Article

Cryopreservation of Whole Rat Livers by Vitrification and Nanowarming

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Abstract

Liver cryopreservation has the potential to enable indefinite organ banking. This study investigated vitrification—the ice-free cryopreservation of livers in a glass-like state—as a promising alternative to conventional cryopreservation, which uniformly fails due to damage from ice formation or cracking. Our unique “nanowarming” technology, which involves perfusing biospecimens with cryoprotective agents (CPAs) and silica-coated iron oxide nanoparticles (sIONPs) and then, after vitrification, exciting the nanoparticles via radiofrequency waves, enables rewarming of vitrified specimens fast enough to avoid ice formation and uniformly enough to prevent cracking from thermal stresses, thereby addressing the two main failures of conventional cryopreservation. This study demonstrates the ability to load rat livers with both CPA and sIONPs by vascular perfusion, cool them rapidly to an ice-free vitrified state, and rapidly and homogenously rewarm them. While there was some elevation of liver enzymes (Alanine Aminotransferase) and impaired indocyanine green (ICG) excretion, the nanowarmed livers were viable, maintained normal tissue architecture, had preserved vascular endothelium, and demonstrated hepatocyte and organ-level function, including production of bile and hepatocyte uptake of ICG during normothermic reperfusion. These findings suggest that cryopreservation of whole livers via vitrification and nanowarming has the potential to achieve organ banking for transplant and other biomedical applications.

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... Since the chosen radiofrequency waves penetrate the system without significant attenuation, this approach is, in theory, fully scalable to human organs. So far, we and others have published on nanowarming of volumes up to 80 mL CPA mL and 30 mL biological nanowarming at rates up to 50 -100 °C/min [13][14][15][16][17][18] (see Table S1). ...
... We next evaluated the ability to uniformly rewarm liter-scale volumes from the cryogenic vitrified state (< -120°C). Although past studies have rewarmed whole rat and rabbit organs using a 15 kW RF coil (AMF Life Systems) [13,16,18] or other commercial RF coils [15,17], these studies were typically limited to < 30 mL, with only a few nearing ~100 mL volumes (Table S1) [6]. As noted earlier, uniformity in rewarming will depend on the uniformity of the applied field, assuming practically uniform IONP distributions (i.e., through vascular perfusion to the capillary beds). ...
... Sucrose, prepared according to their formulations and composition as listed in Table S2. The preparation of the CPAs was completed by weight-volume percent (M22, VS55) and volumevolume percent (EG+sucrose) using a volumetric flask, as previously reported [13,18]. These CPAs were chosen due to their recent study in multiple organ systems, such as VS55 in rat hearts and kidneys [13,[15][16][17], M22 in rabbit kidneys [33], and 40%v/v EG+0.6M ...
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Organ banking by vitrification could revolutionize transplant medicine. However, vitrification and rewarming have never been demonstrated at the human organ scale. Using modeling and experimentation, we tested the ability to vitrify and rewarm 0.5–3 L volumes of three common cryoprotective agent (CPA) solutions: M22, VS55, and 40% EG+0.6M Sucrose. We first demonstrated our ability to avoid ice formation by convectively cooling faster than the critical cooling rates of these CPAs while also maintaining adequate uniformity to avoid cracking. Vitrification success was then verified by visual, thermometry, and x-ray μCT inspection. M22 and EG+sucrose were successfully vitrified in 0.5 L bags, but only M22 was vitrified at 3 L. VS55 did not vitrify at any tested volumes. As additional proof of principle, we successfully vitrified a porcine liver (~1L) after perfusion loading with 40% EG+0.6M Sucrose. Uniform volumetric rewarming was then achieved in up to 2 L volumes (M22 with ~5 mgFe/mL iron-oxide nanoparticles) using nanowarming, reaching a rate of ~88 °C/min with a newly developed 120 kW radiofrequency (RF) coil operating at 35kA/m and 360kHz. This work demonstrates that human organ scale vitrification and rewarming is physically achievable, thereby contributing to technology that enables human organ banking.
... Despite liver transplantation being the best option for many liver diseases [89,108], the supply of organs falls short of demand [109]. Hypothermic storage limits viability to 12-24 h, restricting the time available for screening and matching. ...
... Slow freezing causes ice crystal formation, making it ineffective for liver preservation due to the organ's complexity. Vitrification is promising but faces challenges related to heat and mass transfer, toxicity, and rewarming [109,112]. Long-term storage and auxiliary transplantation of a pig liver using directional freezing have been reported [93]. ...
... Long-term storage and auxiliary transplantation of a pig liver using directional freezing have been reported [93]. Nanowarming with radio frequency magnetic fields and magnetic nanoparticles improves warming rates and uniformity of warming [113], successfully vitrifying and rewarming the largest organ to date-a rat liver-using ethylene glycol and sucrose [109]. However, scaling this method to human organs is limited by batch size and cost [108]. ...
Article
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Recent years have witnessed significant advancements in the cryopreservation of various tissues and cells, yet several challenges persist. This review evaluates the current state of cryopreservation, focusing on contemporary methods, notable achievements, and ongoing difficulties. Techniques such as slow freezing and vitrification have enabled the successful preservation of diverse biological materials, including embryos and ovarian tissue, marking substantial progress in reproductive medicine and regenerative therapies. These achievements highlight improved post-thaw survival and functionality of cryopreserved samples. However, there are remaining challenges such as ice crystal formation, which can lead to cell damage, and the cryopreservation of larger, more complex tissues and organs. This review also explores the role of cryoprotectants and the importance of optimizing both cooling and warming rates to enhance preservation outcomes. Future research priorities include developing new cryoprotective agents, elucidating the mechanisms of cryoinjury, and refining protocols for preserving complex tissues and organs. This comprehensive overview underscores the transformative potential of cryopreservation in biomedicine, while emphasizing the necessity for ongoing innovation to address existing challenges.
... Nanoparticle drug carriers can provide receptor-mediated cell targeting through the coating of ligands on the nanoparticle specific to cell receptors, endocytosis after specific targeting and selective release of drugs to the endothelium [48][49][50] (Table 1). The use of nanoparticles also provides image-guided (MRI and MPI) targeting validation [51][52][53][54][55]. Thus, targeting endothelial cells through ex vivo perfusion of coated nanoparticle drug carriers is an attractive strategy that can reduce reperfusion injury, initiating an innate immune response to improve graft survival of marginal donors. ...
... This would enable the improved modeling of HSP expression kinetics and the validation of nanoparticle localization through imaging modalities such as MRI and MPI. The washout of nanoparticles from livers has been demonstrated in rat models during hypothermic perfusion [55]. The experimental validation of washout during hyperthermic treatments is needed, as the hypermetabolic state of cells during hyperthermia could result in some endocytosis of nanoparticles, depending on the treatment time and flow velocity. ...
... For example, Rapamycin has demonstrated effective immunosuppression; however, its water insolubility makes it challenging to develop formulations [107]. Several publications cited in this review demonstrate the enhanced stability and efficacy of Rapamycin in a nanoparticle conjugate [55,71,72]. ...
Article
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Liver transplantation is the only treatment for hepatic insufficiency as a result of acute and chronic liver injuries/pathologies that fail to recover. Unfortunately, there remains an enormous and growing gap between organ supply and demand. Although recipients on the liver transplantation waitlist have significantly higher mortality, livers are often not allocated because they are (i) classified as extended criteria or marginal livers and (ii) subjected to longer cold preservation time (>6 h) with a direct correlation of poor outcomes with longer cold ischemia. Downregulating the recipient’s innate immune response to successfully tolerate a graft having longer cold ischemia times or ischemia-reperfusion injury through induction of immune tolerance in the graft and the host would significantly improve organ utilization and post-transplant outcomes. Broadly, technologies proposed for development aim to extend the life of the transplanted liver through post-transplant or recipient conditioning. In this review, we focus on the potential benefits of nanotechnology to provide unique pre-transplant grafting and recipient conditioning of extended criteria donor livers using immune tolerance induction and hyperthermic pre-conditioning.
