Zebrafish as a high throughput model for organ preservation and transplantation research.

Despite decades of effort, the preservation of complex organs for transplantation remains a significant barrier that exacerbates the organ shortage crisis. Progress in organ preservation research is significantly hindered by suboptimal research tools that force investigators to sacrifice translatability over throughput. For instance, simple model systems, such as single cell monolayers or co-cultures, lack native tissue structure and functional assessment, while mammalian whole organs are complex systems with confounding variables not compatible with high-throughput experimentation. In response, diverse fields and industries have bridged this experimental gap through the development of rich and robust resources for the use of zebrafish as a model organism. Through this study, we aim to demonstrate the value zebrafish pose for the fields of solid organ preservation and transplantation, especially with respect to experimental transplantation efforts. A wide array of methods were customized and validated for preservation-specific experimentation utilizing zebrafish, including the development of assays at multiple developmental stages (larvae and adult), methods for loading and unloading preservation agents, and the development of viability scores to quantify functional outcomes. Using this platform, the largest and most comprehensive screen of cryoprotectant agents (CPAs) was performed to determine their toxicity and efficiency at preserving complex organ systems using a high subzero approach called partial freezing (i.e., storage in the frozen state at -10°C). As a result, adult zebrafish cardiac function was successfully preserved after 5 days of partial freezing storage. In combination, the methods and techniques developed have the potential to drive and accelerate research in the fields of solid organ preservation and transplantation.

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 2 h 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 24 h with either SNMP (n = 3) or SCS (n = 3) before heterotopic allotransplantation. Recipient animals received immunosuppression and were followed up for 14 days. 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 2 weeks 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.

Polyethylene glycol and caspase inhibitor emricasan alleviate cold injury in primary rat hepatocytes.

Current methods of storing explanted donor livers at 4 °C in University of Wisconsin (UW) solution result in loss of graft function and ultimately lead to less-than-ideal outcomes post transplantation. Our lab has previously shown that supplementing UW solution with 35-kilodalton polyethylene glycol (PEG) has membrane stabilizing effects for cold stored primary rat hepatocytes in suspension. Expanding on past studies, we here investigate if PEG has the same beneficial effects in an adherent primary rat hepatocyte cold storage model. In addition, we investigated the extent of cold-induced apoptosis through treating cold-stored hepatocytes with pan caspase inhibitor emricasan. In parallel to storage at the current cold storage standard of 4 °C, we investigated the effects of lowering the storage temperature to -4 °C, at which the storage solution remains ice-free due to the supercooling phenomenon. We show the addition of 5 % PEG to the storage medium significantly reduced the release of lactate dehydrogenase (LDH) in plated rat hepatocytes and a combinatorial treatment with emricasan maintains hepatocyte viability and morphology following recovery from cold storage. These results show that cold-stored hepatocytes undergo multiple mechanisms of cold-induced injury and that PEG and emricasan treatment in combination with supercooling may improve cell and organ preservation.

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

INTRODUCTION: Multiple 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. METHODS: Partial 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. RESULTS: Overall, 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. CONCLUSIONS: This 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.

BACKGROUND: 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. METHODS: 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. RESULTS: 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). CONCLUSION: 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.

Activation of autophagy during normothermic machine perfusion of discarded livers is associated with improved hepatocellular function.

Liver transplantation is hampered by a severe shortage of donor organs. Normothermic machine perfusion (NMP) of donor livers allows dynamic preservation in addition to viability assessment before transplantation. Little is known about the injury and repair mechanisms induced during NMP. To investigate these mechanisms, we examined gene and protein expression changes in a cohort of discarded human livers, stratified by hepatocellular function, during NMP. Six human livers acquired through donation after circulatory death (DCD) underwent 12 h of NMP. Of the six livers, three met predefined criteria for adequate hepatocellular function. We applied transcriptomic profiling and protein analysis to evaluate temporal changes in gene expression during NMP between functional and nonfunctional livers. Principal component analysis segregated the two groups and distinguished the various perfusion time points. Transcriptomic analysis of biopsies from functional livers indicated robust activation of innate immunity after 3 h of NMP followed by enrichment of prorepair and prosurvival mechanisms. Nonfunctional livers demonstrated delayed and persistent enrichment of markers of innate immunity. Functional livers demonstrated effective induction of autophagy, a cellular repair and homeostasis pathway, in contrast to nonfunctional livers. In conclusion, NMP of discarded DCD human livers results in innate immune-mediated injury, while also activating autophagy, a presumed mechanism for support of cellular repair. More pronounced activation of autophagy was seen in livers that demonstrated adequate hepatocellular function.NEW & NOTEWORTHY We demonstrate that ischemia-reperfusion injury occurs in all livers during NMP, though there are notable differences in gene expression between functional and nonfunctional livers. We further demonstrate that activation of the liver's repair and homeostasis mechanisms through autophagy plays a vital role in the graft's response to injury and may impact liver function. These findings indicate that liver autophagy might be a key therapeutic target for rehabilitating the function of severely injured or untransplantable livers.

