The inability to preserve vascular organs beyond several hours contributes to the scarcity of organs for transplantation
. Standard hypothermic preservation at +4 °C (refs.
) 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 livers
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.
•Static cold storage is a key bottleneck for all regenerative medicine therapies.•Machine perfusion is an important emerging organ preservation technology.•Subzero preservation holds the promise to ...enable organ banking.•Machine perfusion and subzero preservation can be used in synergy.
Improving organ preservation and extending the preservation time would have game-changing effects on the current practice of organ transplantation. Machine perfusion has emerged as an improved preservation technology to expand the donor pool, assess graft viability and ensure adequate graft function. However, its efficacy in extending the preservation time is limited. Subzero organ preservation does hold the promise to significantly extend the preservation time and recent advances in cryobiology bring it closer to clinical translation. In this review, we aim to broaden the perspective in the field from a focus on these individual technologies to that of a systems engineering. This would enable the creation of a preservation process that integrates the benefits of machine perfusion with those of subzero preservation, with the ultimate goal to provide on demand availability of donor organs through organ banking.
Preservation of human organs at subzero temperatures has been an elusive goal for decades. The major complication hindering successful subzero preservation is the formation of ice at temperatures ...below freezing. Supercooling, or subzero non-freezing, preservation completely avoids ice formation at subzero temperatures. We previously showed that rat livers can be viably preserved three times longer by supercooling as compared to hypothermic preservation at +4 °C. Scalability of supercooling preservation to human organs was intrinsically limited because of volume-dependent stochastic ice formation at subzero temperatures. However, we recently adapted the rat preservation approach so it could be applied to larger organs. Here, we describe a supercooling protocol that averts freezing of human livers by minimizing air-liquid interfaces as favorable sites of ice nucleation and uses preconditioning with cryoprotective agents to depress the freezing point of the liver tissue. Human livers are homogeneously preconditioned during multiple machine perfusion stages at different temperatures. Including preparation, the protocol takes 31 h to complete. Using this protocol, human livers can be stored free of ice at -4 °C, which substantially extends the ex vivo life of the organ. To our knowledge, this is the first detailed protocol describing how to perform subzero preservation of human organs.
Loss of hepatocyte viability and metabolic function after cryopreservation is still a major issue. Although vitrification is a promising alternative, it has generally been proven to be unsuitable for ...vitrification of large cell volumes which is required for clinical applications. Here, we propose a novel bulk droplet (3–5 mm diameter) vitrification method which allows high throughput volumes (4 mL/min), while using a low preincubated CPA concentration (15% v/v) to minimize toxicity and loss of cell viability and function. We used rapid (1.25 s) osmotic dehydration to concentrate a low preincubated intracellular CPA concentration ahead of vitrification, without the need of fully equilibrating toxic CPA concentrations. We compared direct postpreservation viability, long-term viability, and metabolic function of bulk droplet vitrified, cryopreserved, and fresh hepatocytes. Simulations and cooling rate measurements confirmed an adequate concentration of the intracellular CPA concentration (up to 8.53 M) after dehydration in combination with high cooling rates (960–1320 °C/min) for successful vitrification. In comparison to cryopreserved hepatocytes, bulk droplet vitrified hepatocytes had a significantly higher viability, directly after preservation and after 1 day in culture. Moreover, bulk droplet vitrified hepatocytes had evidently better morphology and showed significantly higher metabolic activity than cryopreserved hepatocytes in long-term collagen sandwich cultures. In conclusion, we developed a novel bulk droplet vitrification method of which we validated the theoretical background and demonstrated the feasibility to use this method to vitrify large cell volumes. Moreover, we showed that this method results in improved hepatocyte viability and metabolic function as compared to cryopreservation.
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.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
Despite decades of effort, the preservation of complex organs for transplantation remains a significant barrier that exacerbates the organ shortage crisis. Progress in organ preservation research is ...significantly hindered by suboptimal research tools that force investigators to sacrifice translatability over throughput. For instance, simple model systems, such as single cell monolayers or co‐cultures, lack native tissue structure and functional assessment, while mammalian whole organs are complex systems with confounding variables not compatible with high‐throughput experimentation. In response, diverse fields and industries have bridged this experimental gap through the development of rich and robust resources for the use of zebrafish as a model organism. Through this study, we aim to demonstrate the value zebrafish pose for the fields of solid organ preservation and transplantation, especially with respect to experimental transplantation efforts. A wide array of methods were customized and validated for preservation‐specific experimentation utilizing zebrafish, including the development of assays at multiple developmental stages (larvae and adult), methods for loading and unloading preservation agents, and the development of viability scores to quantify functional outcomes. Using this platform, the largest and most comprehensive screen of cryoprotectant agents (CPAs) was performed to determine their toxicity and efficiency at preserving complex organ systems using a high subzero approach called partial freezing (i.e., storage in the frozen state at −10°C). As a result, adult zebrafish cardiac function was successfully preserved after 5 days of partial freezing storage. In combination, the methods and techniques developed have the potential to drive and accelerate research in the fields of solid organ preservation and transplantation.
Zebrafish as a high‐throughput, relevant model organism for heart cryopreservation research.
Genovar distributions of Chlamydia trachomatis based on ompA typing differ between men who have sex with men (MSM) and heterosexuals. We investigated clonal relationships using a high resolution ...typing method to characterize C. trachomatis types in these two risk groups.
C. trachomatis positive samples were collected at the STI outpatient clinic in Amsterdam between 2008 and 2010 and genotyped by multilocus sequence typing. Clusters were assigned using minimum spanning trees and these were combined with epidemiological data of the hosts.
We typed 526 C. trachomatis positive samples: 270 from MSM and 256 from heterosexuals. Eight clusters, containing 10-128 samples were identified of which 4 consisted of samples from MSM (90%-100%), with genovars D, G, J, and L2b. The other 4 clusters consisted mainly of samples from heterosexuals (87%-100%) with genovars D, E, F, I, and J. Genetic diversity was much lower in the MSM clusters than in heterosexual clusters. Significant differences in number of sexual partners and HIV-serostatus were observed for MSM-associated clusters.
C. trachomatis transmission patterns among MSM and heterosexuals were largely distinct. We hypothesize that these differences are due to sexual host behavior, but bacterial factors may play a role as well.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The global shortage of donor organs has made it crucial to deeply understand and better predict donor liver viability. However, biomarkers that effectively assess viability of marginal grafts for ...organ transplantation are currently lacking. Here, we showed that hepatocytes, sinusoidal endothelial, stellate, and liver-specific immune cells were released into perfusates from Lewis rat livers as a result of cold ischemia and machine perfusion. Perfusate comparison analysis of fresh livers and cold ischemic livers showed that the released cell profiles were significantly altered by the duration of cold ischemia. Our findings show for the first time that parenchymal cells are released from organs under non-proliferative pathological conditions, correlating with the degree of ischemic injury. Thus, perfusate cell profiles could serve as potential biomarkers of graft viability and indicators of specific injury mechanisms during organ handling and transplantation. Further, parenchymal cell release may have applications in other pathological conditions beyond organ transplantation.
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