Split-Liver Ex Situ Machine Perfusion: A Novel Technique for Studying Organ Preservation and Therapeutic Interventions.

Ex situ machine perfusion is a promising technology to help improve organ viability prior to transplantation. However, preclinical studies using discarded human livers to evaluate therapeutic interventions and optimize perfusion conditions are limited by significant graft heterogeneity. In order to improve the efficacy and reproducibility of future studies, a split-liver perfusion model was developed to allow simultaneous perfusion of left and right lobes, allowing one lobe to serve as a control for the other. Eleven discarded livers were surgically split, and both lobes perfused simultaneously on separate perfusion devices for 3 h at subnormothermic temperatures. Lobar perfusion parameters were also compared with whole livers undergoing perfusion. Similar to whole-liver perfusions, each lobe in the split-liver model exhibited a progressive decrease in arterial resistance and lactate levels throughout perfusion, which were not significantly different between right and left lobes. Split liver lobes also demonstrated comparable energy charge ratios. Ex situ split-liver perfusion is a novel experimental model that allows each graft to act as its own control. This model is particularly well suited for preclinical studies by avoiding the need for large numbers of enrolled livers necessary due to the heterogenous nature of discarded human liver research.

Metabolic and lipidomic profiling of steatotic human livers during ex situ normothermic machine perfusion guides resuscitation strategies.

There continues to be a significant shortage of donor livers for transplantation. One impediment is the discard rate of fatty, or steatotic, livers because of their poor post-transplant function. Steatotic livers are prone to significant ischemia-reperfusion injury (IRI) and data regarding how best to improve the quality of steatotic livers is lacking. Herein, we use normothermic (37°C) machine perfusion in combination with metabolic and lipidomic profiling to elucidate deficiencies in metabolic pathways in steatotic livers, and to inform strategies for improving their function. During perfusion, energy cofactors increased in steatotic livers to a similar extent as non-steatotic livers, but there were significant deficits in anti-oxidant capacity, efficient energy utilization, and lipid metabolism. Steatotic livers appeared to oxidize fatty acids at a higher rate but favored ketone body production rather than energy regeneration via the tricyclic acid cycle. As a result, lactate clearance was slower and transaminase levels were higher in steatotic livers. Lipidomic profiling revealed ω-3 polyunsaturated fatty acids increased in non-steatotic livers to a greater extent than in steatotic livers. The novel use of metabolic and lipidomic profiling during ex situ normothermic machine perfusion has the potential to guide the resuscitation and rehabilitation of steatotic livers for transplantation.

Exploring donor demographics effects on hepatocyte yield and viability: Results of whole human liver isolation from one center.

Due to the growth of cell-based therapeutic alternatives addressing the shortage of livers for transplant, there is necessity for a reliable source of human hepatocytes. In addition, pharmaceutical research often requires human hepatocytes to assess new drug therapies during development or to achieve FDA approval. Whole human livers producing large quantities of cells from the same donor are ideal, enhancing reproducibility for all purposes, while also allowing for capturing variances in drug-metabolism across different demographics for pharmaceutical testing and development but are limited in availability and quality for research purposes. The present study investigates the effect of donor and liver procurement factors of 16 human livers on cell viability and yield, showing that typical exclusion criteria for transplant still produce viable hepatocytes with significant yields. Although limited in number of data points, which should be taken into consideration, the conclusions of this study could be utilized as indications, allowing for expansion of liver selection criteria for hepatocyte isolation and provide the necessary quality hepatocytes in large quantities for the growing pharmaceutical, biomedical, and therapeutic research fields.

Preparation of Decellularized Liver Scaffolds and Recellularized Liver Grafts.

Severe liver disease is the 12th leading cause of death in the USA, with organ transplantation often being the only viable option for treatment. However, due to the shortage of viable donor livers, it is estimated that over 1200 patients died in 2015 while waiting for liver transplantation. This highlights the need for alternative sources of viable organs. In this study, we describe a method that provides the groundwork for the development of functional liver grafts. The approach described here is for removal of cells from intact livers and subsequently repopulating them with functional liver cells. Briefly, rat livers are harvested and subjected to a series of perfusion decellularization steps using an anionic detergent such that an intact decellularized liver matrix (DLM) scaffold with preserved vascular architecture is obtained. Further, we describe methods to recellularize DLM scaffolds with adult primary hepatocytes, creating a liver graft that exhibits hepatic functions in vitro.

Endothelial Dysfunction in Steatotic Human Donor Livers: A Pilot Study of the Underlying Mechanism During Subnormothermic Machine Perfusion.

BACKGROUND: Steatosis is a major risk factor for primary nonfunction in liver transplantations. Steatotic livers recover poorly from ischemia reperfusion injury, in part due to alterations in the microcirculation, although the exact mechanism is unclear. In this study, we tested if there were any alterations in the shear stress sensing Kruppel-like factor 2 (KLF2) and its likely downstream consequences in the ex vivo perfused human liver endothelium, which would imply perturbations in microcirculatory flow in macrosteatotic livers disrupts laminar flow to evaluate if this is a potential therapeutic target for steatotic livers. METHODS: Using a subnormothermic machine perfusion system, 5 macrosteatotic and 4 nonsteatotic human livers were perfused for 3 hours. Flow, resistance, and biochemical profile were monitored. Gene expression levels of nitric oxide synthase 3 (eNOS), KLF2, and thrombomodulin were determined. Nitric oxide (NO) was measured in the perfusion fluid and activation of eNOS was measured with Western blotting. RESULTS: Flow dynamics, injury markers, and bile production were similar in both groups. Kruppel-like factor 2 expression was significantly higher in nonsteatotic livers. Western blotting analyses showed significantly higher levels of activated eNOS in nonsteatotic livers, consistent with an increase in NO production over time. Macrosteatotic livers showed decreased KLF2 upregulation, eNOS activity, and NO production during machine perfusion. CONCLUSIONS: These results indicate a perturbed KLF2 sensing in steatotic livers, which aligns with perturbed microcirculatory state. This may indicate endothelial dysfunction and contribute to poor posttransplantation outcomes in fatty livers, and further studies to confirm by evaluation of flow and testing treatments are warranted.

