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Ischemia is a major limiting factor in Vascularized Composite Allotransplantation (VCA) as irreversible muscular injury can occur after as early as 4–6 h of static cold storage (SCS). Organ preservation technologies have led to the development of storage protocols extending rat liver ex vivo preservation up to 4 days. Development of such a protocol for VCAs has the added challenge of inherent ice nucleating factors of the graft, therefore, this study focused on developing a robust protocol for VCA supercooling. Rodent partial hindlimbs underwent subnormothermic machine perfusion (SNMP) with several loading solutions, followed by a storage solution with cryoprotective agents (CPA) developed for VCAs. Storage occurred in suspended animation for 24h and VCAs were recovered using SNMP with modified Steen. This study shows a robust VCA supercooling preservation protocol in a rodent model. Further optimization is expected to allow for its application in a transplantation model, which would be a breakthrough in the field of VCA preservation. [ABSTRACT FROM AUTHOR]

Current methods of storing explanted donor livers at 4 °C in University of Wisconsin (UW) solution result in loss of graft function and ultimately lead to less-than-ideal outcomes post transplantation. Our lab has previously shown that supplementing UW solution with 35-kilodalton polyethylene glycol (PEG) has membrane stabilizing effects for cold stored primary rat hepatocytes in suspension. Expanding on past studies, we here investigate if PEG has the same beneficial effects in an adherent primary rat hepatocyte cold storage model. In addition, we investigated the extent of cold-induced apoptosis through treating cold-stored hepatocytes with pan caspase inhibitor emricasan. In parallel to storage at the current cold storage standard of 4 °C, we investigated the effects of lowering the storage temperature to −4 °C, at which the storage solution remains ice-free due to the supercooling phenomenon. We show the addition of 5 % PEG to the storage medium significantly reduced the release of lactate dehydrogenase (LDH) in plated rat hepatocytes and a combinatorial treatment with emricasan maintains hepatocyte viability and morphology following recovery from cold storage. These results show that cold-stored hepatocytes undergo multiple mechanisms of cold-induced injury and that PEG and emricasan treatment in combination with supercooling may improve cell and organ preservation. [ABSTRACT FROM AUTHOR]

Stable aqueous supercooling has shown significant potential as a technique for human tissue preservation, food cold storage, conservation biology, and beyond, but its stochastic nature has made its translation outside the laboratory difficult. In this work, we present an isochoric nucleation detection (INDe) platform for automated, high-throughput characterization of aqueous supercooling at >1 mL volumes, which enables statistically-powerful determination of the temperatures and time periods for which supercooling in a given aqueous system will remain stable. We employ the INDe to investigate the effects of thermodynamic, surface, and chemical parameters on aqueous supercooling, and demonstrate that various simple system modifications can significantly enhance supercooling stability, including isochoric (constant-volume) confinement, hydrophobic container walls, and the addition of even mild concentrations of solute. Finally, in order to enable informed design of stable supercooled biopreservation protocols, we apply a statistical model to estimate stable supercooling durations as a function of temperature and solution chemistry, producing proof-of-concept supercooling stability maps for four common cryoprotective solutes. [ABSTRACT FROM AUTHOR]

In recent years there have been several advancements in organ preservation that have yet to see widespread clinical translation. While static cold storage (SCS) at 2 °C–4 °C continues to be the state-of-the-art strategy, it contributes to the current shortage of transplantable organs due to the limited preservation times it affords combined with the limited ability of marginal grafts to tolerate SCS. The era of optimizing storage solutions to minimize SCS-induced hypothermic injury has largely plateaued in its improvements, resulting in a shift towards the use of machine perfusion systems to provide continuous metabolic support, or the use of sub-zero storage temperatures to leverage the protection brought forth by a reduction in metabolic demand. Many of the rigors that organs are subjected to at low sub-zero temperatures (−80 °C to −196 °C) commonly used for mammalian cell preservation have yet to be surmounted, and therefore the focus of this article lies on an intermediate range of storage temperatures (0 °C to −20 °C) where much success has been seen in the past two decades. Numerous mechanisms leveraged by organisms capable of withstanding prolonged periods at these temperatures through either avoiding or tolerating the formation of ice has provided a foundation for some of the more promising efforts, and thus we aim to contextualize the translation of these nature-derived strategies to mammalian organ preservation. [ABSTRACT FROM AUTHOR]

