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.

Anti-apoptotic treatment of warm ischemic male rat livers in machine perfusion improves symptoms of ischemia-reperfusion injury.

Background: Liver donation after cardiac death (DCD) makes up a small percentage of the organs used in transplantation and poses a higher risk of graft loss compared to donation after brain death (DBD); this is a result of ischemia reperfusion for which the exact injury mechanisms are currently not fully understood. However, reperfusion injury has been shown to lead to necrosis as well as apoptosis through oxidative stress and mitochondrial dysfunction. In this work, we propose that use of the pro-survival, anti-apoptotic CEPT cocktail in post-ischemia normothermic machine perfusion (NMP) may improve recovery in rat livers subjected to extended durations of warm ischemia. Materials and Methods: Livers procured from male Lewis rats were subjected to 90 min of warm ischemia, followed by 6 h of NMP where they were treated either with the survival-enhancing anti-apoptotic cocktail (CEPT), the vehicle (DMSO) or the base media with no additives. Results: The CEPT-treated group exhibited lower expression of hepatic injury biomarkers, and improvement in a range of hepatocellular symptoms associated with the hepatic parenchyma, biliary epithelium and the sinusoidal endothelium, including recovery of bile secretion and lowered vascular resistance. Conclusions: This study's findings suggest apoptosis plays a more significant role in ischemia-reperfusion injury than previously understood, and provide useful insight for further investigation of the specific underlying mechanisms and development of novel treatment methods.

Polyethylene Glycol and Caspase Inhibitor Emricasan Alleviates 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 leads 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.

Anti-apoptotic treatment of warm ischemic male rat livers in machine perfusion improves symptoms of ischemia-reperfusion injury.

Liver donation after cardiac death (DCD) makes up a small percentage of the donor pool and poses a higher risk of graft loss compared to donation after brain death (DBD); this is a result of ischemia reperfusion for which the exact injury mechanisms are currently not fully understood. However, reperfusion injury has been shown to lead to necrosis as well as apoptosis at the cellular level. In this work, we propose that use of the pro-survival, anti-apoptotic CEPT cocktail in post-ischemia normothermic machine perfusion (NMP) may improve recovery in rat livers subjected to extended durations of warm ischemia. Livers procured from male lewis rats were subjected to 90 minutes of warm ischemia, followed by 6 hours of NMP where they were treated with the survival-enhancing anti-apoptotic cocktail (CEPT), the vehicle (DMSO) or the base media with no additives. The CEPT-treated group exhibited lower expression of hepatic injury biomarkers, and improvement in a range of hepatocellular functions associated with the hepatic parenchyma, biliary epithelium and especially the sinusoidal endothelium. This study's findings provide useful insight for further investigation of the extent of apoptotic contribution to ischemia reperfusion injury (IRI).

Quantifying Cell-Derived Changes in Collagen Synthesis, Alignment, and Mechanics in a 3D Connective Tissue Model.

Dysregulation of extracellular matrix (ECM) synthesis, organization, and mechanics are hallmark features of diseases like fibrosis and cancer. However, most in vitro models fail to recapitulate the three-dimensional (3D) multi-scale hierarchical architecture of collagen-rich tissues and as a result, are unable to mirror native or disease phenotypes. Herein, using primary human fibroblasts seeded into custom fabricated 3D non-adhesive agarose molds, a novel strategy is proposed to direct the morphogenesis of engineered 3D ring-shaped tissue constructs with tensile and histological properties that recapitulate key features of fibrous connective tissue. To characterize the shift from monodispersed cells to a highly-aligned, collagen-rich matrix, a multi-modal approach integrating histology, multiphoton second-harmonic generation, and electron microscopy is employed. Structural changes in collagen synthesis and alignment are then mapped to functional differences in tissue mechanics and total collagen content. Due to the absence of an exogenously added scaffolding material, this model enables the direct quantification of cell-derived changes in 3D matrix synthesis, alignment, and mechanics in response to the addition or removal of relevant biomolecular perturbations. To illustrate this, the effects of nutrient composition, fetal bovine serum, rho-kinase inhibitor, and pro- and anti-fibrotic compounds on ECM synthesis, 3D collagen architecture, and mechanophenotype are quantified.

Partial freezing of rat livers extends preservation time by 5-fold.