Optimization of Ex Vivo Machine Perfusion and Transplantation of Vascularized Composite Allografts.

BACKGROUND: Machine perfusion is gaining interest as an efficient method of tissue preservation of Vascularized Composite Allografts (VCA). The aim of this study was to develop a protocol for ex vivo subnormothermic oxygenated machine perfusion (SNMP) on rodent hindlimbs and to validate our protocol in a heterotopic hindlimb transplant model. METHODS: In this optimization study we compared three different solutions during 6 h of SNMP (n = 4 per group). Ten control limbs were stored in a preservation solution on Static Cold Storage [SCS]). During SNMP we monitored arterial flowrate, lactate levels, and edema. After SNMP, muscle biopsies were taken for histology examination, and energy charge analysis. We validated the best perfusion protocol in a heterotopic limb transplantation model with 30-d follow up (n = 13). As controls, we transplanted untreated limbs (n = 5) and hindlimbs preserved with either 6 or 24 h of SCS (n = 4 and n = 5). RESULTS: During SNMP, arterial outflow increased, and lactate clearance decreased in all groups. Total edema was significantly lower in the HBOC-201 group compared to the BSA group (P = 0.005), 4.9 (4.3-6.1) versus 48.8 (39.1-53.2) percentage, but not to the BSA + PEG group (P = 0.19). Energy charge levels of SCS controls decreased 4-fold compared to limbs perfused with acellular oxygen carrier HBOC-201, 0.10 (0.07-0.17) versus 0.46 (0.42-0.49) respectively (P = 0.002). CONCLUSIONS: Six hours ex vivo SNMP of rodent hindlimbs using an acellular oxygen carrier HBOC-201 results in superior tissue preservation compared to conventional SCS.

Evolving utilization of donation after circulatory death livers in liver transplantation: The day of DCD has come.

Compared to donation after brain death (DBD), livers procured for transplantation from donation after circulatory death (DCD) donors experience more ischemia-reperfusion injury and higher rates of ischemic cholangiopathy due to the period of warm ischemic time (WIT) following withdrawal of life support. As a result, utilization of DCD livers for liver transplant (LT) has generally been limited to short WITs and younger aged donor grafts, causing many recovered DCD organs to be discarded without consideration for transplant. This study assesses how DCD liver utilization and outcomes have changed over time, using OPTN data from adult, first-time, deceased donor, whole-organ LTs between January 1995 and December 2019. Results show that increased clinical experience with DCD LT has translated into increased use of livers from DCD donors, shorter ischemic times, shorter lengths of hospitalization after transplant, and lower rates of retransplantation. The data also reveal that over the past decade, the rate of increase in DCD LTs conducted in the United States has outpaced that of DBD. Together, these trends signal an opportunity for the field of liver transplantation to mitigate the organ shortage by capitalizing on DCD liver allografts that are currently not being utilized.

Viability testing of discarded livers with normothermic machine perfusion: Alleviating the organ shortage outweighs the cost.

