Subnormothermic Machine Perfusion Research Articles

Enhanced VCA Storage: A Pilot Study Demonstrating Supercooling in Orthotopic Rodent Hindlimb Transplantation

The field of vascularized composite allograft (VCA) transplantation has seen steady, rapid growth, with new innovations driving the evolution from experimental procedures to more standardized therapies. With this expansion comes challenges with graft allocation, preservation, and postoperative graft rejection. Here, we outline the first example of subzero nonfreezing (SZNF), supercooled storage of a whole rat hindlimb with orthotopic transplantation. Rat hindlimbs were procured, loaded, and supercooled for 48 hours at -4°C (n = 4), after which, they were recovered. The loading and recovery phase were performed using subnormothermic machine perfusion (SNMP) during which viability markers (glucose and oxygen consumption, lactate, and resistance) were tracked. Control limbs underwent static cold storage (SCS). After ex vivo validation, the model was piloted in a transplant model, comparing 48 hours of SZNF (n = 1), 48 hours of SCS (n = 1), and 72 hours of SCS (n = 1), which demonstrated no survival beyond postoperative day 4 in the SCS models, and survival until the end of study (postoperative day [POD] 28) in the SZNF model. This study demonstrates the promise of this model in future studies on long-term VCA preservation.

366.3: Integrated organ viability assessment method for hypothermic and subnormothermic machine perfusion for liver transplantation.

366.3: Integrated organ viability assessment method for hypothermic and subnormothermic machine perfusion for liver transplantation.

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

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

Subnormothermic Oxygenated Machine Perfusion (24 h) in DCD Kidney Transplantation.

Ex vivo kidney perfusion is an evolving platform that demonstrates promise in preserving and rehabilitating the kidney grafts. Despite this, there is little consensus on the optimal perfusion conditions. Hypothermic perfusion offers limited functional assessment, whereas normothermic perfusion requires a more complex mechanical system and perfusate. Subnormothermic machine perfusion (SNMP) has the potential to combine the advantages of both approaches but has undergone limited investigation. Therefore, the present study sought to determine the suitability of SNMP for extended kidney preservation. SNMP at 22-25 °C was performed on a portable device for 24 h with porcine kidneys. Graft assessment included measurement of mechanical parameters and biochemical analysis of the perfusate using point-of-care tests. To investigate the viability of kidneys preserved by SNMP, porcine kidney autotransplants were performed in a donation after circulatory death (DCD) model. SNMP was also compared with static cold storage (SCS). Finally, follow-up experiments were conducted in a subset of human kidneys to test the translational significance of findings in porcine kidneys. In the perfusion-only cohort, porcine kidneys all displayed successful perfusion for 24 h by SNMP, evidenced by stable mechanical parameters and biological markers of graft function. Furthermore, in the transplant cohort, DCD grafts with 30 min of warm ischemic injury demonstrated superior posttransplant graft function when preserved by SNMP in comparison with SCS. Finally, human kidneys that underwent 24-h perfusion exhibited stable functional and biological parameters consistent with observations in porcine organs. These observations demonstrate the suitability and cross-species generalizability of subnormothermic machine perfusion to maintain stable kidney perfusion and provide foundational evidence for improved posttransplant graft function of DCD kidneys after SNMP compared with SCS.

Machine Perfusion Enables 24-h Preservation of Vascularized Composite Allografts in a Swine Model of Allotransplantation.

The current gold standard for preserving vascularized composite allografts (VCA) is 4°C static cold storage (SCS), albeit muscle vulnerability to ischemia can be described as early as after 2h of SCS. Alternatively, machine perfusion (MP) is growing in the world of organ preservation. Herein, we investigated the outcomes of oxygenated acellular subnormothermic machine perfusion (SNMP) for 24-h VCA preservation before allotransplantation in a swine model. Six partial hindlimbs were procured on adult pigs and preserved ex vivo for 24h with either SNMP (n = 3) or SCS (n = 3) before heterotopic allotransplantation. Recipient animals received immunosuppression and were followed up for 14days. Clinical monitoring was carried out twice daily, and graft biopsies and blood samples were regularly collected. Two blinded pathologists assessed skin and muscle samples. Overall survival was higher in the SNMP group. Early euthanasia of 2 animals in the SCS group was linked to significant graft degeneration. Analyses of the grafts showed massive muscle degeneration in the SCS group and a normal aspect in the SNMP group 2weeks after allotransplantation. Therefore, this 24-h SNMP protocol using a modified Steen solution generated better clinical and histological outcomes in allotransplantation when compared to time-matched SCS.

