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  1. Abstract 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. 
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  2. Abstract

    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.

     
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  3. Mukhopadhyay, Partha (Ed.)
    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. 
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  4. Abstract

    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.

     
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  5. Abstract

    Engineered tissues usually fall short of physiological cell densities and sizes, resulting in limited functional performance. Viability of large tissues is constrained by inadequate diffusion‐driven nutrient exchange. Methods to form large viable tissues are lacking and are constrained by diffusion‐driven nutrient exchange. Here, the use of the Bio‐Pick, Place, and Perfuse (Bio‐P3) is reported, an integrated biofabrication‐bioreactor platform that semiautomatically and rapidly assembles physiologically cell‐dense macrotissues with 100 million cells while being actively perfused. The Bio‐P3 grips, aligns, and stacks prefabricated, scaffold‐free microtissue parts with integrated lumens on a perfusable build‐platform. Parts spontaneously fuse into one continuous macrotissue with perfusable channels. Customizable microtissues are rapidly prepared up to centimeter‐scale with sustained functional performance. Computational models are developed and experimentally validated to elucidate the effects of perfusion rate and tissue geometry on convective nutrient transport in built macrotissues. It is shown that macrotissues constructed from human hepatocellular microtissues maintain geometry and function (albumin and urea secretion) over 5 days. The Bio‐P3 technology fabricates massive solid tissues with high cell numbers and densities to mimic human physiology for preclinical and clinical applications.

     
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