BACKGROUND: Over 700 donor livers are discarded annually in the United States due to high risk of poor graft function. The objective of this study was to determine the impact of using normothermic machine perfusion to identify transplantable livers among those currently discarded. STUDY DESIGN: A series of 21 discarded human livers underwent viability assessment during normothermic machine perfusion. Cross-sectional analysis of the Scientific Registry of Transplant Recipients database and cost analysis was performed to extrapolate the case series to national experience. RESULTS: 21 discarded human livers were included in the perfusion cohort. 11 of 20 (55%) eligible grafts met viability criteria for transplantation. Grafts in the perfusion cohort had a similar donor risk index compared with discarded grafts (n = 1402) outside of New England in 2017 and 2018 (median [IQR]: 2.0 [1.5, 2.4] vs. 2.0 [1.7, 2.3], P = .40). 705 (IQR 677-741) livers were discarded annually in the United States since 2005, translating to the potential for 398 additional transplants nationally. The median cost to identify a transplantable graft with machine perfusion was $28,099 USD. CONCLUSIONS: Normothermic machine perfusion of discarded livers could identify a significant number of transplantable grafts, significantly improving access to liver transplantation.

Pumping new life into old ideas: Preservation and rehabilitation of the liver using ex situ machine perfusion.

Recent advances in machine perfusion technology have reinvigorated the field of liver transplantation with the possibilities of vastly improving the efficiency and safety of the life-saving procedure. With this improved preservation technology, transplant surgeons are now able to use previously untransplantable donor livers without significantly compromising patient outcomes. Early clinical studies demonstrate the ability to extend preservation times and assess a graft's potential viability using normothermic machine perfusion, in addition to restoring the energy supply in donor livers by supporting metabolism through circulation of vital nutrients and blood-based oxygen carriers. Future endeavors for surgeons and scientists should focus on improving criteria to assess viability, optimizing protocols for perfusion research, investigating mechanisms of poor graft viability, and targeting these mechanisms with novel therapies to improve graft function prior to transplantation. Long-term goals include extending preservation times on the scale of days to weeks, enabling long-distance organ sharing, and establishing regional organ perfusion centers to streamline the procurement, perfusion, and transplantation process.

The efficacy of HBOC-201 in ex situ gradual rewarming kidney perfusion in a rat model.

Gradual rewarming from hypothermic to normothermic is a novel perfusion modality with superior outcome to sudden rewarming to normothermic. However, the identification of an oxygen carrier that could function at a temperature range from 4 to 7°C or whether it is necessary to use oxygen carrier during kidney rewarming, remains unresolved. This study was designed to test the use of a hemoglobin-based oxygen carrier (HBOC) during gradual kidney rewarming as an alternative to simple dissolved oxygen. In this study, 10 rat kidneys were randomly divided into the control and the HBOC group. In the control group, no oxygen carrier was used during rewarming perfusion and the perfusion solution was oxygenated only by applying diffused carbogen flow. The protocol mimicked a donor after circulatory death (DCD) kidney transplantation, where after 30 minutes warm ischemia and 120 minutes cold storage in University of Wisconsin solution, the DCD kidneys underwent gradual rewarming from 10 to 37°C during 90 minutes with or without HBOC. This was followed by 30 minutes of warm ischemia in room temperature to mimic the anastomosis time and 120 minutes of reperfusion at 37°C to mimic the early post-transplant state of the graft. The HBOC group demonstrated superior kidney function which was highlighted by higher ultrafiltrate production, better glomerular filtration rate and improved sodium reabsorption. There was no significant difference between the 2 groups regarding the hemodynamics, tissue injury, and adenosine triphosphate levels. In conclusion, this study suggests better renal function recovery in DCD kidneys after rewarming with HBOC compared to rewarming without an oxygen carrier.

Synthetic hemoglobin-based oxygen carriers are an acceptable alternative for packed red blood cells in normothermic kidney perfusion.

Normothermic machine perfusion presents a novel platform for pretransplant assessment and reconditioning of kidney grafts. Maintaining the metabolic activity of a preserved graft at physiologic levels requires an adequate oxygen supply, typically delivered by crystalloid solutions supplemented with red blood cells. In this study, we explored the feasibility of using a synthetic hemoglobin-based oxygen carrier (HBOC) in human kidney normothermic perfusion. Fourteen discarded human kidneys were perfused for 6 hours at a mean temperature of 37°C using a pressure-controlled system. Kidneys were perfused with a perfusion solution supplemented with either HBOC (n = 7) or packed red blood cells (PRBC) (n = 7) to increase oxygen-carrying capacity. Renal artery resistance, oxygen extraction, metabolic activity, energy stores, and histological features were evaluated. Throughout perfusion, kidneys from both groups exhibited comparable behavior regarding vascular flow (P = .66), oxygen consumption (P = .88), and reconstitution of tissue adenosine triphosphate (P = .057). Lactic acid levels were significantly higher in kidneys perfused with PRBC (P = .007). Histological findings were comparable between groups, and there was no evidence of histological damage caused by the HBOC. This feasibility experiment demonstrates that a HBOC solution can offer a logistically more convenient off-the-shelf alternative to PRBC in normothermic machine perfusion of human kidneys.