Effect of Subnormothermic Machine Perfusion on the Preservation of Vascularized Composite Allografts After Prolonged Warm Ischemia.

Warm ischemia time (WIT) and ischemia-reperfusion injury are limiting factors for vascularized composite allograft (VCA) transplantation. Subnormothermic machine perfusion (SNMP) has demonstrated the potential to extend WIT in organ transplantation. This study evaluates the effect of SNMP on VCA viability after prolonged WIT. Rat hindlimbs underwent WIT for 30, 45, 60, 120, 150, or 210 min, followed by 3-h SNMP. Monitoring of perfusion parameters and outflow determined the maximum WIT compatible with limb viability after SNMP. Thereafter, 2 groups were assessed: a control group with inbred transplantation (Txp) after 120 min of WIT and an experimental group that underwent WIT + SNMP + Txp. Graft appearance, blood gas, cytokine levels, and histology were assessed for 21 d. Based on potassium levels, the limit of WIT compatible with limb viability after SNMP is 120 min. Before this limit, SNMP reduces potassium and lactate levels of WIT grafts to the same level as fresh grafts. In vivo, the control group presented 80% graft necrosis, whereas the experimental group showed no necrosis, had better healing (P = 0.0004), and reduced histological muscle injury (P = 0.012). Results of blood analysis revealed lower lactate, potassium levels, and calcium levels (P = 0.048) in the experimental group. Both groups presented an increase in interleukin (IL)-10 and IL-1b/IL-1F2 with a return to baseline after 7 to 14 d. Our study establishes the limit of WIT compatible with VCA viability and demonstrates the effectiveness of SNMP in restoring a graft after WIT ex vivo and in vivo, locally and systemically.

The role of oxygenation in kidney and liver machine perfusion

Background. Organ transplantation is the optimal decision for patients in the end stage of many diseases. Certain conditions are required for the transportation and preservation of a donor organ after explantation, including factors such as temperature, pressure, and preservative solution. All currently available methods of preservation of donor organs are aimed at maximizing the complete preservation of the functional state of the graft from the moment of its removal to implantation and reperfusion in the recipient's body.Aim. The purpose of this review is to provide up-to-date information on the results of the studies performed in order to decide on the preferred method of organ preservation.Material and methods. An analysis of literature sources in English and Russian from 2009 to 2023 on this topic was performed in the databases PubMed, MEDLINE, Google Scholar. The review highlights the results of preclinical (on animal models) and clinical studies, as well as achievements in the field of ex-vivo machine perfusion with an emphasis on machine hypothermic perfusion and modified oxygenated hypothermic machine perfusion, subnormothermic machine perfusion and machine normothermic perfusion.Results. The daily increase in the number of patients in need of organ transplantation delays the timely selection and search for a donor. Organ donation after cardiac death is a promising step in an attempt to overcome the disbalance between the number of patients and organs, but the risk of developing early graft damage increases. The criteria for selecting donors and donor organs are being expanded, as a result, elderly donors and not-optimal grafts are included, but they are less resistant to ischemic damage. In this connection, there is a need for long-term infusion support through machine perfusion.Conclusion. In recent years, research has focused on alternative preservation methods, studying hypothermic, subnormothermic and normothermic machine perfusion. The use of machine perfusion has become the most widespread among kidney transplants and has shown good results. Further development is expected in the field of studying and improving this method of organ preservation, which allows not only transporting, but also improving the functional state of the graft.

Towards Optimizing Sub-Normothermic Machine Perfusion in Fasciocutaneous Flaps: A Large Animal Study.

Machine perfusion has developed rapidly since its first use in solid organ transplantation. Likewise, reconstructive surgery has kept pace, and ex vivo perfusion appears as a new trend in vascularized composite allotransplants preservation. In autologous reconstruction, fasciocutaneous flaps are now the gold standard due to their low morbidity (muscle sparing) and favorable functional and cosmetic results. However, failures still occasionally arise due to difficulties encountered with the vessels during free flap transfer. The development of machine perfusion procedures would make it possible to temporarily substitute or even avoid microsurgical anastomoses in certain complex cases. We performed oxygenated acellular sub-normothermic perfusions of fasciocutaneous flaps for 24 and 48 h in a porcine model and compared continuous and intermittent perfusion regimens. The monitored metrics included vascular resistance, edema, arteriovenous oxygen gas differentials, and metabolic parameters. A final histological assessment was performed. Porcine flaps which underwent successful oxygenated perfusion showed minimal or no signs of cell necrosis at the end of the perfusion. Intermittent perfusion allowed overall better results to be obtained at 24 h and extended perfusion duration. This work provides a strong foundation for further research and could lead to new and reliable reconstructive techniques.