In biotechnological processes such as chromosomal manipulation studies, semen has become a reference in the ploidy verification of the evaluated material. However, the use of fresh samples is limited to the use at field conditions because the analysis is performed under laboratory conditions. Thus, this study aimed to develop a simpler procedure for storing dry semen at 28 °C to reduce cold storage costs. For this, semen samples were evaluated according to established quality semen parameters, a protocol for dry, and 3 sterilization treatments of dry semen were applied to the store. The integrity of the DNA was evaluated every two months, using fresh semen, dry semen (untreated), and particles 3C to compare the peaks by flow cytometry. The results indicated that all samples evaluated before and after drying showed no significant difference in the DNA content. UV-treated semen showed a 1C peak in the histogram up to 180 days of storage and a non-significant difference (P > 0.05) from fresh control in the number of DNA particles up to 120 days and untreated only showed a 1C peak up to 120 days. The developed method may become an interesting procedure to serve as a reference peak for practical flow cytometric analysis, not only in the field of fish biology but also in biomedical and agricultural sciences. Furthermore, dried semen can become a tool for the preservation of genetic material and is a promising low-cost storage technique for biobanking. [ABSTRACT FROM AUTHOR]

Cold transport of epididymides from genetically modified mice is an efficient alternative to the shipment of live animals between research facilities. Mouse sperm from epididymides cold-stored for short periods can maintain viability. We previously reported that cold storage of mouse epididymides in Lifor® perfusion medium prolonged sperm motility and fertilization potential and that the sperm efficiently fertilized oocytes when reduced glutathione was added to the fertilization medium. Cryopreservation usually results in decreased sperm viability; an optimized protocol for cold storage of epididymides plus sperm cryopreservation has yet to be established. Here, we examined the motility and fertilization potential of cryopreserved, thawed (frozen-thawed) sperm from previously cold-stored mouse epididymides. We also examined the protective effect of sphingosine-1-phosphate (S1P) on sperm viability when S1P was added to the preservation medium during cold storage. We assessed viability of frozen-thawed sperm from mouse epididymides that had been cold-transported domestically or internationally and investigated whether embryos fertilized in vitro with these sperm developed normally when implanted in pseudo-pregnant mice. Our results indicate that frozen-thawed sperm from epididymides cold-stored for up to 48 h maintained high fertilization potential. Fertilization potential was reduced after cold storage for 72 h, but not if S1P was included in the cold storage medium. Live pups were born normally to recipients after in vitro fertilization using frozen-thawed sperm from cold-transported epididymides. In summary, we demonstrate an improved protocol for cold-storage of epididymides that can facilitate transport of genetically engineered-mice and preserve sperm viability after cryopreservation.