Bulk Droplet Vitrification: An Approach to Improve Large-Scale Hepatocyte Cryopreservation Outcome.

Loss of hepatocyte viability and metabolic function after cryopreservation is still a major issue. Although vitrification is a promising alternative, it has generally been proven to be unsuitable for vitrification of large cell volumes which is required for clinical applications. Here, we propose a novel bulk droplet (3-5 mm diameter) vitrification method which allows high throughput volumes (4 mL/min), while using a low preincubated CPA concentration (15% v/v) to minimize toxicity and loss of cell viability and function. We used rapid (1.25 s) osmotic dehydration to concentrate a low preincubated intracellular CPA concentration ahead of vitrification, without the need of fully equilibrating toxic CPA concentrations. We compared direct postpreservation viability, long-term viability, and metabolic function of bulk droplet vitrified, cryopreserved, and fresh hepatocytes. Simulations and cooling rate measurements confirmed an adequate concentration of the intracellular CPA concentration (up to 8.53 M) after dehydration in combination with high cooling rates (960-1320 °C/min) for successful vitrification. In comparison to cryopreserved hepatocytes, bulk droplet vitrified hepatocytes had a significantly higher viability, directly after preservation and after 1 day in culture. Moreover, bulk droplet vitrified hepatocytes had evidently better morphology and showed significantly higher metabolic activity than cryopreserved hepatocytes in long-term collagen sandwich cultures. In conclusion, we developed a novel bulk droplet vitrification method of which we validated the theoretical background and demonstrated the feasibility to use this method to vitrify large cell volumes. Moreover, we showed that this method results in improved hepatocyte viability and metabolic function as compared to cryopreservation.

Leveraging the zebrafish to model organ transplantation.

PURPOSE OF REVIEW: The availability of organs for transplant fails to meet the demand and this shortage is growing worse every year. As the cost of not getting a suitable donor organ can mean death for patients, new tools and approaches that allows us to make advances in transplantation faster and provide a different vantage point are required. To address this need, we introduce the concept of using the zebrafish (Danio rerio) as a new model system in organ transplantation. The zebrafish community offers decades of research experience in disease modeling and a rich toolbox of approaches for interrogating complex pathological states. We provide examples of how already existing zebrafish assays/tools from cancer, regenerative medicine, immunology, and others, could be leveraged to fuel new discoveries in pursuit of solving the organ shortage. RECENT FINDINGS: Important innovations have enabled several types of transplants to be successfully performed in zebrafish, including stem cells, tumors, parenchymal cells, and even a partial heart transplant. These innovations have been performed against a backdrop of an expansive and impressive list of tools designed to uncover the biology of complex systems that include a wide array of fluorescent transgenic fish that label specific cell types and mutant lines that are transparent, immune-deficient. Allogeneic transplants can also be accomplished using immune suppressed and syngeneic fish. Each of these innovations within the zebrafish community would provide several helpful tools that could be applied to transplant research. SUMMARY: We highlight some examples of existing tools and assays developed in the zebrafish community that could be leveraged to overcome barriers in organ transplantation, including ischemia-reperfusion, short preservation durations, regeneration of marginal grafts, and acute and chronic rejection.

Nuclear receptors control pro-viral and antiviral metabolic responses to hepatitis C virus infection.