Comprehensive review of the application of MP and the potential for graft modification.

Following procurement, the liver graft is exposed to an ischemic period that triggers several pathophysiologic changes in response to oxygen deprivation. Therefore, the goal during organ preservation is to attenuate such response and provide an adequate environment that prepares the graft for its metabolic reactivation following implantation. This has been widely achieved via static cold storage preservation, where the maintenance of the graft using cold preservation solutions reduce its metabolic activity and confer cytoprotection until transplantation. However, despite being the gold standard for organ preservation, static cold storage holds several disadvantages. In addition, the ongoing organ shortage has led to the use of unconventional grafts that could benefit from therapies pre-transplant. Organ preservation via machine perfusion systems appears as a promising solution to address both. Here, we aim to present a state-of-the-art narrative review regarding liver graft modification options using machine perfusion systems in combination with adjuvant strategies including immunomodulation, gene therapy and pharmacotherapy. Available reports are scarce and mostly on experimental animal models. Most of the literature reflects the use of normothermic or subnormothermic machine perfusion devices given that these particular type of machine allows for a metabolically active organ, and therefore facilitates its modification. Although limited, promising findings in available reports suggest that organ preservation using machine perfusion system when combined with alternative therapies can be feasible and safe strategies for graft modification. Further research on clinical settings are needed to better elucidate the true effect of graft modification pre-transplant on short- and long-term graft and patient survival. There is a long way ahead to develop guidelines and approve these novel therapies for clinical practice. However, the path looks promising.

(866) Subnormothermic Machine Perfusion of Neonatal and Small Pediatric Sized Hearts

(866) Subnormothermic Machine Perfusion of Neonatal and Small Pediatric Sized Hearts

Development of a rat forelimb vascularized composite allograft (VCA) perfusion protocol.

Vascularized composite allografts (VCAs) refer to en bloc heterogenous tissue that is transplanted to restore form and function after amputation or tissue loss. Rat limb VCA has emerged as a robust translational model to study the pathophysiology of these transplants. However, these models have predominately focused on hindlimb VCAs which does not translate anatomically to upper extremity transplantation, whereas the majority of clinical VCAs are upper extremity and hand transplants. This work details our optimization of rat forelimb VCA procurement and sub-normothermic machine perfusion (SNMP) protocols, with results in comparison to hindlimb perfusion with the same perfusion modality. Results indicate that compared to hindlimbs, rat forelimbs on machine perfusion mandate lower flow rates and higher acceptable maximum pressures. Additionally, low-flow forelimbs have less cellular damage than high-flow forelimbs based on oxygen uptake, edema, potassium levels, and histology through 2 hours of machine perfusion. These results are expected to inform future upper extremity VCA preservation studies.

The role of antifreeze glycoprotein (AFGP) and polyvinyl alcohol/polyglycerol (X/Z-1000) as ice modulators during partial freezing of rat livers.

The current liver organ shortage has pushed the field of transplantation to develop new methods to prolong the preservation time of livers from the current clinical standard of static cold storage. Our approach, termed partial freezing, aims to induce a thermodynamically stable frozen state at high subzero storage temperatures (-10°C to -15°C), while simultaneously maintaining a sufficient unfrozen fraction to limit ice-mediated injury. Using glycerol as the main permeating cryoprotectant agent, this research first demonstrated that partially frozen rat livers showed similar outcomes after thawing from either -10°C or -15°C with respect to subnormothermic machine perfusion metrics. Next, we assessed the effect of adding ice modulators, including antifreeze glycoprotein (AFGP) or a polyvinyl alcohol/polyglycerol combination (X/Z-1000), on the viability and structural integrity of partially frozen rat livers compared to glycerol-only control livers. Results showed that AFGP livers had high levels of ATP and the least edema but suffered from significant endothelial cell damage. X/Z-1000 livers had the highest levels of ATP and energy charge (EC) but also demonstrated endothelial damage and post-thaw edema. Glycerol-only control livers exhibited the least DNA damage on Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining but also had the lowest levels of ATP and EC. Further research is necessary to optimize the ideal ice modulator cocktail for our partial-freezing protocol. Modifications to cryoprotective agent (CPA) combinations, including testing additional ice modulators, can help improve the viability of these partially frozen organs.

