003eBlood samples and bile samples were collected every hour during the experimental time course. Blood gases and bile samples from the donor liver were drawn and analyzed every hour during the experimental time course, on an ABL 90 FLEX blood gas analyzer (Radiometer Medical ApS, Br\u0026oslash;nsh\u0026oslash;j, Denmark).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eTissue Sampling/Processing\u003c/h2\u003e\u003cp\u003eBiopsies were taken randomly within the same liver lobe for all biological replicates repeatedly during the experimental timeline. Samples were fixed in 4% paraformaldehyde for 48 hours before transfer to 0.01% sodium azide in PBS. For hematoxylin and eosin (H\u0026amp;E) and fluorescent staining samples were embedded in paraffin wax after dehydration in ethanol series. Hematoxylin and eosin staining (Merck Millipore, Germany) was performed on 4 \u0026micro;m sections. Sections (cut using a microtome) were de-paraffinized, starting with xylene (100%), followed by immersion in a graded ethanol series (from 99.99% to 70%) and ending with rinses in distilled water. After H\u0026amp;E staining (Histolab, Askim, Sweden), slides were dehydrated in ethanol, 70%, 96% and 99.99%, and finally with xylene (100%). Sections were mounted in Pertex (Histolab, Gothenburg, Sweden).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eFluorescent Staining\u003c/h2\u003e\u003cp\u003eStaining was carried out directly on glass slides using a PAP pen to create a hydrophobic barrier. After permeabilization and blocking slices were incubated with primary antibody (IBA1, 1:750; CK19, 1:250) overnight at 4 degrees Celsius. Following washes samples were incubated with secondary antibody (goat anti-rabbit 568, 1:1000) for 90 minutes at 4 degrees Celsius. Finally, samples were incubated with DAPI (1:1000) and tomato lectin (1:500) for 30 minutes at room temperature before final washes and mounting with fluoromount-G. All imaging was carried out using a Nikon confocal A1RHD.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eImage Acquisition\u003c/h2\u003e\u003cp\u003eFor H\u0026amp;E staining, imaging was carried out on an Olympus light microscope. Representative images of each sample were captured using a 4x, 10x, and 20x objective. For fluorescent staining, imaging was carried out on a Nikon A1RHD confocal. All images were captured at a resolution of 2765\u003csup\u003e2\u003c/sup\u003e pixels (total field of view (FOV)\u0026thinsp;=\u0026thinsp;2.8mm\u003csup\u003e2\u003c/sup\u003e), at a resolution of 0.61\u0026micro;m/pixel, using a 20x objective, NA-0.75, RI-1.0. Four random FOV\u0026rsquo;s were imaged per sample. Laser and gain settings were kept constant across samples. Nikon files were exported as 8-bit TIFF images, one per channel, for analysis.\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eConfocal Image Analysis\u003c/h2\u003e\u003cp\u003eFor fluorescent staining, images were analyzed using Fiji\u003csup\u003e\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u003c/sup\u003e. Macros were written for all analytics carried out in this manuscript. Briefly, both IBA1 and CK19 coverage were analyzed by thresholding the image and analyzing the signal coverage area as a % of the whole FOV.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eHistopathological Scoring\u003c/h2\u003e\u003cp\u003eH\u0026amp;E images, at 4x, 10x and 20x magnifications, from all timepoints (healthy control, NMP-end and CC-end) were compiled into a blinded slide deck. The compiled images were sent to a pathologist for scoring where the tissue was assessed according to the BANFF criteria\u003csup\u003e\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e, accounting for portal inflammation, bile ductal inflammation/damage and venous endothelial inflammation, with additional scoring for degree of edema and hemorrhage.\u003c/p\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003eCytokine Evaluation\u003c/h2\u003e\u003cp\u003eCytokine evaluation was carried out on tissue homogenate at the end of machine perfusion, and end of cross circulation. A porcine kit for IL-6 and IL-10 was used (EPX090-60829-901, Thermo Fisher Scientific) according to the manufacturer\u0026rsquo;s instructions. The kit was run using a Bioplex-200system (BioRad, Hercules, CA, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\u003ch2\u003eTUNEL Staining, Imaging \u0026amp; Quantification\u003c/h2\u003e\u003cp\u003eTUNEL Assay Kit BrdU Red was used (Abcam, ab66110). 10 \u0026micro;m FFPE liver sections were de-paraffinised and rehydrated according to standard protocols. Rehydrated samples were permeabilised for 10 min at room temperature in 3% triton-x-100 after which staining was carried out according to suppliers instructions. Before mounting samples were counterstained with tomato lectin-488. Imaging was carried out on Nikon ti2 epifluorescence microscope. Samples were imaged blinded and 4 random FOVs were acquired from each biopsy. Analysis was carried out in Fiji and TUNEL positive cells were automatically detected using a pre-developed macro.