... The extension of vitrification to organs was shown by Fahy and colleagues in the mid-1980s, when they showed that rabbit kidneys that had been perfused with sufficiently concentrated CPA solutions could be cooled to an ice-free vitrified state. Vitrification of other organs, including rat kidneys (93), hearts (94,95), livers (96), porcine blood vessels (97), and ovaries (98), has also been demonstrated more recently. However, rewarming vitrified organs while preserving viability and function remains challenging. ...
... Since RF waves penetrate biomaterials efficiently, and the nanoparticles are distributed throughout the vasculature, the process of nanowarming is essentially independent of system size and can scale from rodent to human organs. The physical success of this approach has been demonstrated for the blood vessels, heart, kidney, and liver in animal models (93)(94)(95)(96)(97). However, future work will be needed to minimize CPA toxicity for improved organ function and scaling various aspects of the protocols to human organ size. ...
Article
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Organ transplantation remains the only treatment option for patients with end-stage organ failure. The last decade has seen a flurry of activity in improving organ preservation technologies, which promise to increase utilization in a dramatic fashion. They also bring the promise of extending the preservation duration significantly, which opens the doors to sharing organs across local and international boundaries and transforms the field. In this work, we review the recent literature on machine perfusion of livers across various protocols in development and clinical use, in the context of extending the preservation duration. We then review the next generation of technologies that have the potential to further extend the limits and open the door to banking organs, including supercooling, partial freezing, and nanowarming, and outline the opportunities arising in the field for researchers in the short and long term.
... While SCS at 4°C limits preservation time to 8-10 hours, further depression in temperature has the potential to allow inde nite storage (vitri cation) [3,16,17]. Several preservation strategies were developed to take advantage of temperature-induced metabolic depression for the extension of organ storage [18][19][20]. For example, high-subzero techniques such as supercooling and isochoric preservation, whereby ice nucleation is avoided through careful maintenance of mechanical stability and volume, can facilitate multi-day storage [19,21,22]. ...
... For example, high-subzero techniques such as supercooling and isochoric preservation, whereby ice nucleation is avoided through careful maintenance of mechanical stability and volume, can facilitate multi-day storage [19,21,22]. Alternately, vitri cation can stop biological time altogether, reaching temperatures as low as − 196°C, enabling inde nite storage [20]. However, these approaches are limited, with supercooling requiring highly stable storage conditions to prevent ice nucleation, isochoric preservation requiring technologically advanced storage systems for maintenance of constant volume, and vitri cation requiring high-powered radiofrequency coils for rapid nanowarming to enable the warming rates necessary to avoid ice nucleation [23,24]. ...
Preprint
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Preserving organs at subzero temperatures with halted metabolic activity holds the potential to prolong preservation and expand the donor organ pool for transplant. Our group recently introduced partial freezing, a novel approach in high-subzero storage at -15°C, enabling 5 days storage of rodent livers through precise control over ice nucleation and unfrozen fraction. However, increased vascular resistance and tissue edema suggested a need for improvements to extend viable preservation. Here, we describe an optimized partial freezing protocol with key optimizations including increased concentration of propylene glycol to reduce ice recrystallization and maintained osmotic balance through an increase in bovine serum albumin, all while minimizing sheer stress during cryoprotectant unloading with an acclimation period. These approaches ensured the viability during preservation and recovery processes, promoting liver function and ensuring optimal preservation. This was evidenced by increased oxygen consumption, decreased vascular resistance and edema. Ultimately, we show that using the optimized protocol, livers can be stored for 10 days with comparable vascular resistance and lactate levels to 5 days, outperforming the viability of time-matched cold stored livers as the current gold standard. This study represents a significant advancement in expanding organ availability through prolonged preservation and thereby revolutionizing transplant medicine.
... In the past, we and others have shown that nanowarming can rewarm vitrified organs (including kidneys) from animal models with physical success, but only partial biological recovery and no transplant data [17][18][19][20] . We found that CPA damage, not physical injury from vitrification and nanowarming, was the limiting step for biologic and functional recovery. ...
... Normothermic machine perfusion of kidneys was performed as we have reported for livers 19 with brief modifications. The arterial perfusate was a modified Krebs-Henseleit Bicarbonate Buffer (KHB, 120 mM NaCl, 4.7 mM KCl, 1.2 mM KH 2 PO 4 ,1.5 mM CaCl 2 , 1.2 mM MgSO 4 , 25 mM NaHCO 3 , 0.1 mM EDTA) that was supplemented with 5.54 mM glucose, 5 g/L BSA, 1 g/L amino acids, and 0.5 g/L creatinine. ...
Article
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Banking cryopreserved organs could transform transplantation into a planned procedure that more equitably reaches patients regardless of geographical and time constraints. Previous organ cryopreservation attempts have failed primarily due to ice formation, but a promising alternative is vitrification, or the rapid cooling of organs to a stable, ice-free, glass-like state. However, rewarming of vitrified organs can similarly fail due to ice crystallization if rewarming is too slow or cracking from thermal stress if rewarming is not uniform. Here we use “nanowarming,” which employs alternating magnetic fields to heat nanoparticles within the organ vasculature, to achieve both rapid and uniform warming, after which the nanoparticles are removed by perfusion. We show that vitrified kidneys can be cryogenically stored (up to 100 days) and successfully recovered by nanowarming to allow transplantation and restore life-sustaining full renal function in nephrectomized recipients in a male rat model. Scaling this technology may one day enable organ banking for improved transplantation.
... Specimens have to be stored below −130 °C to avoid devitrification. Functional cryopreservation by vitrification is an active field of basic research, and has been successfully applied to the rat kidney and liver [31,32]. Functional cryopreservation has not yet been demonstrated for the whole adult mammalian brain, let alone body [33][34][35][36][37][38][39][40]. ...
Article
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Structural brain preservation (SBP) and classical cryonics are techniques aimed at preserving the human brain for potential future applications. Reluctant public discourse around these techniques may be explained with intuitive aversions identified by moral psychology. In the first part of the paper, we conjecture the existence of a self-sustaining cycle of moral condemnation of SBP and classical cryonics due to quick, affect-laden moral intuitions. In the second part, we propose an alternative framing of SBP and classical cryonics through a thought experiment featuring a time machine metaphor called "Schrödinger’s chrono-cat", which might avoid triggering aversive moral intuitions and foster public discourse. We discuss the limitations of this framing and its consequences.
... Since then, the scope of vitrification has broadened to include applications in reproductive medicine and organ preservation [177][178][179][180]. For example, rat livers have been vitrified and demonstrated preserved tissue architecture after thawing [158], and small animal hearts and kidneys have also been successfully vitrified [156,181]. A notable achievement by Fahy's team was the successful vitrification of a rabbit kidney, which functioned for 48 days posttransplant, despite challenges such as persistently elevated creatinine levels and lethargy [182]. ...