Continuous versus Pulsatile Flow in 24-Hour Vascularized Composite Allograft Machine Perfusion in Swine: A Pilot Study

IntroductionMultiple perfusion systems have been investigated on vascularized composite allografts, with various temperatures and different preservation solutions, most using continuous flow (CF). However, physiological flow is pulsatile and provides better outcomes in kidney and lung ex vivo perfusions. The objective of this pilot study is to compare pulsatile flow (PF) with CF in our 24-h subnormothermic machine perfusion protocol for swine hindlimbs. MethodsPartial hindlimbs were harvested from Yorkshire pigs and perfused with a modified Steen solution at 21°C for 24 h either with CF (n = 3) or with pulsatile flow (PF) at 60 beats/min (n = 3). Perfusion parameters, endothelial markers, and muscle biopsies were assessed at different timepoints. ResultsOverall, lactate levels were significantly lower in the PF group (P = 0.001). Glucose uptake and potassium concentration were similar in both groups throughout perfusion. Total nitric oxide levels were significantly higher in the PF group throughout perfusion (P = 0.032). Nitric oxide/endothelin-1 ratio also tends to be higher in the PF group, reflecting a potentially better vasoconductivity with PF, although not reaching statistical significance (P = 0.095). Arterial resistances were higher in the PF group (P < 0.001). Histological assessment did not show significant difference in muscular injury between the two groups. Weight increased quicker in the CF group but reached similar values with the PF after 24 h. ConclusionsThis pilot study suggests that PF may provide superior preservation of vascularized composite allografts when perfused for 24 h at subnormothermic temperatures, with potential improvement in endothelial function and decreased ischemic injury.

Exceeding the Limits of Static Cold Storage in Limb Transplantation Using Subnormothermic Machine Perfusion.

For 50 years, static cold storage (SCS) has been the gold standard for solid organ preservation in transplantation. Although logistically convenient, this preservation method presents important constraints in terms of duration and cold ischemia-induced lesions. We aimed to develop a machine perfusion (MP) protocol for recovery of vascularized composite allografts (VCA) after static cold preservation and determine its effects in a rat limb transplantation model. Partial hindlimbs were procured from Lewis rats and subjected to SCS in Histidine-Tryptophan-Ketoglutarate solution for 0, 12, 18, 24, and 48 hours. They were then either transplanted (Txp), subjected to subnormothermic machine perfusion (SNMP) for 3 hours with a modified Steen solution, or to SNMP + Txp. Perfusion parameters were assessed for blood gas and electrolytes measurement, and flow rate and arterial pressures were monitored continuously. Histology was assessed at the end of perfusion. For select SCS durations, graft survival and clinical outcomes after transplantation were compared between groups at 21 days. Transplantation of limbs preserved for 0, 12, 18, and 24-hour SCS resulted in similar survival rates at postoperative day 21. Grafts cold-stored for 48 hours presented delayed graft failure (p = 0.0032). SNMP of limbs after 12-hour SCS recovered the vascular resistance, potassium, and lactate levels to values similar to limbs that were not subjected to SCS. However, 18-hour SCS grafts developed significant edema during SNMP recovery. Transplantation of grafts that had undergone a mixed preservation method (12-hour SCS + SNMP + Txp) resulted in better clinical outcomes based on skin clinical scores at day 21 post-transplantation when compared to the SCS + Txp group (p = 0.01613). To date, VCA MP is still limited to animal models and no protocols are yet developed for graft recovery. Our study suggests that ex vivo SNMP could help increase the preservation duration and limit cold ischemia-induced injury in VCA transplantation.

Analysis of DCD porcine kidney graft viability during sub-normothermic perfusion using magnetic resonance imaging and spectroscopy