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\u003ch2\u003eWet-dry Ratio\u003c/h2\u003e\u003cp\u003eHepatic oedema was assessed by measuring the wet weight to dry weight ratio in liver tissue from the distal liver segments from four hours of NMP and after completed CC. Liver tissue pieces were weighed, lyophilised for 24 hours, and then weighed again. The ratio between the wet and dry weight was then calculated.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\u003ch2\u003eMass spectrometry analysis\u003c/h2\u003e\u003cp\u003eProteins from bile and liver tissue were extracted and digested with trypsin (Promega, Madison, WI, USA) using a S-Trap digestion protocol (ProTifi, Fairport, New York, USA). Mass spectra were acquired through the data-independent acquisition (DIA) method on a timsTOF HT instrument coupled to an EVOSEP one and analyzed using DIA-NN v1.8.1\u003csup\u003e80\u003c/sup\u003e. Downstream analyses were performed in RStudio v2024.09.01, using R v4.4.2. Differentially expressed proteins were determined through thresholds q-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (FDR-corrected p-value) and log2-fold change estimated from bootstrapping methods\u003csup\u003e\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec29\" class=\"Section2\"\u003e\u003ch2\u003eUltrahigh-Content Imaging\u003c/h2\u003e\u003cp\u003eImaging was carried out on 10\u0026micro;m FFPE sections using the automated cyclic staining platform, MACSima\u0026trade; (Miltenyi Biotec, Bergisch Gladbach, Germany) with MACSwell 4 frames. Prior to imaging, sections underwent antigen retrieval by boiling in TEC buffer for 30 minutes. Before samples were loaded into the MACSima prestaining with DAPI (1:10) was done for 10 minutes followed by 3 washes in running buffer. Finally, each well was filled with 250\u0026micro;L running buffer before loading into MACSima\u0026trade;. Antibodies used from this readout were a combination of those produced and pre-validated by Miltenyi Biotec for ultrahigh-content imaging, as well as antibodies from external sources. All antibodies were directly conjugated with either FITC, PE, APC, Alexa-fluor-488 or Alexa-fluor-635. Staining conditions which include antibody dilution, incubation time, staining cycle sequence, photobleaching, and exposure time were kept constant across all runs. Image acquisition took place with a 20x long working distance objective (NA-0.45).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eMACSima Image Analysis\u003c/h3\u003e\n\u003cp\u003eImage pre-processing and analysis was carried out using MACS-iQ View software (Miltenyi Biotech), which includes stitching, spatial alignment and registration, and subtraction of residual intensities from autofluorescence. Single cell segmentation was carried out using the \u003cem\u003eAdvanced Morphology for Tissue\u003c/em\u003e algorithm based on DAPI as a reference channel with a \u003cem\u003eDonut\u003c/em\u003e method to segment the cytoplasm. Cell classification was achieved using a histogram-based gating strategy for each marker.\u003c/p\u003e\u003cdiv id=\"Sec31\" class=\"Section2\"\u003e\u003ch2\u003eStatistics\u003c/h2\u003e\u003cp\u003eData collection was conducted with Microsoft Excel. Data were analyzed using GraphPadPrism10. Normality was difficult to assess due to the small sample sizes, however based on histogram assessment all data were considered normal. Data were analyzed using either a one-way ANOVA with multiple comparisons using Tukey\u0026rsquo;s test, when comparing more than two groups, or a t-test when comparing two groups. Significance was defined as p\u0026thinsp;\u0026lt;\u0026thinsp;0.05(*), p\u0026thinsp;\u0026lt;\u0026thinsp;0.01(**), p\u0026thinsp;\u0026lt;\u0026thinsp;0.001(***), p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001(****).\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e Ethics: All experimental procedures were performed according to ethical approval by the ethical Committee on Animal Research (Dnr 5.2.18\u0026ndash;8927/16) and conducted according to the CODEX guidelines by the Swedish Research Council, Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes and Regulation (EU) 2019/1010 on the alignment of reporting obligations and complies with ARRIVE guidelines.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFUNDING\u003c/h2\u003e\u003cp\u003eGrants awarded by the Marianne and Marcus Wallenberg Foundation, the Knut and Alice Wallenberg Foundation, and the ALF Foundation were used for this research.