The objectives of this study were to identify the presence of different spermatozoa subpopulations (SPs) according to their kinematic characteristics in the sperm of common carp and to test the effects of cryopreservation and prolonged (6-day) storage at room temperature (RT; 23 °C) and 4 °C on spermatozoa motility and subsequently on SP dynamics. Two-step clustering analyses identified three motile SPs based on their kinematic properties: SP1 contained spermatozoa with low velocity and low/moderate STR/LIN values (slow non-linear SP); SP2 was comprised of spermatozoa with high velocities and high STR/LIN values (fast linear SP); SP3 was characterized with high VCL, and moderate LIN/STR (fast non-linear SP); and an additional SP0 was added comprising immotile spermatozoa. Total motility, progressive motility and VCL decreased after cryopreservation to approximately 50% of their value in fresh sperm, while the frequency of SPs characterized by high values of motility parameters declined in favor of those with low motility values and SP0. Motility values of fresh and cryopreserved spermatozoa which were washed with fresh extender after thawing decreased significantly after 24 h of storage at RT and after 72 h of storage at 4 °C, while cryopreserved sperm which remained in the original cryomedium faced a steep decline in motility after only 2 h of storage. As subpopulation frequencies followed this dynamic, this indicates that cryopreserved sperm should be washed with fresh extender in order to obtain favorable sperm kinematic properties after freezing. • Common carp sperm is not homogenous and contains subpopulations. • A total of three subpopulations with different kinematic properties were identified. • Cryopreservation and cold storage influenced the subpopulation dynamics. • Washing of sperm after cryopreservation enables longer preservation of quality. [ABSTRACT FROM AUTHOR]

In feline species, cooled transport of ovaries can be employed without detrimental effects to retrieve immature oocytes intended for in vitro embryo production purposes. Indeed, this is the most common way to collect gametes from gonads of wild, valuable animals after they die or are castrated far from specialized laboratories. However, fresh retrieved gametes are generally used, and their cryosensitivity is not known. This study employed ovariectomy-derived domestic cat gonads as a model for wild felids, and aimed to compare the yield and developmental competence of Cryotop-vitrified oocytes (VOs) collected and cryopreserved right after ovary excision (In loco -VOs) or after 24 h cooled transport of ovaries (Shipped-VOs). The number of collected oocytes was higher in In loco -VOs than in Shipped-VOs (mean ± SD: 8 ± 3.36 vs 5.6 ± 3.1, p = 0.05). In vitro embryo production resulted in similar maturation (35% for both vitrified groups, p = 1) and fertilization rates (In loco -VOs: 29.1%; Shipped-VOs: 22.2%; p = 0.295), but showed a difference in cleavage (In loco -VOs: 25.6%; Shipped-VOs: 14.5%; p = 0.0495). No differences were found in further embryo development. Taken together, results suggested that delayed oocyte vitrification after cooled transport of organs was feasible and allowed embryo development. However, the number of collected oocytes and the cleavage rate of matured oocytes were higher when oocyte vitrification was performed without delay after ovary excision, and this should be considered in gamete conservation programs for endangered felids. • Oocyte vitrification after cooled transport of ovaries allowed embryo development. • After ovary transport, retrieved oocytes were fewer than at ovary excision. • In vitro maturation of vitrified oocytes was not influenced by ovary transport. • Cleavage was lower in oocytes vitrified after ovaries cooled transport. [ABSTRACT FROM AUTHOR]

Abstract At refrigeration temperature, mouse embryos can retain their developmental ability for a couple of days. Previous research reports have focused on the effect of cool temperature on the development of 2-cell stage embryos, morulae or blastocysts and determined that the embryo still has the ability to produce offspring after about 48 h storage at refrigeration temperature. Here we examined whether refrigeration temperature affects the development of the eight-cell stage and if the stored eight-cell stage embryo can still be used as a host embryo for ES cell injection. Our results show that eight-cell stage embryos can develop into blastocysts and yield pups after cold storage for 24 and 48 h. After ES cell injection, stored eight-cell stage embryos can support ES cells developing to F0 pups. In summary, cool storage can preserve the developmental ability of eight-cell stage embryos for at least 48 h, allowing transportation of the embryos at refrigeration temperature between different labs and their subsequent use as host embryos for ES cell injection. [ABSTRACT FROM AUTHOR]

