Persufflation (or gaseous oxygen perfusion) as a method of organ preservation

Improved preservation techniques have the potential to improve transplant outcomes by better maintaining donor organ quality and by making more organs available for allotransplantation. Persufflation, (PSF, gaseous oxygen perfusion) is potentially one such technique that has been studied for over a century in a variety of tissues, but has yet to gain wide acceptance for a number of reasons. A principal barrier is the perception that ex vivo PSF will cause in vivo embolization post-transplant. This review summarizes the extensive published work on heart, liver, kidney, small intestine and pancreas PSF, discusses the differences between anterograde and retrograde PSF, and between PSF and other conventional methods of organ preservation (static cold storage, hypothermic machine perfusion). Prospective implications of PSF within the broader field of organ transplantation, and in the specific application with pancreatic islet isolation and transplant are also discussed. Finally, key issues that need to be addressed before PSF becomes a more widely utilized preservation strategy are summarized and discussed.     

Induction of heme oxygenase-1 improves cold preservation effect of liver graft

We have examined the protective effect and mechanisms of heme oxygenase-1 (HO-1) induction in rat liver model of ex vivo cold ischemia preservation using cobalt protoporphyrin (CoPP) as HO-1 inducer and zinc protoporphyrin (ZnPP) as HO-1 inhibitor. There was a decrease in both aspartate transaminase and lactate dehydrogenase activities and in malondialdehyde level in liver of the CoPP-treated group compared with controls (p < 0.05). In the CoPP-treated rats, the histological signs of reperfusion injury were much lower than in control. Up-regulation of HO-1 expression was also associated with reduced levels of tumor necrosis factor α and interleukin-6. Markedly fewer apoptotic liver cells (determined by TUNEL assay) could be detected in CoPP-treated group compared with the control group. These protective effects were prevented by administration of ZnPP. In conclusion, induction of HO-1 provides protection against liver injury during cold ischemia preservation and improves the preservation of liver graft. The mechanisms underlying these beneficial effects include reduction of oxidative injury and of inflammatory response and prevention of apoptosis. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 5, pp. 674–681.     

Inherent toxicity of organ preservation solutions to cultured hepatocytes

Organ preservation solutions have been designed to protect grafts against the injury inflicted by cold ischemia. However, toxicity of University of Wisconsin (UW) solution during rewarming has been reported. Therefore, we here assessed the toxicity of UW, histidine-tryptophan-ketoglutarate (HTK), Euro-Collins, histidine-lactobionate (HL), sodium-lactobionate-sucrose and Celsior solutions in cultured hepatocytes under hypothermic (4 °C), intermediate (21 °C) and physiological (37 °C) conditions. Marked toxicity of UW, HTK, HL and Euro-Collins solutions was observed at both 37 and 21 °C. With the exception of UW solution, these solutions also increased cell injury during cold incubation (LDH release after 18 h at 4 °C: HTK 76 ± 2%, Euro-Collins 78 ± 17%, HL 81 ± 15%; control: Krebs-Henseleit buffer 20 ± 6%). Testing of individual components using modified Krebs-Henseleit buffers suggested that histidine and phosphate are responsible for (part of) this toxicity. These potential toxicities should be taken into account in the development of future preservation solutions.     

Acid-base buffering in organ preservation solutions as a function of temperature: new parameters for comparing buffer capacity and efficiency

Control of acidity and preventing intracellular acidosis are recognized as critical properties of an effective organ preservation solution. Buffer capacity and efficiency are therefore important for comparing the relative merits of preservation fluids for optimum hypothermic storage, but these parameters are not available for the variety of organ preservation solutions of interest in transplantation today. Moreover, buffer capacity is dependent upon both concentration and pH such that buffer capacity is not easily predicted for a complex solution containing multiple buffer species. Using standard electrometric methods to measure acid dissociation constants, this study was undertaken to determine the maximum and relative buffer capacities of a variety of new and commonly used hypothermic preservation solutions as a function of temperature. The reference data provided by these measurements show that comparative buffer capacity and efficiency vary widely between the commonly used solutions. Moreover, the fluids containing zwitterionic sulfonic acid buffers such as Hepes possess superior buffering for alpha-stat pH regulation in the region of physiological importance.     

Optimizing the concentration of hydroxyethylstarch in a novel intestinal-specific preservation solution

Introduction

Our lab has developed an effective nutrient-rich solution that facilitates energy production and control of oxidative stress during static cold storage of the intestine; however, the requirement for oncotic agents, such as hydroxyethylstarch (HES), has not been evaluated. This study investigated the effectiveness and requirement for HES in an intraluminal preservation solution during a clinically relevant period of cold storage.