Article
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Organ transplantation, a critical treatment for end-stage organ failure, has witnessed significant advancements due to the integration of improved surgical techniques, im-munosuppressive therapies, and donor-recipient matching. This review explores the progress of organ preservation, focusing on the shift from static cold storage (SCS) to advanced machine perfusion techniques such as hypothermic (HMP) and normo-thermic machine perfusion (NMP). Although SCS has been the standard approach, its limitations in preserving marginal organs and preventing ischemia-reperfusion injury (IRI) have led to the adoption of HMP and NMP. HMP, which is now the gold standard for high-risk donor kidneys, reduces metabolic activity and improves posttransplant outcomes. NMP allows real-time organ viability assessment and reconditioning, especially for liver transplants. Controlled oxygenated rewarming further minimizes IRI by addressing mitochondrial dysfunction. The review also highlights the potential of cryo-preservation for long-term organ storage, despite challenges with ice formation. These advances are crucial for expanding the donor pool, improving transplant success rates, and addressing organ shortages. Continued innovation is necessary to meet the growing demands of transplantation and save more lives.
... such as supercooling and isochoric preservation, whereby ice nucleation is avoided through careful maintenance of mechanical stability and volume, can facilitate multi-day storage 19,21,22 . Alternately, vitrification can stop biological time altogether, reaching temperatures as low as − 196 °C and enabling indefinite storage 20 . However, these approaches are limited, with supercooling requiring highly stable storage conditions to prevent ice nucleation, isochoric preservation requiring technologically advanced storage systems for maintenance of constant volume, and vitrification requiring high-powered radiofrequency coils for rapid nanowarming to enable the warming rates necessary to avoid ice nucleation 23,24 . ...
Article
Full-text available
Preserving organs at subzero temperatures with halted metabolic activity holds the potential to prolong preservation and expand the donor organ pool for transplant. Our group recently introduced partial freezing, a novel approach in high-subzero storage at -15 °C, enabling 5-day storage of rodent livers through precise control over ice nucleation and unfrozen fraction. However, increased vascular resistance and tissue edema suggested a need for improvements to extend viable preservation. Here, we describe an optimized partial freezing protocol with key optimizations, including an increased concentration of polyethylene glycol (PEG) to enhance membrane stability while minimizing shear stress during cryoprotectant unloading with an acclimation period and a maintained osmotic balance through an increase in bovine serum albumin (BSA). These approaches ensured the viability during preservation and recovery processes, promoting liver function and ensuring optimal preservation. This was evidenced by increased oxygen consumption, decreased vascular resistance, and edema. Ultimately, we show that using the optimized protocol, livers can be stored for 10 days with comparable vascular resistance and lactate levels to 5 days, outperforming the viability of time-matched static cold stored (SCS) livers as the current gold standard. This study represents a significant advancement in expanding organ availability through prolonged preservation, thereby revolutionizing transplant medicine.
... remarkable predictive power with knowledge only of the individual molar masses and volumes of the pure components at room temperature and pressure, data that are broadly available for most materials miscible with water. This ability to accurately predict homogeneous nucleation temperatures as a function of readily available solute properties may save researchers significant experimental effort in future characterization of binary aqueous solutions, with applications in the study of supercooling-and vitrification-based cryopreservation processes, [18][19][20][21][22] agricultural cold storage processes, [23][24][25] atmospheric nucleation processes, 26 etc. More generally, the physical interpretation of the role of entropy provided here may help to establish a theoretical foundation from which to enable the rational design and predictive synthesis of aqueous solutions with desired nucleation behaviors. ...
Article
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The nucleation of ice from aqueous solutions is a process essential to myriad environmental and industrial processes, but the physical factors affecting the capacity of different solutes to depress the homogeneous nucleation temperature of ice are yet poorly understood. In this work, we demonstrate that for many binary aqueous solutions of non-ionic solutes, this depression is dominated by the entropy of the liquid phase. Employing the classic Turnbull interpretation of the interfacial free energy γ∼TSliquid−Ssolid and estimating solution entropies with a Flory-style modification of the ideal entropy of mixing that accounts for solute size effects, we demonstrate that mixing entropy alone predicts experimental homogeneous nucleation temperatures across a wide variety of non-ionic solutions. We anticipate that this physical insight will not only enhance a fundamental understanding of homogeneous nucleation processes across fields but also open new avenues to the rational design of aqueous solutions for desired nucleation behaviors.
... Nonetheless, prolonged storage is well known to increase the risk of graft dysfunction that contributes to complications post-transplantation [7,9]. Encouragingly, groups have made considerable progress developing improved preservation protocols to minimize preservation-related injury and prolong storage duration in ex vivo whole livers [10,11,12,13,14,15]. However, although these studies have increased our knowledge of how speci c preservation protocols can lead to greater liver graft outcomes, whole organ studies provide limited options for deeper investigations into mechanisms of cellular survival or damage. ...
Preprint
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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.
... The rewarming rate increased with the increase in nanoparticle concentration. Nanoparticles, including mesoporous silicacoated iron oxide nanoparticles, 50 microporous silica-coated iron oxide nanoparticles, 78 and commercial iron oxide nanoparticles coated with phosphonate, 79 were later used for rewarming organs, including rat hearts, kidney, and livers, 35,51,52 effectively demonstrating the potential nanowarming application in human organ cryopreservation. Zhao applied novel RF magnetic rewarming, and the addition of iron oxide nanoparticles significantly increased the rewarming and cell survival rate. ...
Article
Cryopreservation is the most effective technology for the long-term preservation of biological materials, including cells, tissues, and even organs in the future. The process of cooling and rewarming is essential to the successful preservation of biological materials. One of the critical problems in the development of cryopreservation is the optimization of effective rewarming technologies. This article reviewed rewarming methods, including traditional boundary rewarming commonly used for small-volume biological materials and other advanced techniques that could be potentially feasible for organ preservation in the future. The review focused on various rewarming technique principles, typical applications, and their possible limitations for cryopreservation of biological materials. This article introduced nanowarming methods in the progressing optimization and the possible difficulties. The trends of novel rewarming methods were discussed, and suggestions were given for future development.
... Successful vascular CPA perfusion protocols have been developed and optimized through years of primarily empirical testing. The first vitrification of a whole organ (rabbit kidney) was achieved by Fahy et al. in [7] and has been more recently demonstrated in additional organ systems [8,12,38,39]. Decades of effort in the kidney has refined a general protocol that allows for successful loading and unloading with a functioning kidney at the end. We aim to build off the principles demonstrated through those efforts, and leverage established biological modeling concepts developed in cells and tissues, to demonstrate a general mathematical formulation that allows the systematic optimization of CPA perfusion in organs. ...
Article
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To extend the preservation of donor hearts beyond the current 4–6 h, this paper explores heart cryopreservation by vitrification—cryogenic storage in a glass‐like state. While organ vitrification is made possible by using cryoprotective agents (CPA) that inhibit ice during cooling, failure occurs during convective rewarming due to slow and non‐uniform rewarming which causes ice crystallization and/or cracking. Here an alternative, “nanowarming”, which uses silica‐coated iron oxide nanoparticles (sIONPs) perfusion loaded through the vasculature is explored, that allows a radiofrequency coil to rewarm the organ quickly and uniformly to avoid convective failures. Nanowarming has been applied to cells and tissues, and a proof of principle study suggests it is possible in the heart, but proper physical and biological characterization especially in organs is still lacking. Here, using a rat heart model, controlled machine perfusion loading and unloading of CPA and sIONPs, cooling to a vitrified state, and fast and uniform nanowarming without crystallization or cracking is demonstrated. Further, nanowarmed hearts maintain histologic appearance and endothelial integrity superior to convective rewarming and indistinguishable from CPA load/unload control hearts while showing some promising organ‐level (electrical) functional activity. This work demonstrates physically successful heart vitrification and nanowarming and that biological outcomes can be expected to improve by reducing or eliminating CPA toxicity during loading and unloading.