Abstract Cold storage is a common procedure for liver preservation in a transplant setting. However, during cold ischemia, the liver suffers molecular alterations that can affect its performance. Also, deleterious mechanisms set forth in the storage phase are exacerbated during reperfusion. This study aimed to identify liver proteins associated with injury during cold storage and/or normothermic reperfusion using the isolated perfused rat liver model. Livers from male rats were subjected to either (1) cold storage for 24 h, (2) ex vivo normothermic reperfusion for 90 min or (3) cold storage for 24 h followed by ex vivo normothermic reperfusion for 90 min. Then, the livers were homogenized and proteins were extracted. Protein expression between each experimental group and the control (freshly resected livers) was compared by two-dimensional (2D) gel electrophoresis. Protein identification was carried out by matrix‐assisted laser desorption/ionization time‐of‐flight spectrometry (MALDI‐TOF/TOF) using MASCOT as the search engine. 23 proteins were detected with significantly altered levels of expression among the different treatments, including molecular chaperones, antioxidant enzymes, and proteins involved in energy metabolism. Some of them have been postulated as biomarkers for liver damage while others had been identified in other organs subjected to ischemia and reperfusion injury. The whole data set will be a useful resource for studying deleterious molecular mechanisms that result in diminished liver function during storage and subsequent reperfusion. Graphical abstract Image 1 [ABSTRACT FROM AUTHOR]

Abstract Background There are currently two approaches to hypothermic preservation for most solid organs: static or dynamic. Cold storage is the main method used for static storage (SS), while hypothermic pulsatile perfusion (HPP) and other machine perfusion-based methods, such as normothermic machine perfusion and oxygen persufflation, are the methods used for dynamic preservation. HPP is currently approved for kidney transplantation. Methods We evaluated, for the first time, the feasibility of HPP on 11 human pancreases contraindicated for clinical transplantation because of advanced age and/or history of severe alcoholism and/or abnormal laboratory tests. Two pancreases were used as SS controls, pancreas splitting was performed on 2 other pancreases for SS and HPP and 7 pancreases were tested for HPP. HPP preservation lasted 24 h at 25 mmHg. Resistance index was continuously monitored and pancreas and duodenum histology was evaluated every 6 h. Results The main finding was the complete absence of edema of the pancreas and duodenum at all time-points during HPP. Insulin, glucagon and somatostatin staining was normal. Resistance index decreased during the first 12 h and remained stable thereafter. Conclusion 24 h hypothermic pulsatile perfusion of marginal human pancreas-duodenum organs was feasible with no deleterious parenchymal effect. These observations encourage us to further develop this technique and evaluate the safety of HPP after clinical transplantation. [ABSTRACT FROM AUTHOR]