Methods

Rat intestines were procured, including an intravascular flush with University of Wisconsin solution followed by a ‘back table’ intraluminal flush with a nutrient-rich preservation solution containing varying amounts of HES (n = 6 per group): Group 1, 0%; Group 2, 2.5%; Group 3, 5%; Group 4, 10%. Energetics, oxidative stress, and morphology were assessed over a 24 h time-course of cold storage.

Results

Overall, the 5% HES solution, Group 3, demonstrated superior energetic status (ATP and total adenylates) compared to all groups, P < 0.05. Malondialdehyde levels indicated a reduction in oxidative stress in Groups 3 and 4 (P < 0.05). After 12 h, median modified Parks’ grades for Groups 2 and 3 were significantly lower than Groups 1 and 4, P < 0.05.

Conclusion

Our data suggests that when employing an intraluminal preservation solution for static organ storage, oncotic support is a fundamental requirement; 5% HES is optimal.     

Current techniques and the future of lung preservation

Although lung transplant remains the only option for patients suffering from end-stage lung failure, donor supply is insufficient to meet demand. Static cold preservation is the most common method to preserve lungs in transport to the recipient; however, this method does not improve lung quality and only allows for 8 h of storage. This results in lungs which become available for donation but cannot be used due to failure to meet physiologic criteria or an inability to store them for a sufficient time to find a suitable recipient. Therefore, lungs lost due to failure to meet physiological or compatibility criteria may be mitigated through preservation methods which improve lung function and storage durations. Ex situ lung perfusion (ESLP) is a recently developed method which allows for longer storage times and has been demonstrated to improve lung function such that rejected lungs can be accepted for donation. Although greater use of ESLP will help to improve donor lung utilization, the ability to cryopreserve lungs would allow for organ banking to better utilize donor lungs. However, lung cryopreservation research remains underrepresented in the literature despite its unique advantages for cryopreservation over other organs. Therefore, this review will discuss the current techniques for lung preservation, static cold preservation and ESLP, and provide a review of the cryopreservation challenges and advantages unique to lungs.     

Assessment of hepatic integrity after ischemic preservation by isolated perfusion in vitro: the role of albumin

Isolated perfusion of rat livers (IPRL) represents an attractive set-up to be used as a an evaluative tool in the easy and reproducible assessment of liver injury, allowing for screening of new approaches to organ preservation without the expenditure of actual transplantation experiments. Depending on the pathology under investigation, controversy exists concerning the inclusion of albumin in the IPRL. The present study evaluates the use of bovine serum albumin (BSA), simultaneously comparing its effect on healthy and ischemically challenged livers in the same model. Rat livers were excised, flushed via portal vein with Histidine-Tryptophan-Ketoglutarate (HTK) solution and preserved for up to 18 h in HTK at 4 degrees C. Perfusion was performed with Krebs-Henseleit buffer with or without addition of 3% BSA. Control preparations were perfused without prior ischemic storage. In the described model, stability of the preparations was documented for up to 120 min of isolated perfusion and addition of 3% BSA had no adverse effects on the viability of nonischemic livers. While liver perfusion without albumin was inappropriate to reveal alterations in parenchymal or vascular integrity after 18 h of cold preservation, albumin in the perfusate significantly and gradually unmasked differences between nonischemic liver preparations and livers stored ischemically for 8 or 18 h. It could be shown that BSA did have a significant modulatory effect on hepatic induction of apoptosis after ischemia in reducing cleavage of caspase 3. The implementation of albumin is advocated since experimental results are pivotally influenced by the presence or absence of this physiologically constitutive compound in the perfusate.     

Liver integrity after warm ischemia in situ and brief preservation ex vivo: the value of aerobic post-conditioning