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The inability to preserve vascular organs beyond several hours contributes to the scarcity of organs for transplantation1,2. Standard hypothermic preservation at +4 °C (refs. 1,3) limits liver preservation to less than 12 h. Our group previously showed that supercooled ice-free storage at -6 °C can extend viable preservation of rat livers4,5 However, scaling supercooling preservation to human organs is intrinsically limited because of volume-dependent stochastic ice formation. Here, we describe an improved supercooling protocol that averts freezing of human livers by minimizing favorable sites of ice nucleation and homogeneous preconditioning with protective agents during machine perfusion. We show that human livers can be stored at -4 °C with supercooling followed by subnormothermic machine perfusion, effectively extending the ex vivo life of the organ by 27 h. We show that viability of livers before and after supercooling is unchanged, and that after supercooling livers can withstand the stress of simulated transplantation by ex vivo normothermic reperfusion with blood.
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A plethora of human hepatocyte-based in vitro systems for toxicity testing has been developed over the past few years. These systems are either directly derived from liver tissue or have been generated through stem cell technology. A wide variety of parameters is currently used to demonstrate the acquisition of in vivo-like hepatocellular physiology and toxicity in such novel in vitro systems. This frequently leads to flawed claims regarding applicability and may impede comparison between in vitro systems. A possible solution lies in defining a set of consensus criteria for proper benchmarking. A proposal for characterization of hepatocyte-based in vitro systems for toxicity screening is made in this paper and consists of testing critical features of viability, morphology, functionality and toxicity.
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Liver transplantation represents the standard treatment for people with an end-stage liver disease and some liver-based metabolic disorders; however, shortage of liver donor tissues limits its availability. Furthermore, whole liver replacement eliminates the possibility of using native liver as a possible target for future gene therapy in case of liver-based metabolic defects. Cell therapy has emerged as a potential alternative, as cells can provide the hepatic functions and engraft in the liver parenchyma. Various options have been proposed, including human or other species hepatocytes, hepatocyte-like cells derived from stem cells or more futuristic alternatives, such as combination therapies with different cell types, organoids and cell–biomaterial combinations. In this review, we aim to give an overview of the cell therapies developed so far, highlighting preclinical and/or clinical achievements as well as the limitations that need to be overcome to make them fully effective and safe for clinical applications. This article is part of the theme issue ‘Designer human tissue: coming to a lab near you’.
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Liver transplantation is a highly successful treatment, but is severely limited by the shortage in donor organs. However, many potential donor organs cannot be used; this is because sub-optimal livers do not tolerate conventional cold storage and there is no reliable way to assess organ viability preoperatively. Normothermic machine perfusion maintains the liver in a physiological state, avoids cooling and allows recovery and functional testing. Here we show that, in a randomized trial with 220 liver transplantations, compared to conventional static cold storage, normothermic preservation is associated with a 50% lower level of graft injury, measured by hepatocellular enzyme release, despite a 50% lower rate of organ discard and a 54% longer mean preservation time. There was no significant difference in bile duct complications, graft survival or survival of the patient. If translated to clinical practice, these results would have a major impact on liver transplant outcomes and waiting list mortality.
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In 2016 over 5 million reconstructive procedures were performed in the USA. The recent successes of clinical vascularized composite allotransplantations (VCA), hand and face transplantations included, established the tremendous potential of these life-enhancing reconstructions. Nevertheless, Due to limited availability and lifelong immunosuppression, application is limited. Long term banking of composite transplants may increase the availability of esthetically compatible parts with partial or complete HLA matching, reducing the risk of rejection and the immunosuppressive burden. The study purpose was to develop efficient protocols for the cryopreservation and transplantation of a complete rodent limb. Directional freezing is a method in which a sample is cooled at a constant velocity linear temperature gradient enabling precise control of the process and ice crystal formation. Vitrification is an alternative cryopreservation method in which the sample solidifies without the formation of ice crystals. Testing both methods on a rat hindlimb composite tissue transplantation model, we found reliable, reproducible and stable ways to preserve composite tissue. We believe that with further research and development cryopreservation may lead to composite tissue "banks". This may lead to a paradigm shift from few and far apart emergent surgeries to wide scale, well planned, and better controlled elective surgeries. This article is protected by copyright. All rights reserved.
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The OPTN Deceased Donor Potential Study (DDPS), funded by the Health Resources and Services Administration, characterized the current pool of potential deceased donors and estimated changes through 2020. The goal was to inform policy development and suggest practice changes designed to increase the number of donors and organ transplants. Donor estimates used filtering methodologies applied to datasets from the OPTN, the National Center for Health Statistics, and the Agency for Healthcare Research and Quality and used these estimates with the number of actual donors to estimate the potential donor pool through 2020. Projected growth of the donor pool was 0.5% per year through 2020. Potential donor estimates suggested unrealized donor potential across all demographic groups, with the most significant unrealized potential (70%) in the 50 to 75 year old age group and potential Donation after Circulatory Death (DCD) donors. Actual transplants that may be realized from potential donors in these categories is constrained by confounding medical comorbidities not identified in administrative databases and by limiting utilization practices for organs from DCD donors. Policy, regulatory, and practice changes encouraging organ procurement and transplantation of a broader population of potential donors may be required to increase transplant numbers in the United States. This article is protected by copyright. All rights reserved.
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Work over several decades has laid solid foundations for the advancement of liver cell therapy. To date liver cell therapy in people has taken the form of hepatocyte transplantation for metabolic disorders with a hepatic basis, and for acute or chronic liver failure. Although clinical trials using various types of autologous cells have been implemented to promote liver regeneration or reduce liver fibrosis, clear evidence of therapeutic benefits have so far been lacking. Cell types that have shown efficacy in preclinical models include hepatocytes, liver sinusoidal endothelial cells, mesenchymal stem cells, endothelial progenitor cells, and macrophages. However, positive results in animal models have not always translated through to successful clinical therapies and more realistic preclinical models need to be developed. Studies defining the optimal repopulation by transplanted cells, including routes of cell transplantation, superior engraftment and proliferation of transplanted cells, as well as optimal immunosuppression regimens are required. Tissue engineering approaches to transplant cells in extrahepatic locations have also been proposed. The derivation of hepatocytes from pluripotent or reprogramed cells raises hope that donor organ and cell shortages could be overcome in the future. Critical hurdles to be overcome include the production of hepatocytes from pluripotent cells with equal functional capacity to primary hepatocytes and long-term phenotypic stability in vivo. Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
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The realization of long-term human organ preservation will have groundbreaking effects on the current practice of transplantation. Herein we present a new technique based on subzero nonfreezing preservation and extracorporeal machine perfusion that allows transplantation of rat livers preserved for up to four days, thereby tripling the viable preservation duration.
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Species differences in drug metabolism and drug toxicity are well-established phenomenon. As a result, the classical paradigm of preclinical testing of drug candidates in animals may not be appropriate. One preclinical approach to evaluate human drug properties, especially ADMET (absorption, disposition, metabolism, elimination, and toxicity) properties, is to apply in vitro experimental systems with relevant human properties. The latest advances include the use of human hepatocytes to evaluate hepatic uptake, metabolism, efflux and toxicity. Successful cryopreservation of human hepatocytes to retain high viability, metabolic capacity, as well as the ability to be cultured allow routine application of this relevant experimental system. This review summarizes the latest findings on human hepatocytes isolation, cryopreservation, culturing, as well as application in the evaluation of metabolic stability, metabolite profiling, hepatic uptake and efflux, metabolic drug-drug interactions, and drug toxicity. The use of hepatocyte to evaluate the role of metabolism in drug toxicity represents a major advance in drug toxicity evaluation. The use of the novel integrated discrete multiple organ co-culture (IdMOC) system allows the evaluation of the role of hepatic metabolism on nonhepatic toxicity.