Abstract We have developed a novel, intraluminal preservation solution that is tailored to the metabolic requirements of the intestine. This organ-specific solution addresses many of the problems associated with low temperature organ storage including energy, oxidative and osmotic stresses. However, conservation of energy levels remains one of the most difficult obstacles to overcome due to the inherent sensitivity of the mucosa to ischemia. Creatine-loading has become a popular and scientifically proven method of augmenting energy reserves in athletes performing anaerobic burst work activities. We hypothesized that if we could develop a method that was able to augment cellular energy levels, the structure and function of the mucosa would be more effectively preserved. The purpose of this study was to determine if creatine-loading is a feasible and effective strategy for preserving the intestine. Our data indicate that creatine loading has significant impact on energy levels during storage with corresponding improvements in mucosal structure and function. Both of our rodent models, a) continuous perfusion for 4 h and b) a single flush with our intraluminal preservation solution supplemented with 50 mM creatine, demonstrated significant improvements in creatine phosphate, ATP, Energy Charge and ATP/AMP following cold storage (P < 0.05). Notably, after 10 h creatine phosphate was 324% greater in Creatine-treated tissues compared to Controls (P < 0.05). Preferential utilization of glutathione in the Creatine group was effective at controlling oxidative injury after 10 h storage (P < 0.05). Improvements in barrier function and electrophysiology with creatine-treatment reflected superior mucosal integrity after 10 h storage; Permeability and Transepithelial resistance measurements remained at fresh tissue values. This was in stark contrast to Control tissues in which permeability rose to >300% of fresh tissue values (P < 0.005) and transepithelial resistance dropped by 95% (P < 0.005). After 10 h storage, Park's grading of histologic injury reflected extensive villus denudation (grade 4) in control tissues compared to healthy tissue (grade 0) in the Creatine group. This study demonstrates that a strategy of creatine supplementation of our intraluminal preservation solution facilitates the preservation of the intestinal mucosa during storage. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the impact of prolonged storage at 4 °C on survival of cat preantral follicles (PAFs) pre- and post-vitrification. Ovaries were obtained from 12 queens and transported at 4 ºC within 2–6 h. Parts of the ovaries were stored for an additional 24 h or 72 h. The ovarian cortex was dissected, analyzed for viability (neutral red - NR) and morphology (histology - HE and ultrastructural analysis by TEM) and vitrified. We used 2 mm biopsy punches to obtain equal size pieces as the experimental units. After NR assessment, each sample was fixed and embedded in paraffin for HE staining to determine the number of morphologically intact follicles. Another 2 mm piece of ovary was subjected to TEM. NR viability assessment and HE results showed a similar tendency with PAF survival postvitrification even after prolonged cooling at 24 h and 72 h. With TEM, integrity of mitochondria, plasma and basal membranes as well as the presence of pre-granulose cells of PAFs were documented postvitrification for the control group and 24 h prolonged storage group, but not after 72 h storage. Our results showed that cat PAFs can survive prolonged storage followed by vitrification. The described set of techniques are applicable towards creating a gamete bank for endangered feline species. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A growing number of fish species are endangered due to human activities. A short- or long-time preservation of gametes could conserve genetic resources of threatened fish species. The aim of this study was to evaluate a hypothermic condition for short-term preservation of spermatogonia and oogonia cells isolated from immature transgenic rainbow trout, Oncorhynchus mykiss , and to determine the maximum time point for further transplantation. Viability rate of germ cells was investigated after isolation and during storage at 4 °C up to 24 h. Dulbecco's modification of Eagle's medium supplemented with Hepes fetal bovine serum and l -glutamine was used as hypothermic storage media. The results showed that while viability decreased following 24 h storage, the remaining viable cells did not vary morphologically as well as GFP intensity retained similar to those observed in freshly isolated cells. The hypothermal storage study indicated that culture medium is suitable for preserving germ cells in the short periods of time. Simplicity, easily available culture media and low cost provide new insight into hypothermic conditions for preserving and transporting of germ cells for next applied and basic studies. [ABSTRACT FROM AUTHOR]

Yellow catfish ( Pelteobagrus fulvidraco ) is a promising aquaculture species in China with an increasing market demand. To serve the growing demand of male broodstock for artificial fertilization and the preservation of valuable strains for selective breeding, we tried to develop a species-specific cryopreservation protocol for yellow catfish sperm in this study. Important factors such as cryoprotectant, freezing height above the liquid nitrogen (LN) surface, dilution ratio, equilibration time, thawing temperature and cool storage before freezing were standardized. Among the cryoprotectants tested here, 10% Me 2 SO was the most suitable for sperm cryopreservation. Freezing at 7 cm above the LN surface for 10 min yielded the highest post-thaw motility. Further evaluation showed that dilution ratio of 1:3 and 1:5 produced higher post-thaw motility than semen diluted at 2:1, 1:1, 1:9 or 1:19. Equilibration times from 0 to 30 min did not cause significant differences in both equilibrated and post-thaw motility. Also, cool storage up to 24 h did not affect the suitability of sperm for cryopreservation. After thawing, sperm could be stored at 4 °C for 2 h without a reduction in motility parameters. With the combination of optimized freezing conditions, the fertilization and hatching rate of cryopreserved sperm were 87.1 ± 5.2% and 78.5 ± 7.4%, respectively, which were similar to those of fresh sperm (91.8 ± 3.5% and 83.7 ± 2.5%). In general, the cryopreservation protocol optimized here would facilitate breeding practice and hatchery operation in this economically important fish. [ABSTRACT FROM AUTHOR]