Abstract Objective Better preservation strategies for the storage of donation after circulatory death grafts could increase the number of kidneys available and improve patient survival. Warm (22°C and 37°C) ex-vivo perfusion has emerged as an alternative to hypothermia and a feasible strategy to recover/repair organs, but the underlying mechanism remains elusive. Here, using magnetic resonance imaging spectroscopy (MRIs), we evaluated kidney viability, and adenosine triphosphate (ATP) production during sub-normothermic ex-vivo kidney perfusion (22°C + O2) versus hypothermic machine perfusion (4°C and 4°C + O2) in a porcine kidney autotransplantation model. Methods To mimic donation after circulatory death (DCD), kidneys from 8-month-old pigs underwent 60 minutes of warm ischemia, prior to procurement. Kidneys were then perfused ex-vivo at 4°C with (4°C + O2), and without oxygen (4°C) or at 22°C (22°C + O2) before autotransplantation. During the ex-vivo perfusion, and after transplantation we assessed energy metabolites using MRIs. In addition, we performed Gadolinum (Gd) perfusion sequences. Each sample underwent histopathological analyzing and scoring. mRNA expression was analyzed on renal biopsies at various time points. Results Using MRI, we found that in pig kidney, total ATP content was 4 times higher during ex-vivo perfusion at sub-normothermic temperature compared to cold perfusion, with or without oxygen. At 22°C, ATP levels gradually increased up to 10 hrs of perfusion, then progressively declined. Similarly, AMP content was increased in SNOP perfused organs, then slowly consumed. over time. In addition, 22°C + O2 improved cortical and medullary perfusion (Gd elimination). Finally, sub-normothermic ex-vivo perfused graft had lower grade of histological damages 1 hour after transplantation compared to cold perfused organs (injury score 22°C + O2 : 8.8–12.2, 4°C : 13.5–18.8, 4°C + O2 : 17.5–18.5). Conclusion In kidneys, sub-normothermic perfusion improved graft viability when compared with hypothermic perfusions. These results suggest that sub-normothermic ex-vivo kidney perfusion might dampen the negative effect of warm ischemia and promote kidney metabolism such as ATP production. Future clinical studies will define the benefits of sub-normothermic ex-vivo kidney perfusion in improving kidney graft function, and patient's survival.

Experimental long-term sub-normothermic machine perfusion for non-allocable human liver grafts: first data towards feasibility

Summary Background Patients with end-stage liver disease can only be cured by liver transplantation. Due to the gap between demand and supply, surgeons are forced to use expanded criteria donor (ECD) organs, which are more susceptible to ischemia–reperfusion injury (IRI). Therefore, enhanced storing techniques are required. Machine perfusion (MP) has moved into the spotlight of research because of its feasibility for investigating liver function prior to implantation. However, as the perfect MP protocol has not yet been found, we aimed to investigate the potential of sub-normothermic (SN)MP in this field. Methods Non-allocable human livers were subjected to 24 h of SNMP at 21 °C after delivery to the study team. Perfusion was performed with Custodiol® (Dr. Franz Köhler Chemie, Bensheim, Germany) or Belzer MPS® (Bridge to Life Europe, London, UK) and perfusate liver parameters were determined. For determination of biliary conditions, pH, glucose, and HCO3- levels were measured. Results Liver parameters were slightly increased irrespective of perfusate or reason for liver rejection during 24 h of perfusion. Six livers failed to produce bile completely, whereas the remaining 10 livers produced between 2.4 ml and 179 ml of bile. Biliary carbonate was increased in all but one liver. The bile-glucose-to-perfusate-glucose ratio was near 1 for most of the organs and bile pH was above 7 in all but one case. Conclusion This study provides promising data on the feasibility of long-term SNMP as a tool to gain time during MP to optimize ECD organs to decrease the gap between organ demand and supply. Long-term (24 h) sub-normothermic liver machine perfusion seems to be possible, although some adjustments to the protocol might be necessary to improve the general outcome. This has so far been shown for normothermic machine perfusion, bearing some drawbacks compared to the sub-normothermic variant.

Optimal Conditions for Oxygenated Subnormothermic Machine Perfusion for Liver Grafts Using a Novel Perfusion Device

Liver transplantation from donors after cardiac death (DCD) resolves donor shortages. We investigated the optimal time for subnormothermic oxygenated perfusion in DCD liver transplantation. Ten F1 pigs (body weight: 27-32 kg) were allocated to 2 groups: the heart beating group (n=6), from which livers were retrieved while the heart was beating, and the donation after cardiac death (DCD) group (n=4), in which liver retrieval was performed on pigs under apnea-induced cardiac arrest for 20 minutes. In both groups, the livers were kept in cold storage for 2 hours after retrieval and perfused with a subnormothermic oxygenated Krebs-Henseleit buffer for 120 minutes. We used a novel perfusion device, which can set maximum perfusion pressures of arteries and portal vein, developed by Asahikawa Medical University and Chuo Seiko Co. Bile production, liver enzymes, and inflammatory cytokines were measured and the sinusoidal space, using tissue specimens taken from liver grafts, was measured at 30, 60, 90, and 120 minutes after the start of perfusion. Bile production peaked at 90 minutes. Significantly higher levels of liver enzymes and inflammatory cytokines were found in the DCD group (P < .05). The release of liver enzymes peaked at 60 minutes and that of inflammatory cytokines peaked at 90 minutes. The hepatic sinusoidal space was wide at 90 minutes and narrowed after 120 minutes. The results suggest that subnormothermic oxygenation perfusion may maintain optimal graft condition until around 90 minutes and perfusion for more than 120 minutes may be counterproductive.