We evaluated the respective effects of warm ischemic injury in non-heart-beating donor (NHBD) grafts and/or cold ischemia time on liver viability. Eventually, the restorative potential of oxygenated hypothermic perfusion after cold storage should be investigated. Livers were retrieved from male Wistar rats and preserved with HTK-solution for 6 h or 18 h by cold storage (CS). Organ retrieval took place either prior to (ctrl.) or 30 min after cardiac arrest (NHBD). Compared to 6 h CS of ctrl. livers, enzyme leakage and functional recovery (oxygen consumption, ammonia clearance, bile production) upon warm reperfusion were massively deteriorated after 18 h CS in NHBD-livers. By contrast, 6 h CS of NHBD resulted in an only limited impairment of all parameters, which was found quite similar to the results in ctrl. after 18 h CS. Induction of cellular apoptosis (cleavage PARP) was found equally influenced by preceding warm ischemia (NHBD) or extended times of CS, but significantly triggered only by the combination of both events. After 6 h of CS, 1 h of oxygenated hypothermic machine perfusion (‘post-conditioning’) was able to bring the performance of NHBD-liver into line with the controls. Based on this work, we concluded that a limited time of warm ischemia in the donor only multiplied graft injury after long-term CS, but does not need to preclude acceptable results if reperfusion is initiated after short periods of CS. Moreover, conditioning of those grafts is effective even 1 h prior to implantation and may help to judge liver viability according to adequate parameters after hypothermic machine perfusion has been established.     

类器官的应用研究进展

类器官是利用干细胞的自我更新和分化能力,在体外培养形成的一种微小组织器官类似物,在很大程度上具有体内相应器官的功能。迄今为止,在3D培养条件下,已经成功培养出多种类器官如肺、胃、肠、肝和肾等类器官。它们不仅可作为组织器官的替代品用于药物和临床研究,还可用于体内器官移植。本文综述了类器官在药物毒性检测、药效评价和新药筛选中的作用以及利用类器官建立疾病模型、研究组织器官发育和类器官在精准医疗、再生医学中的价值。     

Monocyte subsets in bone marrow grafts may contribute to a low incidence of acute graft‐vs‐host disease for young donors

Young donors are associated with a lower cumulative incidence of acute graft‐vs‐host disease (aGVHD) after allogenic haematopoietic stem cell transplantation (allo‐HSCT) than old donors. Although grafts are harvested from healthy donors, it is unclear whether donor age is associated with aGVHD occurrence owing to its effect on cell compositions in grafts. Moreover, the differences in monocyte subsets in grafts between young and old donors and the association between monocyte subsets in bone marrow (BM) grafts and aGVHD remain to be elucidated. In the current study, non‐classical monocytes and the CD4⁺/CD8⁺ T cell ratio were remarkably decreased in BM grafts in donors <30 years old. Multivariate analysis further revealed that the level of non‐classical monocytes in BM grafts (≥0.31 × 10⁶/kg) was an independent risk factor for the occurrence of II‐IV aGVHD. In summary, our data indicate that non‐classical monocytes in BM grafts may help identify patients at high risk for aGVHD after allo‐HSCT. Although further validation is required, our results suggest that the low level of non‐classical monocytes and a low ratio of CD4⁺/CD8⁺ T cell in BM grafts may be correlated with the lower incidence of aGVHD in young donors.     

Recommendations on the use of alcohols for preservation of ant specimens (Hymenoptera,Formicidae)

Summary Collectors use a variety of concentrations and types of alcohols to preserve ant specimens. We evaluated existing literature, experimental evidence, and expert myrmecological advice to determine what kind and concentration of alcohol will result in the best preserved specimens for card-point mounting and DNA extraction. For our experimental evaluation, we killed and stored Solenopsis invicta, Camponotus floridanus, and Dorymyrmex bureni workers in isopropanol and ethanol at four concentrations (70, 85, 95, 100%) over three time periods (24 h, 1 month, 6 months). We then compared specimen condition and amenability to manipulation for mounting on card points. Specimens stored in either 95% isopropanol or 95% ethanol for time periods longer than 24 h produced the best specimens for mounting. A literature review revealed that DNA is best preserved in 95–100% ethanol due to the ability of ethanol to more rapidly penetrate cellular membranes and deactivate DNase activity than other primary alcohols. We recommend that general collections of adult ant specimens should be killed and stored in 95% ethanol. Following this recommendation will result in ant specimens that are easier to mount for museum collections and better preserved for molecular studies. A variety of other killing and preservation techniques relevant to the study of ants are also discussed.Received 8 July 2003; revised 23 October 2003; accepted 27 October 2003.     