Somatic embryogenesis (SE) is considered as the most-effective method for vegetative propagation of Norway spruce ( Picea abies L. Karst). For mass propagation, a cryopreservation method able to handle large numbers of embryogenic tissues (ETs) reliably and at low costs is needed. The aim of the present study was to compare pretreatments, cryoprotectants and slow-cooling devices for cryopreservation of Norway spruce ETs, with 12 variations of methods and a total of 136 spruce genotypes. Secondly, possible applications for cold storage of mature somatic embryos were studied with the aim of developing a flexible time window for embling production. At best, 100% of the embryogenic lines were recovered following cryopreservation, but the results varied among the sets of lines. Also physiological condition of the tissues, pre-treatment and cryoprotectant applied, as well as the slow-cooling device used were found to affect the recovery. The best option for cryopreservation of Norway spruce is to select fresh growth from young ETs as samples, pretreat them on semi-solid medium with increasing sucrose concentration (0.1 M for 24 h; 0.2 M for another 24 h), apply a mixture of polyethylene glycol 6000, glucose, and dimethylsulfoxide, 10% w/v each, as cryoprotectant and use a programmable freezer with a slow cooling rate (0.17 °C/min). On average, 87% of the genotypes can be recovered, without any effect on their genetic fidelity, as shown by microsatellite markers and embryo production capacity. Mature somatic embryos of Norway spruce can also be safely cold-stored at +4 °C, without adverse effects on their germination ability. [ABSTRACT FROM AUTHOR]

Hypothermic machine perfusion (HMP) and static cold storage (SCS) are the two methods used to preserve deceased donor kidneys prior to transplant. This study seeks to characterise the metabolic profile of HMP and SCS porcine kidneys in a cardiac death donor model. Twenty kidneys were cold flushed and stored for two hours following retrieval. Paired kidneys then underwent 24 h of HMP or SCS or served as time zero controls. Metabolite quantification in both storage fluid and kidney tissue was performed using one dimensional 1 H NMR spectroscopy. For each metabolite, the net gain for each storage modality was determined by comparing the total amount in each closed system (i.e. total amount in storage fluid and kidney combined) compared with controls. 26 metabolites were included for analysis. Total system metabolite quantities following HMP or SCS were greater for 14 compared with controls (all p < 0.05). In addition to metabolic differences with control kidneys, the net metabolic gain during HMP was greater than SCS for 8 metabolites (all p < 0.05). These included metabolites related to central metabolism (lactate, glutamate, aspartate, fumarate and acetate). The metabolic environments of both perfusion fluid and the kidney tissue are strikingly different between SCS and HMP systems in this animal model. The total amount of central metabolites such as lactate and glutamate observed in the HMP kidney system suggests a greater degree of de novo metabolic activity than in the SCS system. Maintenance of central metabolic pathways may contribute to the clinical benefits of HMP. [ABSTRACT FROM AUTHOR]

Cold storage of two-cell embryos at refrigerated temperatures is a useful means to ship genetically engineered mice. We previously reported that M2 medium maintained the developmental ability of two-cell embryos for 48 h at 4 °C, and offspring were obtained from embryos transported by a courier service under refrigerated temperatures. The limitation of 48 h practically restricts the shipping destination of the embryos. To enhance the applicability of the cold-storage technique, prolonging the time to maintain developmental ability of the embryos is required. Oxidative stress may be a cause of the declining developmental ability of cold-stored embryos. However, the effect of oxidative stress on developmental ability of embryos has not been investigated. We examined intracellular glutathione (GSH) levels of cold-stored two-cell embryos to evaluate the effect of oxidative and investigated the efficacy of adding N-acetyl cysteine (NAC) to the preservation medium on the developmental ability of cold-stored embryos and transported two-cell embryos at refrigerated temperatures. Intracellular GSH levels of two-cell embryos decreased by cold storage for longer than 72 h, whereas NAC recovered this reduction and improved the developmental ability of embryos cold-stored for 96 h. In the transport experiment, the developmental rate of transported two-cell embryos to offspring was increased by adding NAC to the preservation medium. We found that NAC prolonged the storage period of two-cell embryos and maintained the developmental ability by alleviating the reduction of intracellular GSH. These findings will improve the technique of cold-storage of two-cell embryos to facilitate efficient transport of genetically engineered mice worldwide. [ABSTRACT FROM AUTHOR]