Current status and future of subnormothermic machine perfusion preservation

Current status and future of subnormothermic machine perfusion preservation

What is the evidence for oxygenation during kidney preservation for transplantation in 2021? A scoping review.

The main objective of static cold storage is to reduce cellular metabolic demands to extend the period of ischaemia prior to transplantation. Hypothermia does not halt metabolism and the absence of oxygen causes a cellular shift toward anaerobic respiratory pathways. There is emerging evidence that the introduction of oxygenation during organ preservation may help ameliorate the degree of ischaemia reperfusion injury and improve post-transplantation outcomes. This review aims to appraise and summarise all published literature that utilises oxygenation to improve kidney preservation for purposes of transplantation. We performed a scoping review of the literature using the bibliographic databases Embase and MEDLINE. The final date for searches was 20 March 2021. All research studies included were those that reported oxygen delivery during kidney preservation as well as providing a description of the oxygenation technique. 17 human and 48 animal studies met the inclusion criteria. The oxygen delivery methods investigated included hypothermic oxygenated machine perfusion (HOPE), oxygen carriers, two-layer method, venous systemic persufflation, hyperbaric oxygenation, normothermic machine perfusion and sub-normothermic machine perfusion. The COMPARE trial was the only study carried out with the most methodological robustness being a randomised, double blind, controlled, phase III trial that investigated the efficacy of HOPE versus HMP. A variety of studies reflect the evolution of oxygenation with useful lessons and encouraging outcomes. The first in human studies investigating HOPE and oxygen carriers are most robustly investigated strategies for oxygenation during kidney preservation and are, therefore, the best clinical references.

Mitochondrial Consequences of Organ Preservation Techniques during Liver Transplantation.

Allograft ischemia during liver transplantation (LT) adversely affects the function of mitochondria, resulting in impairment of oxidative phosphorylation and compromised post-transplant recovery of the affected organ. Several preservation methods have been developed to improve donor organ quality; however, their effects on mitochondrial functions have not yet been compared. This study aimed to summarize the available data on mitochondrial effects of graft preservation methods in preclinical models of LT. Furthermore, a network meta-analysis was conducted to determine if any of these treatments provide a superior benefit, suggesting that they might be used on humans. A systematic search was conducted using electronic databases (EMBASE, MEDLINE (via PubMed), the Cochrane Central Register of Controlled Trials (CENTRAL) and Web of Science) for controlled animal studies using preservation methods for LT. The ATP content of the graft was the primary outcome, as this is an indicator overall mitochondrial function. Secondary outcomes were the respiratory activity of mitochondrial complexes, cytochrome c and aspartate aminotransferase (ALT) release. Both a random-effects model and the SYRCLE risk of bias analysis for animal studies were used. After a comprehensive search of the databases, 25 studies were enrolled in the analysis. Treatments that had the most significant protective effect on ATP content included hypothermic and subnormothermic machine perfusion (HMP and SNMP) (MD = −1.0, 95% CI: (−2.3, 0.3) and MD = −1.1, 95% CI: (−3.2, 1.02)), while the effects of warm ischemia (WI) without cold storage (WI) and normothermic machine perfusion (NMP) were less pronounced (MD = −1.8, 95% CI: (−2.9, −0.7) and MD = −2.1 MD; CI: (−4.6; 0.4)). The subgroup of static cold storage (SCS) with shorter preservation time (< 12 h) yielded better results than SCS ≥ 12 h, NMP and WI, in terms of ATP preservation and the respiratory capacity of complexes. HMP and SNMP stand out in terms of mitochondrial protection when compared to other treatments for LT in animals. The shorter storage time at lower temperatures, together with the dynamic preservation, provided superior protection for the grafts in terms of mitochondrial function. Additional clinical studies on human patients including marginal donors and longer ischemia times are needed to confirm any superiority of preservation methods with respect to mitochondrial function.