Viruses lack the basic machinery needed to replicate and therefore must hijack the host's metabolism to propagate. Virus-induced metabolic changes have yet to be systematically studied in the context of host transcriptional regulation, and such studies shoul offer insight into host-pathogen metabolic interplay. In this work we identified hepatitis C virus (HCV)-responsive regulators by coupling system-wide metabolic-flux analysis with targeted perturbation of nuclear receptors in primary human hepatocytes. We found HCV-induced upregulation of glycolysis, ketogenesis and drug metabolism, with glycolysis controlled by activation of HNF4α, ketogenesis by PPARα and FXR, and drug metabolism by PXR. Pharmaceutical inhibition of HNF4α reversed HCV-induced glycolysis, blocking viral replication while increasing apoptosis in infected cells showing virus-induced dependence on glycolysis. In contrast, pharmaceutical inhibition of PPARα or FXR reversed HCV-induced ketogenesis but increased viral replication, demonstrating a novel host antiviral response. Our results show that virus-induced changes to a host's metabolism can be detrimental to its life cycle, thus revealing a biologically complex relationship between virus and host.

Advances in machine perfusion, organ preservation, and cryobiology: potential impact on vascularized composite allotransplantation.

PURPOSE OF REVIEW: In this review, we discuss novel strategies that allow for extended preservation of vascularized composite allografts and their potential future clinical implications for the field of vascularized composite allotransplantation (VCA). RECENT FINDINGS: The current gold standard in tissue preservation - static cold preservation on ice - is insufficient to preserve VCA grafts for more than a few hours. Advancements in the field of VCA regarding matching and allocation, desensitization, and potential tolerance induction are all within reasonable reach to achieve; these are, however, constrained by limited preservation time of VCA grafts. Although machine perfusion holds many advantages over static cold preservation, it currently does not elongate the preservation time. More extreme preservation techniques, such as cryopreservation approaches, are, however, specifically difficult to apply to composite tissues as the susceptibility to ischemia and cryoprotectant agents varies greatly by tissue type. SUMMARY: In the current scope of extended preservation protocols, high subzero approaches of VCA grafts will be particularly critical enabling technologies for the implementation of tolerance protocols clinically. Ultimately, advances in both preservation techniques and tolerance induction have the potential to transform the field of VCA and eventually lead to broad applications in reconstructive transplantation.

Liver ex situ machine perfusion preservation: A review of the methodology and results of large animal studies and clinical trials.

Ex vivo machine perfusion (MP) is a promising way to better preserve livers prior to transplantation. Currently, no methodology has a verified benefit over simple cold storage. Before becoming clinically feasible, MP requires validation in models that reliably predict human performance. Such a model has been found in porcine liver, whose physiological, anatomical, and immunological characteristics closely resemble the human liver. Since the 1930s, researchers have explored MP as preservation, but only recently have clinical trials been performed. Making this technology clinically available holds the promise of expanding the donor pool through more effective preservation of extended criteria donor (ECD) livers. MP promises to decrease delayed graft function, primary nonfunction, and biliary strictures, which are all common failure modes of transplanted ECD livers. Although hypothermic machine perfusion (HMP) has become the standard for kidney ex vivo preservation, the precise settings and clinical role for liver MP have not yet been established. In research, there are 2 schools of thought: normothermic machine perfusion, closely mimicking physiologic conditions, and HMP, to maximize preservation. Here, we review the literature for porcine ex vivo MP, with an aim to summarize perfusion settings and outcomes pertinent to the clinical establishment of MP. Liver Transplantation 23 679-695 2017 AASLD.

Subzero organ preservation: the dawn of a new ice age?

PURPOSE OF REVIEW: Herein, we review the field of subzero organ preservation with a focus on recent developments in hepatic supercooling. RECENT FINDINGS: Organ preservation is making a rapid shift from the decade old standard of storage on ice toward techniques that improve organ availability as well as preservation time. Long-term organ preservation would have tremendous benefits to the organ transplantation field, including better organ allocation, donor-recipient matching, as well as reduced preservation injury, and subsequent improvement of donor organ use. The formation of ice has proven an important limiting factor and novel techniques attempt to control or prevent freezing using cryoprotective agents, and highly controlled cooling regimens. Various techniques have been employed over the previous decades, including true organ freezing, vitrification, and subzero nonfreezing or supercooling. For most techniques, successful transplantation following long-term subzero preservation has remained elusive. Supercooling, however, recently delivered the first promising results, yielding survival after up to 4 days of supercooled preservation at -6°C. SUMMARY: As the field of organ preservation undergoes significant development, the field of subzero preservation also receives renewed interest. Although many obstacles remain to be overcome to make subzero preservation feasible, novel techniques are beginning to show their potential in achieving long-term preservation.