Liver transplantation is currently the preferred treatment option for end-stage liver disease. Donation after cardiac death was a common practice in the early years of organ donation before brain death criteria were established. Those organs were subjected to variable periods of warm ischemia that might intensify cold ischemia/reperfusion injuries. In the present, shortage of brain dead donors has led to the reassessment of organ donation after cardiac death. Since many cytoprotective roles have been describe for H 2 S during ischemia/reperfusion on a variety of tissues, we hypothesized that graft exposure to this bioactive gas might improve preservation of non-heart beating donated organs. Therefore, to establish a rat model of donation post-cardiac arrest and using this approach to judge H 2 S delivery effects on graft hypothermic preservation, were the main objectives of this investigation. Cardiopulmonary arrest was induced in sedated rats by overload of potassium (K + ). Livers were surgically removed and subsequently stored in HTK Solution (Histidine–tryptophan–ketoglutarate) at 0–4 °C. After 24 h of hypothermic preservation, livers were rewarmed in an ex vivo model. Three experimental groups were established as follows: I – Livers procured before cardiac death and cold stored 24 h in HTK (BCD); II – Livers procured after cardiac death (45 min) and cold stored 24 h in HTK (ACD); III – Livers procured after cardiac death (45 min) and cold stored 24 h in HTK + 10 μM Sodium Sulfide (Na 2 S) (ACD-SS). Data suggest that after 45 min of warm ischemia, viability parameters assessed during reperfusion in the ex vivo model were significantly impaired. Real time PCR revealed that after ex vivo reperfusion there is an increased expression of HO-1 and TNF-α and a modest drop in Bcl-2 mRNA, which could be interpreted as the cellular response to the hypoxic insult sustained during warm ischemia. On the other hand, warm ischemic livers exposed to H 2 S during cold storage, improved microcirculation, morphology and viability parameters during ex vivo reperfusion and showed significant modulation of HO-1 mRNA expression. In conclusion, HTK supplementation with Na 2 S arose as a potential treatment to recover non-heart beating harvested organs. Furthermore, an appropriate model of cardiac dead liver donors was successfully developed. [ABSTRACT FROM AUTHOR]

Cold storage (at 4 °C) offers a compromise between the benefits and disadvantages of cooling. It allows storage of organs or cells for later use that would otherwise quickly succumb to warm ischemia, but comprises cold ischemia that, when not controlled properly, can result in severe damage as well by both similar and unique mechanisms. We hypothesized that polyethylene glycol (PEG) 35 kDa would ameliorate these injury pathways and improve cold primary hepatocyte preservation. We show that reduction of the storage temperature to below zero by means of supercooling, or subzero non-freezing, together with PEG supplementation increases the viable storage time of primary rat hepatocytes in University of Wisconsin (UW) solution from 1 day to 4 days. We find that the addition of 5% PEG 35 kDa to the storage medium prevents cold-induced lipid peroxidation and maintains hepatocyte viability and functionality during storage. These results suggest that PEG supplementation in combination with supercooling may enable a more optimized cell and organ preservation. [ABSTRACT FROM AUTHOR]