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Cryopreservation would potentially very much facilitate the inventory control and distribution of laboratory-produced organs and tissues. Although simple freezing methods are effective for many simple tissues, bioartificial organs and complex tissue constructs may be unacceptably altered by ice formation and dissolution. Vitrification, in which the liquids in a living system are converted into the glassy state at low temperatures, provides a potential alternative to freezing that can in principle avoid ice formation altogether. The present report provides a brief overview of the problem of renal vitrification. We report here the detailed case history of a rabbit kidney that survived vitrification and subsequent transplantation, a case that demonstrates both the fundamental feasibility of complex system vitrification and the obstacles that must still be overcome, of which the chief one in the case of the kidney is adequate distribution of cryoprotectant to the renal medulla. Medullary equilibration can be monitored by monitoring urine concentrations of cryoprotectant, and urine flow rate correlates with vitrification solution viscosity and the speed of equilibration. By taking these factors into account and by using higher perfusion pressures as per the case of the kidney that survived vitrification, it is becoming possible to design protocols for equilibrating kidneys that protect against both devitrification and excessive cryoprotectant toxicity.
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This is the first report on low-temperature preservation of self-assembled cell aggregates by vitrification, which is both a time- and cost-effective technology. We developed an effective protocol for vitrification (ice-free cryopreservation) of hepatocyte spheroids that employs rapid stepwise exposure to cryoprotectants (10.5 min) at room temperature and direct immersion into liquid nitrogen (-196 degrees C). For this, three vitrification solutions (VS) were formulated and their effects on vitrified-warmed spheroids were examined. Cryopreservation using ethylene glycol (EG)-sucrose VS showed excellent preservation capability whereby highly preserved cell viability and integrity of vitrified spheroids were observed, through confocal and scanning electron microscopy imaging, when compared to untreated control. The metabolic functions of EG-sucrose VS-cryopreserved spheroids, as assessed by urea production and albumin secretion, were not significantly different from those of control within the same day of observation. In both the vitrification and control groups, albumin secretion was consistently high, ranging from 47.57 +/- 14.39 to 70.38 +/- 11.29 microg/10(6) cells and from 56.84 +/- 14.48 to 71.79 +/- 16.65 microg/10(6) cells, respectively, and urea production gradually increased through the culture period. The efficacy of vitrification procedure in preserving the functional ability of hepatocyte spheroids was not improved by introduction of a second penetrating cryoprotectant, 1,2-propanediol (PD). Spheroids cryopreserved with EG-PD-sucrose VS showed maintained cell viability; however, in continuous culture, levels of both metabolic functions were lower than those cryopreserved with EG-sucrose VS. EG-PD VS, in which nonpenetrating cryoprotectant (sucrose) was excluded, provided poor protection to spheroids during cryopreservation. This study demonstrated that sucrose plays an important role in the effective vitrification of self-assembled cell aggregates. In a broad view, the excellent results obtained suggest that the developed vitrification strategy, which is an alternative to freezing, may be effectively used as a platform technology in the field of cell transplantation.
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Preservation of vascularized organs, such as the liver, is limited to 24 h before destructive processes disqualify them for transplantation. This narrow window of opportunity prevents the performance of optimal pathogen screening and matching tests and possibly results in the need for retransplantation. Numerous problems are associated with freezing and thawing a whole liver while preserving its viability upon thawing, including complicated geometry, poor heat transfer, release of latent heat, and the difficulty of generating a uniform cooling rate. On the basis of our past success with sheep ovaries, we have now applied our novel freezing technique to a larger solid organ, the liver. Whole rat and pig livers were frozen and thawed using directional solidification apparatus, and viability of these livers was tested by means of integrity and functionality in vitro and in auxiliary liver transplantation. The thawed rat and porcine livers were intact and demonstrated >80% viability. Histology revealed normal architecture. Bile production and blood flow following auxiliary transplantation were normal as well. Our encouraging results in applying this novel cryopreservation technique in rat and pig livers suggest that this method may enable better human organ donor-recipient matching in the future.
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Cells can endure storage at low temperatures such as--196 degrees C for centuries. The challenge is to determine how they can survive both the cooling to such temperatures and the subsequent return to physiological conditions. A major factor is whether they freeze intracellularly. They do so if cooling is too rapid, because with rapid cooling insufficient cell water is removed osmotically to eliminate supercooling. Equations have been developed that describe the kinetics of this water loss and permit one to predict the likelihood of intracellular freezing as a function of cooling rate. Such predictions agree well with observations. Although the avoidance of intracellular freezing is usually necessary for survival, it is not sufficient. Slow freezing itself can be injurious. As ice forms outside the cell, the residual unfrozen medium forms channels of decreasing size and increasing solute concentration. The cells lie in the channels and shrink in osmotic response to the rising solute concentration. Prior theories have ascribed slow freezing injury to the concentration of solutes or the cell shrinkage. Recent experiments, however, indicate that the damage is due more to the decrease in the size of the unfrozen channels. This new view of the mechanism of slow freezing injury ought to facilitate the development of procedures for the preservation of complex assemblages of cells of biological, medical, and agricultural significance.
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We have used microencapsulated hepatocytes as model to develop a method of vitreous cryopreservation of large quantities of cell-containing constructs. The method included a pre-equilibration procedure in which the amount of penetrating cryoprotectant was gradually increased by 15% in each step. The optimal vitrification solution consists of 40% ethylene glycol and 0.6M sucrose. The concentration of 1M sucrose used for the first dilution solution with subsequent decrease of sucrose concentration to 0.7 M sucrose and by 0.2-0.15M for each subsequent step. This sucrose dilution procedure had no adverse effect on cell functions. Three cooling rates (400 degrees C/min and above) and three warming rates (650 degrees C/min and above), in combination with the proposed vitrification solution, were equally effective. The optimization of the procedure and solutions allow microencapsulated hepatocytes to be preserved with almost 100% retention of cell functions and no detectable damage to the fragile microcapsules. The de-linking of the cooling/warming rates with the effectiveness of vitrification potentially paves the way for large scale cryopreservation of complex tissue engineered constructs.
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Cryoprotective agents (CPAs) are routinely applied in cryopreservation protocols to achieve the vitrified state thereby avoiding the damaging effects of ice crystals. Once the CPA has been added, the system needs to cool at a rate ≥ critical cooling rate (CCR) to avoid ice crystallization and successfully enter the vitrified state. Subsequently, upon warming the system needs to meet or exceed a critical warming rate (CWR), often one to two orders of magnitude higher than the CCR, to avoid ice formation and return the system to physiological temperatures for use. Many experimental and theoretical studies have been published on CCRs and CWRs, and correlation for these rates as a function of concentration has been explored for some single component CPAs, but not the CPA cocktails which are commonly used in tissue and organ cryopreservation. In this paper, we summarize the available data of CCRs and CWRs for a variety of CPAs, and suggest a convenient mathematical expression for CCR and CWR that can guide general use for cryoprotective protocol, but also highlights the critical need for further study on CPA cocktails and tissue systems in which CPAs may behave differently and/or may not be fully equilibrated to the loaded CPA.