The limited preservation duration of organs has contributed to the shortage of organs for transplantation. Recently, a tripling of the storage duration was achieved with supercooling, which relies on temperatures between -4 and -6 °C. However, to achieve deeper metabolic stasis, lower temperatures are required. Inspired by freeze-tolerant animals, we entered high-subzero temperatures (-10 to -15 °C) using ice nucleators to control ice and cryoprotective agents (CPAs) to maintain an unfrozen liquid fraction. We present this approach, termed partial freezing, by testing gradual (un)loading and different CPAs, holding temperatures, and storage durations. Results indicate that propylene glycol outperforms glycerol and injury is largely influenced by storage temperatures. Subsequently, we demonstrate that machine perfusion enhancements improve the recovery of livers after freezing. Ultimately, livers that were partially frozen for 5-fold longer showed favorable outcomes as compared to viable controls, although frozen livers had lower cumulative bile and higher liver enzymes.

The effect of blood cells retained in rat livers during static cold storage on viability outcomes during normothermic machine perfusion.

In transplantation, livers are transported to recipients using static cold storage (SCS), whereby livers are exposed to cold ischemic injury that contribute to post-transplant risk factors. We hypothesized that flushing organs during procurement with cold preservation solutions could influence the number of donor blood cells retained in the allograft thereby exacerbating cold ischemic injury. We present the results of rat livers that underwent 24 h SCS after being flushed with a cold University of Wisconsin (UW) solution versus room temperature (RT) lactated ringers (LR) solution. These results were compared to livers that were not flushed prior to SCS and thoroughly flushed livers without SCS. We used viability and injury metrics collected during normothermic machine perfusion (NMP) and the number of retained peripheral cells (RPCs) measured by histology to compare outcomes. Compared to the cold UW flush group, livers flushed with RT LR had lower resistance, lactate, AST, and ALT at 6 h of NMP. The number of RPCs also had significant positive correlations with resistance, lactate, and potassium levels and a negative correlation with energy charge. In conclusion, livers exposed to cold UW flush prior to SCS appear to perform worse during NMP, compared to RT LR flush.

Non-invasive quantification of the mitochondrial redox state in livers during machine perfusion.

Ischemia reperfusion injury (IRI) is a critical problem in liver transplantation that can lead to life-threatening complications and substantially limit the utilization of livers for transplantation. However, because there are no early diagnostics available, fulminant injury may only become evident post-transplant. Mitochondria play a central role in IRI and are an ideal diagnostic target. During ischemia, changes in the mitochondrial redox state form the first link in the chain of events that lead to IRI. In this study we used resonance Raman spectroscopy to provide a rapid, non-invasive, and label-free diagnostic for quantification of the hepatic mitochondrial redox status. We show this diagnostic can be used to significantly distinguish transplantable versus non-transplantable ischemically injured rat livers during oxygenated machine perfusion and demonstrate spatial differences in the response of mitochondrial redox to ischemia reperfusion. This novel diagnostic may be used in the future to predict the viability of human livers for transplantation and as a tool to better understand the mechanisms of hepatic IRI.

Directing fibroblast self-assembly to fabricate highly-aligned, collagen-rich matrices.

Extracellular matrix composition and organization play a crucial role in numerous biological processes ranging from cell migration, differentiation, survival and metastasis. Consequently, there have been significant efforts towards the development of biomaterials and in vitro models that recapitulate the complexity of native tissue architecture. Here, we demonstrate an approach to fabricating highly aligned cell-derived tissue constructs via the self-assembly of human dermal fibroblasts. By optimizing mold geometry, cell seeding density, and media composition we can direct human dermal fibroblasts to adhere to one another around a non-adhesive agarose peg to facilitate the development of cell-mediated circumferential tension. By removing serum and adding ascorbic acid and l-proline, we tempered fibroblast contractility to enable the formation of stable tissue constructs. Similarly, we show that the alignment of cells and the ECM they synthesize can be modulated by changes to seeding density and that constructs seeded with the lowest number of cells have the highest degree of fibrillar collagen alignment. Finally, we show that this highly aligned, tissue engineered construct can be decellularized and that when re-seeded with fibroblasts, it provides instructive cues which enable cells to adhere to and align in the direction of the remaining collagen fiber network. STATEMENT OF SIGNIFICANCE: Cell and extracellular matrix organization is directly related to biological function including cell signaling and tissue mechanics. Changes to this organization are often associated with injury or disease. The majority of in vitro tissue engineering models investigating cell and matrix organization rely on the addition of stress-shielding exogenous proteins and polymers and, or the application of external forces to promote alignment. Here we present a completely cell-based approach that relies on the development of cell-mediated tension to direct anisotropic cellular alignment and matrix synthesis using human dermal fibroblasts. A major challenge with this approach is excessive cellular contractility that results in necking and failure of the tissue construct. While other groups have tried to overcome this challenge by simply adding more cells, here we show that matrix alignment is inversely related to cell seeding density. To engineer tissue constructs with the highest degree of alignment, we optimized media components to reduce cellular contractility and promote collagen synthesis such that fibroblast toroids remained stable for at least 28 days in culture. We subsequently showed that these collagen-rich tissue constructs could be decellularized while maintaining their collagen microstructure and that cells adhered to and responded to the decellularized cell-derived matrix by aligning and elongating along the collagen fibers. The complexity of cell-derived matrices has been shown to better recapitulate in vivo tissue architecture and composition. This study provides a straight-forward approach to fabricating instructive cell-derived matrices with a high degree of uniaxial alignment generated purely by cell-mediated tension.