Kinetics of the use of cryopreserved autologous stem cell grafts: a GITMO-SIDEM survey

Background aimsHematopoietic stem cell cryopreservation significantly contributed to autologous stem cell transplantation (ASCT). Cryopreserved stem cell units (SCU) are expected to be used soon after harvesting for most purposes, but, in a number of cases, they remain stored for some time, creating an increasing load for SCU depositories. Disposal policies vary widely in each center, and the existing guidelines are insufficient.MethodsWe conducted a survey of seven Gruppo Italiano Trapianto di Midollo Osseo centers to investigate the outcome of SCU harvested from January 2005 to December 2009 for ASCT. The data from 1603 collections were gathered, for a total of 5822 SCU.ResultsIn our cohort, 79% of patients collected >5 × 10⁶ CD34+ cells/kg, and 3.4% collected <2 × 10⁶ CD34+ cells/kg. Up to 21% of all the patients and 42% of those with acute leukemia did not undergo reinfusion, and 37% of the cryopreserved SCU were excess, resulting from patients not reinfusing or partially reinfusing. Less than one-third of the excess SCU was disposed, and the major causes of disposal were death and, in a minority of cases, withdrawal of the indication for ASCT. In our analysis, very few first reinfusions occurred after 2 years, and those after 5 years were exceptional. Through the use of a multivariate analysis, we sought to identify the risk factors for collection non-use, independent of the centers' policies. Non-use of SCU was significantly associated with patients with acute leukemia, collections of <2 × 10⁶ CD34/kg and lower age groups.ConclusionsThese data serve as a valid basis to support rational recommendations for cost-effective storage and disposal of SCU.     

Assessment of the cryoprotectant concentration inside a bulky organ for cryopreservation using X-ray computed tomography

Cryoprotection of bulky organs is crucial for their storage and for subsequent transplantation. In this work we demonstrate the capability of the X-ray computed tomography (CT) as a non-invasive method to measure the cryoprotectant (cpa) concentration inside a tissue or an organ, specifically for the case of dymethil sulfoxide (Me₂SO). It is remarkable that the use of Me₂SO has been leader in techniques of cells and tissues cryopreservation. Although CT technologies are mainly based in density differences, and many cpas are alcohols with densities similar to water, the use of very low energies as acceleration voltage (∼70 kV) and the sulfur atom in the molecule of Me₂SO makes possible the visualization of this cpa inside tissues. As result we obtain a CT signal proportional to the Me₂SO concentration with a spatial resolution up to 50 μm in the case of our device.     

The influence of noradrenaline on the process of protein/peptide secretion in the mammalian pineal organ

Summary From studies conducted with the pineal organ of the mouse, it was ascertained that for the in vitro investigation of secretory processes (synthesis and release) of proteic/peptidic compound(s), a culture time of 5 to 14 days is optimal. A 5-day organ culture was therefore chosen to study the effects of noradrenaline on these secretory processes.Addition of noradrenaline to the culture medium provokes, in pineal explants of the normal mouse and the eyeless mouse, an inhibition of the secretory process, characterized by the formation of granular vesicles. In the hamster and rat, however, opposite results were obtained. Moreover, it appears that noradrenaline, at least in the rat, may also be involved in the regulation of the ependymal-like secretory process.The present results indicate clearly that noradrenaline (thus, the sympathetic innervation) is implicated in the regulation of the production of proteic/peptidic hormonal agents, but that the effect of this neurotransmitter is species-specific. This could explain the numerous contradictory results reported in the literature.IBRO/UNESCO fellow     

The role of clay minerals in the preservation of Precambrian organic-walled microfossils

Precambrian organic-walled microfossils (OWMs) are primarily preserved in mudstones and shales that are low in total organic carbon (TOC). Recent work suggests that high TOC may hinder OWM preservation, perhaps because it interferes with chemical interactions involving certain clay minerals that inhibit the decay of microorganisms. To test if clay mineralogy controls OWM preservation, and if TOC moderates the effect of clay minerals, we compared OWM preservational quality (measured by pitting on fossil surfaces and the deterioration of wall margins) to TOC, total clay, and specific clay mineral concentrations in 78 shale samples from 11 lithologic units ranging in age from ca. 1650 to 650 million years ago. We found that the probability of finding well-preserved microfossils positively correlates with total clay concentrations and confirmed that it negatively correlates with TOC concentrations. However, we found no evidence that TOC influences the effect of clay mineral concentrations on OWM preservation, supporting an independent role of both factors on preservation. Within the total clay fraction, well-preserved microfossils are more likely to occur in shales with high illite concentrations and low berthierine/chamosite concentrations; however, the magnitude of their effect on preservation is small. Therefore, there is little evidence that bulk clay chemistry is important in OWM preservation. Instead, we propose that OWM preservation is largely regulated by physical properties that isolate organic remains from microbial degradation such as food scarcity (low TOC) and low sediment permeability (high total clay content): low TOC increases the diffusive distances between potential carbon sources and heterotrophic microbes (or their degradative enzymes), while high clay concentrations reduce sediment pore space, thereby limiting the diffusion of oxidants and degradative enzymes to the sites of decay.     