Article
Background Transplantation of livers obtained from donors after circulatory death is associated with an increased risk of nonanastomotic biliary strictures. Hypothermic oxygenated machine perfusion of livers may reduce the incidence of biliary complications, but data from prospective, controlled studies are limited. Methods In this multicenter, controlled trial, we randomly assigned patients who were undergoing transplantation of a liver obtained from a donor after circulatory death to receive that liver either after hypothermic oxygenated machine perfusion (machine-perfusion group) or after conventional static cold storage alone (control group). The primary end point was the incidence of nonanastomotic biliary strictures within 6 months after transplantation. Secondary end points included other graft-related and general complications. Results A total of 160 patients were enrolled, of whom 78 received a machine-perfused liver and 78 received a liver after static cold storage only (4 patients did not receive a liver in this trial). Nonanastomotic biliary strictures occurred in 6% of the patients in the machine-perfusion group and in 18% of those in the control group (risk ratio, 0.36; 95% confidence interval [CI], 0.14 to 0.94; P=0.03). Postreperfusion syndrome occurred in 12% of the recipients of a machine-perfused liver and in 27% of those in the control group (risk ratio, 0.43; 95% CI, 0.20 to 0.91). Early allograft dysfunction occurred in 26% of the machine-perfused livers, as compared with 40% of control livers (risk ratio, 0.61; 95% CI, 0.39 to 0.96). The cumulative number of treatments for nonanastomotic biliary strictures was lower by a factor of almost 4 after machine perfusion, as compared with control. The incidence of adverse events was similar in the two groups. Conclusions Hypothermic oxygenated machine perfusion led to a lower risk of nonanastomotic biliary strictures following the transplantation of livers obtained from donors after circulatory death than conventional static cold storage. (Funded by Fonds NutsOhra; DHOPE-DCD ClinicalTrials.gov number, NCT02584283.)
Article
Objective: Normothermic machine perfusion (NMP) enables optimized ex-vivo preservation of a donor liver in a normal physiologic state. The impact of this emerging technology on donor liver utilization has yet to be assessed. Summary background data: NMP of the donor liver and ex-vivo enhancement of its function has been envisioned for decades, however only with recent technological advances have devices been suitable for transition to clinical practice. The present study examines the effect NMP on liver utilization in the United States. Methods: The United Network for Organ Sharing database was queried to identify deceased donor livers procured from 2016 to 2019 (n = 30596). Donor livers were divided by preservation method: standard cold-static preservation (COLD, n = 30,368) versus NMP (n = 228). Donor and recipient risk factors, liver disposition, and discard reasons were analyzed. The primary outcome was liver discard rate between 2 groups. Results: A total of 4037 livers were discarded. The NMP group had a 3.5% discard rate versus 13.3% in the COLD group (P < 0.001), and this was despite NMP donors being older (47.7 vs 39.5 years, P < 0.0001), more frequently donation after cardiac death (DCD) (18% vs 7%, P < 0.001), and having a greater donor risk index (1.6 vs 1.5, P < 0.05). The most common reasons for liver discard in the COLD group were biopsy findings (38%), DCD warm ischemic time (11%), and prolonged preservation time (10%). Survival analysis, following propensity score matching, found no significant difference in 1-year overall survival between recipients of NMP versus COLD livers. Conclusions: NMP reduces the discard rate of procured livers despite its use in donors traditionally considered of more marginal quality. NMP maintains excellent graft and patient survival. Broader application of NMP technology holds the potential to generate a significant number of additional liver grafts for transplantation every year, thus greatly reducing the nationwide disparity between supply and demand.
Chapter
Several reviews of the biophysical principles of cryobiology have been published recently and the interested reader is referred particularly to Mazur (1) for a detailed discussion or to Pegg (2) for an introductory account. In this chapter the science of cryopreservation will be approached in a more practical and applied way. We know that freezing living cells is normally lethal, a fact that is put to practical use in cryosurgery. But we also know that cooling slows the chemical processes both of life and of decay and this has lead to the idea that “suspended animation” might be achieved by cooling. Successful preservation will then depend on reducing the destructive action of ice but allowing the protective effect of low temperatures, such that any damaging effects are greatly outweighed by the protective effects. This is a complicated matter: many structures and processes are temperature-dependent and cooling has complex effects that combine to create conditions that are far removed from normal physiology. When cells are cooled much below 0°C, the effects are normally dominated by the freezing of water, which typically constitutes at least 80% of tissue mass. It was generally thought that the ice crystals were directly responsible for damage rather than the concentration of solutes in the progressively diminishing liquid phase as cooling proceeded.
Article
Cryopreservation has become a central technology in many areas of clinical medicine, biotechnology, and species conservation within both plant and animal biology. Cryoprotective agents (CPAs) invariably play key roles in allowing cells to be processed for storage at deep cryogenic Temperatures and to be recovered with high levels of appropriate functionality. As such, these CPA solutes possess a wide range of metabolic and biophysical effects that are both necessary for their modes of action, and potentially complicating for cell biological function. Early successes with cryopreservation were achieved by empirical methodology for choosing and applying CPAs. In recent decades, it has been possible to assemble objective information about CPA modes of action and to optimize their application to living systems, but there still remain significant gaps in our understanding. This review sets out the current status on the biological and chemical knowledge surrounding CPAs, and the conflicting effects of protection versus toxicity resulting from the use of these solutes, which are often required in molar concentrations, far exceeding levels found in normal metabolism. The biophysical properties of CPAs that allow them to facilitate different approaches to cryogenic storage, including vitrification, are highlighted. The topics are discussed with reference to the historical background of applying CPAs, and the relevance of cryoprotective solutes in natural freeze tolerant organisms. Improved cryopreservation success will be an essential step in many future areas such as regenerative medicine, seed banking, or stem cell technology. To achieve this, we will need to further improve our understanding of cryobiology, where better and safer CPAs will be key requirements.
Article
Purpose of review: Preservation of the liver via normothermic machine perfusion (NMP) is rapidly becoming an area of great academic and clinical interest. This review focuses on the benefits and limitations of NMP and where the role for static cold storage may lie. Recent findings: Clinical studies have recently been published reporting the use of NMP in liver preservation for transplantation. They have described the technology to be well tolerated and feasible with potentially improved posttransplant outcomes. NMP facilitates extended preservation times as well as the potential to increase organ utilization through viability assessment and regeneration. However, this technology is considerably more costly than cold storage and carries significant logistical challenges. Cold storage remains the gold standard preservation for standard criteria livers with good long-term patient and graft survival. Summary: NMP is an exciting new technological advancement in liver preservation, which is likely to have a positive impact in liver transplantation. However, randomized controlled trials are required to justify its inclusion into standard practice and provide evidence to support its efficacy.
Article
Vitrification, a kinetic process of liquid solidification into glass, poses many potential benefits for tissue cryopreservation including indefinite storage, banking, and facilitation of tissue matching for transplantation. To date, however, successful rewarming of tissues vitrified in VS55, a cryoprotectant solution, can only be achieved by convective warming of small volumes on the order of 1 ml. Successful rewarming requires both uniform and fast rates to reduce thermal mechanical stress and cracks, and to prevent rewarming phase crystallization. We present a scalable nanowarming technology for 1- to 80-ml samples using radiofrequency-excited mesoporous silica–coated iron oxide nanoparticles in VS55. Advanced imaging including sweep imaging with Fourier transform and microcomputed tomography was used to verify loading and unloading of VS55 and nanoparticles and successful vitrification of porcine arteries. Nanowarming was then used to demonstrate uniform and rapid rewarming at >130°C/min in both physical (1 to 80 ml) and biological systems including human dermal fibroblast cells, porcine arteries and porcine aortic heart valve leaflet tissues (1 to 50 ml). Nanowarming yielded viability that matched control and/or exceeded gold standard convective warming in 1- to 50-ml systems, and improved viability compared to slow-warmed (crystallized) samples. Last, biomechanical testing displayed no significant biomechanical property changes in blood vessel length or elastic modulus after nanowarming compared to untreated fresh control porcine arteries. In aggregate, these results demonstrate new physical and biological evidence that nanowarming can improve the outcome of vitrified cryogenic storage of tissues in larger sample volumes.