\u003c/p\u003e\u003cp\u003eACKNOWLEDGEMENTS:\u003c/p\u003e\u003cp\u003eWe would like to acknowledge our friend Dr. Leif Pierre, a longstanding perfusionist and researcher without whom this work would not have been possible. Lund University Bioimaging Centre (LBIC) is acknowledged for providing access to the confocal and SEM platforms. Support from the Swedish National Infrastructure for Biological Mass Spectrometry is gratefully acknowledged.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eStarzl, T. E. \u003cem\u003eet al.\u003c/em\u003e Evolution of liver transplantation. \u003cem\u003eHepatology\u003c/em\u003e 2, 614S-636S (1982).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTrotter, J. F. Liver transplantation around the world. \u003cem\u003eCurr Opin Organ Transplant\u003c/em\u003e 22, 123\u0026ndash;127 (2017).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKootstra, G. Categories of non-heart-beating donors. \u003cem\u003eTransplant Proc\u003c/em\u003e 27, 2893\u0026ndash;2894 (1995).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSiddiqui, F., Al-Adwan, Y., Subramanian, J. \u0026amp; Henry, M. L. 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MS-DAP platform for downstream data analysis of label-free proteomics uncovers optimal workflows in benchmark data sets and increased sensitivity in analysis of Alzheimer\u0026rsquo;s biomarker data. \u003cem\u003eJ Proteome Res\u003c/em\u003e 22, 374\u0026ndash;386 (2022).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7871574/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7871574/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDonation after circulatory death (DCD) has significantly increased the number of organs potentially available for transplantation. Livers are more vulnerable than most other solid organs to warm ischemia in the DCD process. The injury induced by warm ischemic time (WIT) is further exacerbated during cold ischemia and reperfusion, resulting in tissue damage caused by the production of reactive oxygen species. These are not only cytotoxic but also perpetuate mitochondrial dysfunction and cell death. To improve the utilization of livers from uncontrolled DCD (uDCD) or controlled DCD (cDCD) donors with prolonged WIT, new strategies to mitigate WIT must be developed.\u003c/p\u003e\u003cp\u003eEx vivo normothermic machine perfusion (NMP) has increased DCD organ utilization and improved the assessment of the viability of organs before transplantation. NMP could also serve as a platform for isolated treatment of organs prior to transplant. An innovative approach to mitigate organ injury is to treat damaged livers with mitochondrial transplantation (MTx). In this study, we tested the efficacy of xenogeneic mitochondrial administration to restore the function of porcine DCD livers with two hours of warm ischemia. DCD livers were explanted and connected to NMP, where mitochondrial transplantation was administered as a bolus dose in the portal vein and hepatic artery at the start of perfusion. After four hours of NMP, treated livers demonstrated a significant increase in bile production, improved bile quality, and restored cytoarchitecture.\u003c/p\u003e\u003cp\u003eTo further assess the transplantation suitability of these livers post-NMP, they were connected to cross-circulation (CC) with a recipient pig. After six hours of CC, untreated livers became oedematous, exhibited significant aspartate aminotransferase elevations in the bile, and showed cellular degeneration, including near-complete loss of Kupffer cells. In contrast, livers treated with MTx maintained function across CC and were comparable to healthy controls. These data validate the efficacy of MTx in restoring function in DCD livers exposed to prolonged ischaemic times, presenting a promising approach to expand the donor pool.\u003c/p\u003e","manuscriptTitle":"Mitochondrial Therapy During Machine Perfusion Restores the Function of DCD Livers: A Cross-Circulation Evaluation for Transplant Suitability","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-14 17:29:30","doi":"10.21203/rs.3.rs-7871574/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"27670c8c-a756-4208-8324-43aa6547d1a9","owner":[],"postedDate":"November 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":57492449,"name":"Health sciences/Medical research/Translational research"},{"id":57492450,"name":"Biological sciences/Biological techniques/Biological models/Animal disease models"}],"tags":[],"updatedAt":"2025-11-14T17:29:30+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-14 17:29:30","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7871574","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7871574","identity":"rs-7871574","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}