The influence of short-term fasting on the quality of small bowel graft preservation

INTRODUCTION: Donor nutritional status may be a determinant of small bowel (SB) quality following storage. In this study, we investigated the effect of donor nutritional status and a proven nutrient-rich preservation solution on graft quality following cold storage. METHODS: Rats were fasted (12-14 h) or non-fasted. SB (n=6) was flushed vascularly with modified University of Wisconsin (UW) solution and flushed luminally with UW or an amino acid-rich (AA) solution as follows: Fasted. UWV, none; UWL, UW solution; AAL, AA solution. Non-fasted. UWV, none; UWL, UW solution; AAL, AA solution. Energetics, peroxidation (malondialdehyde; MDA), glutathione and histology were assessed over 24 h at 4 degrees C. RESULTS: Energetics (ATP, ATP/ADP, and energy charge) were significantly higher in AAL (fasted and non-fasted) groups than other groups. However, there were no differences in energetics parameters between fasted and non-fasted animals in all groups. MDA was higher in fasted groups than non-fasted tissues; interestingly, AAL values were up to 10-fold lower than other groups. Higher glutathione levels were detected in non-fasted AAL tissues. Mucosal integrity was markedly superior in luminally treated tissues (UWL and AAL) in fasted and non-fasted states. Most noteably, AAL tissues from fasted animals exhibited grade 2 injury (villus clefting), whereas normal mucosa was observed in non-fasted tissues (grade 0). CONCLUSION: Luminal flushing and a nutrient-rich preservation solution improve energetics, oxidative stress, and mucosal integrity during storage. Poorer donor nutritional status does not affect energetics throughout storage, but causes mucosal injury as a result of increased oxidative stress, even after a brief period of donor fasting.     

Beneficial effects of the novel marine oxygen carrier M101 during cold preservation of rat and human pancreas

Ischaemia impairs organ quality during preservation in a time‐dependent manner, due to a lack of oxygen supply. Its impact on pancreas and islet transplantation outcome has been demonstrated by a correlation between cold ischaemia time and poor islet isolation efficiency. Our goal in the present study was to improve pancreas and islet quality using a novel natural oxygen carrier (M101, 2 g/L), which has been proven safe and efficient in other clinical applications, including kidney transplantation, and for several pre‐clinical transplantation models. When M101 was added to the preservation solution of rat pancreas during ischaemia, a decrease in oxidative stress (ROS), necrosis (HMGB1), and cellular stress pathway (p38 MAPK)activity was observed. Freshly isolated islets had improved function when M101 was injected in the pancreas. Additionally, human pancreases exposed to M101 for 3 hours had an increase in complex 1 mitochondrial activity, as well as activation of AKT activity, a cell survival marker. Insulin secretion was also up‐regulated for isolated islets. In summary, these results demonstrate a positive effect of the oxygen carrier M101 on rat and human pancreas during preservation, with an overall improvement in post‐isolation islet quality.     

The nervous and circulatory systems of a Cretaceous crinoid: preservation,palaeobiology and evolutionary significance

Featherstars, comatulid crinoids that shed their stalk during their ontogeny, are the most species-rich lineage of modern crinoids and the only ones present in shallow water today. Although they are of considerable palaeontological interest as a ‘success story’ of the Mesozoic Marine Revolution, their fossil record is relatively species-poor and fragmentary. New Spanish fossils of the Cretaceous featherstar Decameros ricordeanus preserve the shape and configuration of nervous and circulatory anatomy in the form of infilled cavities, which we reconstruct from CT scans. The circulatory system of D. ricordeanus was relatively extensive and complex, implying a pattern of coelomic fluid flow that is unique among crinoids, and the peripheral parts of the nervous system include linkages both to the circulatory system and to the surface of the body. A phylogenetic analysis (the first to include both living and fossil featherstars and which includes characters from internal anatomy) recovers D. ricordeanus among the lineage of featherstars that includes Himerometroidea, Tropiometra and ‘Antedonoidea’, among others. D. ricordeanus is larger than almost any modern featherstar, and its elaborate coelomic morphology appears to be a consequence of positive allometry. All featherstars with coelomic diverticula are shown to belong to a single comatulid subclade, and this feature may constitute a synapomorphy of that group. Some preservation of cavities corresponding to soft tissue is probably not exceptional in fossil crinoids, providing an opportunity to study the diversity and evolution of extinct anatomical systems typically only preserved in Lagerstätten.