Nondestructive Methods for Monitoring Cell Removal During Rat Liver Decellularization.

Whole liver engineering holds the promise to create transplantable liver grafts that may serve as substitutes for donor organs, addressing the donor shortage in liver transplantation. While decellularization and recellularization of livers in animal models have been successfully achieved, scale up to human livers has been slow. There are a number of donor human livers that are discarded because they are not found suitable for transplantation, but are available for engineering liver grafts. These livers are rejected due to a variety of reasons, which in turn may affect the decellularization outcome. Hence, a one-size-fit-for all decellularization protocol may not result in scaffolds with consistent matrix quality, subsequently influencing downstream recellularization and transplantation outcomes. There is a need for a noninvasive monitoring method to evaluate the extent of cell removal, while ensuring preservation of matrix components during decellularization. In this study, we decellularized rat livers using a protocol previously established by our group, and we monitored decellularization through traditional destructive techniques, including evaluation of DNA, collagen, and glycosaminoglycan (GAG) content in decellularized scaffolds, as well as histology. In addition, we used computed tomography and perfusate analysis as alternative nondestructive decellularization monitoring methods. We found that DNA removal correlates well with the Hounsfield unit of the liver, and perfusate analysis revealed that significant amount of GAG is removed during perfusion with 0.1% sodium dodecyl sulfate. This allowed for optimization of our decellularization protocol leading to scaffolds that have significantly higher GAG content, while maintaining appropriate removal of cellular contents. The significance of this is the creation of a nondestructive monitoring strategy that can be used for optimization of decellularization protocols for individual human livers available for liver engineering.

Isolation and co-culture of rat parenchymal and non-parenchymal liver cells to evaluate cellular interactions and response.

The liver is a central organ in the human body, and first line of defense between host and external environment. Liver response to any external perturbation is a collective reaction of resident liver cells. Most of the current in vitro liver models focus on hepatocytes, the primary metabolic component, omitting interactions and cues from surrounding environment and non-parenchymal cells (NPCs). Recent studies suggest that contributions of NPCs are vital, particularly in disease conditions, and outcomes of drugs and their metabolites. Along with hepatocytes, NPCs-Kupffer (KC), sinusoidal endothelial (LSEC) and stellate cells (SC) are major cellular components of the liver. Incorporation of primary cells in in vitro liver platforms is essential to emulate the functions of the liver, and its overall response. Herein, we isolate individual NPC cell fractions from rat livers and co-culture them in a transwell format incorporating primary rat hepatocytes with LSECs, SCs, and KCs. Our results indicate that the presence and contributions of multiple cells within the co-culture capture the interactions between hepatocytes and NPC, and modulates the responses to inflammatory stimulus such as LPS. The isolation and co-culture methods could provide a stable platform for creating in vitro liver models that provide defined functionality beyond hepatocytes alone.

Proteomic analysis of naturally-sourced biological scaffolds.

A key challenge to the clinical implementation of decellularized scaffold-based tissue engineering lies in understanding the process of removing cells and immunogenic material from a donor tissue/organ while maintaining the biochemical and biophysical properties of the scaffold that will promote growth of newly seeded cells. Current criteria for evaluating whole organ decellularization are primarily based on nucleic acids, as they are easy to quantify and have been directly correlated to adverse host responses. However, numerous proteins cause immunogenic responses and thus should be measured directly to further understand and quantify the efficacy of decellularization. In addition, there has been increasing appreciation for the role of the various protein components of the extracellular matrix (ECM) in directing cell growth and regulating organ function. We performed in-depth proteomic analysis on four types of biological scaffolds and identified a large number of both remnant cellular and ECM proteins. Measurements of individual protein abundances during the decellularization process revealed significant removal of numerous cellular proteins, but preservation of most structural matrix proteins. The observation that decellularized scaffolds still contain many cellular proteins, although at decreased abundance, indicates that elimination of DNA does not assure adequate removal of all cellular material. Thus, proteomic analysis provides crucial characterization of the decellularization process to create biological scaffolds for future tissue/organ replacement therapies.

Metabolic profiling during ex vivo machine perfusion of the human liver.

As donor organ shortages persist, functional machine perfusion is under investigation to improve preservation of the donor liver. The transplantation of donation after circulatory death (DCD) livers is limited by poor outcomes, but its application may be expanded by ex vivo repair and assessment of the organ before transplantation. Here we employed subnormothermic (21 °C) machine perfusion of discarded human livers combined with metabolomics to gain insight into metabolic recovery during machine perfusion. Improvements in energetic cofactors and redox shifts were observed, as well as reversal of ischemia-induced alterations in selected pathways, including lactate metabolism and increased TCA cycle intermediates. We next evaluated whether DCD livers with steatotic and severe ischemic injury could be discriminated from 'transplantable' DCD livers. Metabolomic profiling was able to cluster livers with similar metabolic patterns based on the degree of injury. Moreover, perfusion parameters combined with differences in metabolic factors suggest variable mechanisms that result in poor energy recovery in injured livers. We conclude that machine perfusion combined with metabolomics has significant potential as a clinical instrument for the assessment of preserved livers.