Article
Purpose: To use contrast based on longitudinal relaxation times (T1 ) or rates (R1 ) to quantify the biodistribution of iron oxide nanoparticles (IONPs), which are of interest for hyperthermia therapy, cell targeting, and drug delivery, within primary clearance organs. Methods: Mesoporous silica-coated IONPs (msIONPs) were intravenously injected into 15 naïve mice. Imaging and mapping of the longitudinal relaxation rate constant at 24 h or 1 week postinjection were performed with an echoless pulse sequence (SWIFT). Alternating magnetic field heating measurements were also performed on ex vivo tissues. Results: Signal enhancement from positive T1 contrast caused by IONPs was observed and quantified in vivo in liver, spleen, and kidney at concentrations up to 3.2 mg Fe/(g tissue wt.) (61 mM Fe). In most cases, each organ had a linear correlation between the R1 and the tissue iron concentration despite variations in intra-organ distribution, degradation, and IONP surface charge. Linear correlation between R1 and volumetric SAR in hyperthermia therapy was observed. Conclusion: The linear dependence between R1 and tissue iron concentration in major organs allows quantitative monitoring of IONP biodistribution in a dosage range relevant to magnetic hyperthermia applications, which falls into the concentration gap between CT and conventional MRI techniques. Magn Reson Med 78:702-712, 2017. © 2016 International Society for Magnetic Resonance in Medicine.
Article
While vitrified cryopreservation holds great promise, practical application has been limited to smaller systems (cells and thin tissues) due to diffusive heat and mass transfer limitations, which are typically manifested as devitrification and cracking failures during thaw. Here then we describe a new approach for rapidly and uniformly heating cryopreserved biospecimens with radiofrequency (RF) excited magnetic nanoparticles (mNPs). Importantly, heating rates can be increased several fold over conventional boundary heating techniques and are independent of sample size. Initial differential scanning calorimetry studies indicate that the addition of the mNPs has minimal impact on the freeze-thaw behavior of the cryoprotectant systems themselves. Then proof-of-principle experiments in aqueous and cryoprotectant solutions demonstrate the ability to heat at rates high enough to mitigate or eliminate devitrification (hundreds of °C/min) and scaled heat transfer modeling is used to illustrate the potential of this innovative approach. Finally, X-ray micro-computed-tomography (micro-CT) is investigated as a planning and quality control tool, where the density-based measurements are able to quantify changes in cryoprotectant concentration, mNP concentration, and the frozen state (i.e. crystallized versus vitrified).
Article
1. Cryopreservation of precision-cut tissue slices (PCTS) would have many advantages for drug development and would encourage more extensive use of the PCTS preparation. 2. Three methods have been studied to date: slow freezing, fast freezing, and vitrification. 3. Slow freezing can be very effective for some PCTS but is devastating to rat liver PCTS. Fast freezing can be successful for rat liver PCTS but is devastating to renal PCTS and has given inconsistent results even for rat liver PCTS. Vitrification has been effective for some slice systems but less effective for rat liver PCTS. Rat liver PCTS appear to be particularly difficult to cryopreserve well. 4. The general cryobiological principles of slow freezing, rapid freezing, and vitrification are reviewed. The empirical literature on the cryopreservation of PCTS has not taken sufficient account of these principles, and may, for example, include the effects of easily preventable osmotic injury. 5. More attention is needed to the effects of cryopreservation on specific cell types within PCTS and to the general integrity and viability of cryopreserved PCTS. Drug metabolism as a sole endpoint of study can be highly misleading. 6. Better application of cryobiological principles may enable improved results in the future.
Article
Successful vitrification of organ slices is hampered by both osmotic stress and chemical toxicity of cryoprotective agents (CPAs). In the present study, we focused on the effect of osmotic stress on the viability of precision-cut liver slices (PCLS) by comparing different CPA solutions and different methods of loading and unloading the slices with the CPAs. For this purpose, we developed a gradient method to load and unload CPAs with the intention of minimizing sudden changes in osmolarity and thereby avoiding osmotic stress in the slices in comparison with the commonly used step-wise loading/unloading approach. With this gradient method, the CPA solution was introduced at a constant rate into a specially designed mixing chamber containing the slices. We showed that immediate mixing of the infused CPA and the chamber constituents occurred, which enabled us to control the CPA concentration to which PCLS were exposed as a function of time.
Article
Whole ovary cryopreservation and transplantation has been proposed as a method for preserving long-term ovarian function. This work reports ovarian function 6years post transplantation of frozen-thawed whole sheep ovaries. Three 9-month-old Assaf sheep underwent unilateral oophorectomy to provide organs for the experiments. After perfusing with cold University of Wisconsin solution supplemented with 10% dimethyl sulphoxide, ovaries were cryopreserved using unidirectional solidification freezing technology. After thawing, ovaries were re-perfused and re-transplanted orthotopically by microvascular re-anastomosis, to the contralateral ovarian pedicle after removing the remaining ovary. Six years following transplantation and after inducing superovulation, the sheep were killed and the ovaries analysed. Two ovaries had normal size and shape showing some recent corpora lutea, while the third showed atrophic changes. A total of 36 antral follicles were counted by transillumination and four germinal vesicle oocytes were aspirated and matured in vitro to metaphase II. Serum progesterone concentrations were indicative of ovulatory activity in one of the three sheep. Histological evaluations revealed normal tissue architecture, intact blood vessels and follicles at various stages. Currently, this is the longest recorded ovarian function after cryopreservation and re-transplantation. Cryopreservation of whole ovaries, using directional freezing combined with microvascular anastomosis, is a promising method for preserving long-term reproductive capacity and endocrine function.
Article
The University of Chicago program in pediatric liver transplantation continues actively to seek innovative surgical solutions to problems related to the management of children with end-stage liver disease. Among the most important problems facing these children is a shortage of donor organs, which results from three factors in addition to the actual supply of pediatric donors: the concentration of pediatric liver disease in the population younger than 2 years; the necessity for a graft that is small enough; and the epidemiology of accidents and other events that lead to organ donation. Transplantation using a liver lobe as a graft overcomes size disparity and shifts the available supply of organs from older donors to younger recipients. This work describes the technical aspects of recent innovations in the use of liver lobes in pediatric transplantation, simple reduced-size liver transplantation (RLT), split-liver transplantation (SLT), orthotopic auxiliary liver grafting (ALT), and transplantation using a living related donor (LRLT), and compares their results. Since November 1986 a total of 61 procedures have been performed in which a liver lobe was used as a graft: 26 RLT; 30 SLT, 25 in children and 5 in adults; 5 LRLT; and 1 ALT. Overall 62% of transplants performed in children have involved using a liver lobe as a graft. The rates of complications are somewhat higher than with whole-liver transplantation, but this may not be entirely the result of the complex procedures. Split liver transplantation is associated with the highest mortality and complication rates. Living related liver transplantation has been associated with complications in donors and recipients, but to date survival is 100%. Orthotopic auxiliary liver transplantation effectively corrected the metabolic defect in one patient with ornithine transcarbamylase deficiency. Overall the various modalities of using graft reduction have resulted in postoperative results similar to those achieved with full-size grafts, while pretransplantation mortality has been limited to less than 2%. Thus the use of grafts as liver lobes accomplishes the goal of reducing global mortality among children with end-stage liver disease, but at the cost of increased surgical complexity and more postoperative complications.