Quantitative Live-Cell Confocal Imaging of 3D Spheroids in a High-Throughput Format.

Accurately predicting the human response to new compounds is critical to a wide variety of industries. Standard screening pipelines (including both in vitro and in vivo models) often lack predictive power. Three-dimensional (3D) culture systems of human cells, a more physiologically relevant platform, could provide a high-throughput, automated means to test the efficacy and/or toxicity of novel substances. However, the challenge of obtaining high-magnification, confocal z stacks of 3D spheroids and understanding their respective quantitative limitations must be overcome first. To address this challenge, we developed a method to form spheroids of reproducible size at precise spatial locations across a 96-well plate. Spheroids of variable radii were labeled with four different fluorescent dyes and imaged with a high-throughput confocal microscope. 3D renderings of the spheroid had a complex bowl-like appearance. We systematically analyzed these confocal z stacks to determine the depth of imaging and the effect of spheroid size and dyes on quantitation. Furthermore, we have shown that this loss of fluorescence can be addressed through the use of ratio imaging. Overall, understanding both the limitations of confocal imaging and the tools to correct for these limits is critical for developing accurate quantitative assays using 3D spheroids.

Quantifying the kinetics and morphological changes of the fusion of spheroid building blocks.

Tissue fusion, whereby two or more spheroids coalesce, is a process that is fundamental to biofabrication. We have designed a quantitative, high-throughput platform to investigate the fusion of multicellular spheroids using agarose micro-molds. Spheroids of primary human chondrocytes (HCH) or human breast cancer cells (MCF-7) were self-assembled for 24 h and then brought together to form an array comprised of two spheroids (one doublet) per well. To quantify spheroid fusogenicity, we developed two assays: (1) an initial tack assay, defined as the minimum amount of time for two spheroids to form a mechanically stable tissue complex or doublet, and (2) a fusion assay, in which we defined and tracked key morphological parameters of the doublets as a function of time using wide-field fluorescence microscopy over a 24 h time-lapse. The initial tack of spheroid fusion was measured by inverting the micro-molds and centrifuging doublets at various time points to assess their connectedness. We found that the initial tack between two spheroids forms rapidly, with the majority of doublets remaining intact after centrifugation following just 30 min of fusion. Over the course of 24 h of fusion, several morphological changes occurred, which were quantified using a custom image analysis pipeline. End-to-end doublet lengths decreased over time, doublet widths decreased for chondrocytes and increased for MCF-7, contact lengths increased over time, and chondrocyte doublets exhibited higher intersphere angles at the end of fusion. We also assessed fusion by measuring the fluorescence intensity at the plane of fusion, which increased over time for both cell types. Interestingly, we observed that doublets moved and rotated in the micro-wells during fusion and this rotation was inhibited by ROCK inhibitor Y-27632 and myosin II inhibitor blebbistatin. Understanding and optimizing tissue fusion is essential for creating larger tissues, organs, or other structures using individual microtissues as building parts.

Harnessing cellular-derived forces in self-assembled microtissues to control the synthesis and alignment of ECM.

The alignment and blend of extracellular matrix (ECM) proteins give a tissue its specific mechanical properties as well as its physiological function. Various tissue engineering methods have taken purified ECM proteins and aligned them into gels, sponges and threads. Although, each of these methods has created aligned ECM, they have had many limitations including loss of hierarchal collagen structure and poor mechanical performance. Here, we have developed a new method to control ECM synthesis using self-assembled cells. Cells were seeded into custom designed, scaffold-free, micro-molds with fixed obstacles that harnessed and directed cell-mediated stresses. Cells within the microtissue reacted to self-generated tension by aligning, elongating, and synthesizing an ECM whose organization was dictated by the strain field that was set by our micro-mold design. We have shown that through cell selection, we can create tissues with aligned collagen II or aligned elastin. We have also demonstrated that these self-assembled microtissues have mechanical properties in the range of natural tissues and that mold design can be used to further tailor these mechanical properties.