Article
A theoretical model is proposed which is used to derive a quantitative relationship between the critical cooling rate and average crystal size at any location within a biological specimen of given shape subject to rapid freezing. The model is applicable to the slamming, plunging or spraying methods of cryofixation provided the ice crystal size is at least 5 times greater than the size of the critical nucleus. Complete vitrification of pure water or aqueous solutions is shown to take place at cooling rates in excess of about 3 × 106 K/s.
Article
In light of the increasing incidence of liver disease and continuing shortage of donor organs, cell-based therapies are gaining attention as promising treatments for liver failure. Currently, several extracorporeal bioartificial liver devices are undergoing clinical evaluation. Their future use will depend on the choice and stabilization of the cellular component. Although cell lines offer a limitless cell source, primary hepatocytes may be preferred because of their broad expression of liver-specific functions. Xenogenic primary cells are available in large quantities, but immunologic and infectious concerns may necessitate the use of human cells or human-derived cells. To improve and maintain functional primary hepatocytes, bioreactor designs must provide architecture that supports cell attachment, cell-cell interaction, cell-matrix interaction, and potential for scale-up. While the safety of BAL devices has been established, there are no uniform standards of efficacy, which may vary with the etiology of the liver failure. Consensus is needed in clinical trial design, including choice of end points, use of controls, and indications for enrollment. Also, a better understanding of the interplay between liver regeneration and BAL therapy will be critical to optimizing the implementation of this modality.
Article
Cryopreservation and cryosurgery are important biomedical applications used to selectively preserve or destroy cellular systems through freezing. Studies using cryomicroscopy techniques, which allow the visualization of the freezing process in single cells, have shown that a drop in viability correlates with the extent of two biophysical events during the freezing process: (a) intracellular ice formation and (b) cellular dehydration. These same biophysical events operate in tissue systems; however, the inability to visualize and quantify the dynamics of the freezing process in tissues has hampered direct correlation of these events with freezing-induced changes in viability. This review highlights two new techniques that use freeze substitution and differential scanning calorimetry to provide dynamic freezing data in tissue. Characteristic dimensions and parameters extracted from these new data are then used in a predictive model of biophysical freezing response in several tissues, including liver and tumor. This approach promises to help guide improved design of both cryopreservation and cryosurgical applications of tissue freezing.
Article
Although hypothermic machine perfusion (HMP) preservation has been shown to improve organ function and to expand the organ donor pool, problems still exist with the current HMP technology for liver preservation. The present study was conducted to investigate endothelial and hepatocellular functions following extended HMP (> r =24 hr) in rat liver model. Following 24-hour hypothermic HMP with University of Wisconsin (UW) solution or 24-hour simple cold storage (SCS), livers were reperfused with Krebs-Henseleit buffer solution at 37 degree C for 30 minutes. Hepatocyte damage and function were assessed by measuring lactate dehydrogenase (LDH) activity, bile production, and indocyanine green (ICG) extraction. Sinusoidal endothelial cell (SEC) function and permeability were determined by hyaluronic acid (HA) uptake and multiple indicator dilution (MID) method, respectively. After 24-hour hypothermic preservation, HMP livers showed lower released LDH levels, higher bile flow rate, and greater hepatic ICG uptake compared with SCS livers. However, LDH levels became significantly higher in HMP than in SCS after 30 minutes of warm perfusion. The increased enzyme levels were accompanied by a significant increase in endothelial permeability to albumin and a decrease in hyaluronic acid uptake in HMP compared to SCS. Liver wet/dry weight ratio confirmed a greater edema in HMP livers than SCS livers. These results suggest that 24-hour hypothermic HMP may help preservation of hepatocyte function, but endothelial cell dysfunction during the cold preservation may play a key role in hepatocyte dysfunction and parenchymal cell death upon reperfusion.
Article
This is the first report of successful cryopreservation and transplantation of an intact ovary in a large animal (sheep). Three of eight transplanted sheep had resumed hormonal cyclicity.
Article
Precision-cut tissue slices of both hepatic and extra-hepatic origin are extensively used as an in vitro model to predict in vivo drug metabolism and toxicity. Cryopreservation would greatly facilitate their use. In the present study, we aimed to improve (1) rapid freezing and warming (200 degrees C/min) using 18% Me(2)SO as cryoprotectant and (2) vitrification with high molarity mixtures of cryoprotectants, VM3 and VS4, as methods to cryopreserve precision-cut rat liver and kidney slices. Viability after cryopreservation and subsequent 3-4h of incubation at 37 degrees C was determined by measuring ATP content and by microscopical evaluation of histological integrity. Confirming earlier studies, viability of rat liver slices was maintained at high levels by rapid freezing and thawing with 18% Me(2)SO. However, vitrification of liver slices with VS4 resulted in cryopreservation damage despite the fact that cryoprotectant toxicity was low, no ice was formed during cooling and devitrification was prevented. Viability of liver slices was not improved by using VM3 for vitrification. Kidney slices were found not to survive cryopreservation by rapid freezing. In contrast, viability of renal medullary slices was almost completely maintained after vitrification with VS4, however vitrification of renal cortex slices with VS4 was not successful, partly due to cryoprotectant toxicity. Both kidney cortex and medullary slices were vitrified successfully with VM3 (maintaining viability at 50-80% of fresh slice levels), using an optimised pre-incubation protocol and cooling and warming rates that prevented both visible ice-formation and cracking of the formed glass. In conclusion, vitrification is a promising approach to cryopreserve precision-cut (kidney) slices.
Article
Cryopreservation is the use of very low temperatures to preserve structurally intact living cells and tissues. Unprotected freezing is normally lethal and this chapter seeks to analyze some of the mechanisms involved and to show how cooling can be used to produce stable conditions that preserve life. The biological effects of cooling are dominated by the freezing of water, which results in the concentration of the solutes that are dissolved in the remaining liquid phase. Rival theories of freezing injury have envisaged either that ice crystals pierce or tease apart the cells, destroying them by direct mechanical action, or that damage is from secondary effects via changes in the composition of the liquid phase. Cryoprotectants, simply by increasing the total concentration of all solutes in the system, reduce the amount of ice formed at any given temperature; but to be biologically acceptable they must be able to penetrate into the cells and have low toxicity. Many compounds have such properties, including glycerol, dimethyl sulfoxide, ethanediol, and propanediol.
Article
Acute liver failure (ALF) is a widespread problem with an unfavorable prognosis. Currently, liver transplantation is the only direct means of treatment for patients in ALF. Due to the scarcity of donor organs, liver support technologies are being developed and clinically tested with the intent of supporting a patient in ALF until the patient regains native liver function or until a donor organ becomes available. Two major categories of devices are currently being tested. Artificial liver support is purely mechanical, including albumin dialysis. Bioartificial devices contain cellular material. No single system has reproducibly demonstrated improvement in patient mortality. However, with the advent of new technology and cell acquisition techniques, further randomized controlled trials will be necessary to determine the role of artificial and bioartificial liver support devices in the treatment of patients with ALF.
Kidney preservation for transportation. Initial perfusion and 30 hours’ ice storage
  • G M Collins
  • M Bravo-Shugarman
  • P I Terasaki
  • GM Collins
Cryopreservation of precision-cut tissue slices
  • G M Fahy
  • N Guan
  • I A De Graaf
  • Y Tan
  • L Griffin
  • G M Groothuis
  • GM Fahy
Scientific Registry of Transplant Recipients National Data Report
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The effect of thermal expansion of ingredients on the cocktails VS55 and DP6
  • J Plitz
  • Y Rabin
  • J R Walsh