Abstract: Biorepositories worldwide collect human serum samples and store them for future research. Currently, hundreds of biorepositories across the world store human serum samples in refrigerators, freezers, or liquid nitrogen without following any specific cryopreservation protocol. This method of storage is both expensive and potentially detrimental to the biospecimens. To decrease both cost of storage and the freeze/thaw stresses, we explored the feasibility of storing archival human serum samples at non-cryogenic temperatures using isothermal vitrification. When biospecimens are vitrified, biochemical reactions can be stopped, the specimen ceases to degrade, and macromolecules can be stabilized without requiring cryogenic storage. In this study, 0.2, 0.4, or 0.8M trehalose; 0, 0.005 or 0.01M dextran; and 0 or 10% (v/v) glycerol was added to human serum samples. The samples were either dried diffusively as sessile droplets or desiccated under vacuum after they are adsorbed onto glass microfiber filters. The glass transition temperatures (T g) of the desiccated samples were measured by temperature-ramp Fourier Transform Infrared (FTIR) spectroscopy. Sera samples vitrified at 4±2°C when 0.8M trehalose and 0.01M dextran were added and the samples were vacuum dried for two hours. Western immunoblotting showed that vitrified serum proteins were minimally degraded when stored for up to one month at 4°C. About 80% of all proteins were recovered after storage at 4°C on glass microfiber filters, and recovery did not decrease with storage time. These results demonstrated the feasibility of long-term storage of vitrified serum at hypothermic (and non-cryogenic) temperatures. [Copyright &y& Elsevier]

Abstract: During hypothermic preservation of cells (0–4°C), metabolism is diminished and energy-dependent transport processes are arrested. The effect of hypothermic preservation of hepatocytes in endocytic transport following rewarming has not been previously reported. We evaluated the uptake of EGF (Epidermal Growth Factor) ligand conjugated to fluorescent Quantum Dots (QDs) probes in rat hepatocytes after 24 and 72h cold storage in University of Wisconsin (UW) solution at 4°C. QDs uptake was visualized during rewarming to 37°C under air or, in a second approach, at the end of rewarming under 5% CO2. After 24h in UW solution, QDs were internalized under both rewarming conditions similar to non-preserved hepatocytes and cells maintained a normal cytoskeleton distribution. However, in hepatocytes preserved 72h none of the cells internalized QDs, which remained bound to the membranes. After rewarming, this group showed diminished actin staining and 60% reduction in ATP levels, while viability was maintained at ∼70%. Our results present evidence that, hypothermic preservation for 72h in UW solution at 4°C does not prevent EGFR (epidermal growth factor receptor) activation but irreversibly impairs endocytic uptake upon EGF stimulation; presumably due to actin cytoskeleton disassembling besides reduced ATP pool. Our approach can be applied on other membrane receptor systems and with other hypothermic preservation solutions to understand the effect of cooling in endocytic transport and to determine the optimal cold storage period. [Copyright &y& Elsevier]

Abstract: The holding time (HT) is the period during which an ejaculate, either in a raw or diluted state, is held at 17°C before further processing for cold-storage. In boars, the HT positively influences select sperm quality parameters of semen cooled from 15 to 5°C, a range in temperature during which plasma membrane remodeling occurs. Objective insight into the effect of HT on plasma membrane organization remains unknown. Therefore, the present work sought to elucidate if HT contributes to minimizing alterations in boar sperm plasma membrane fluidity at the initial step of the cooling process in a cryopreservation practice (holding at 5°C) and in relation with select sperm quality parameters. Nineteen ejaculates from five boars were collected and processed according to different treatments: T1) Fresh diluted semen, 0h at 17°C; T2) Fresh diluted semen, 24h at 17°C (HT); T3) Sperm from T1 in a lactose-egg yolk (LEY) extender, 3h at 5°C; T4) Sperm from T2 in LEY, 3h at 5°C; T5) Sperm from T1 in LEY, 24h at 5°C; T6) Sperm from T2 in LEY, 24h at 5°C. Sperm motility was assessed using CASA, and sperm plasma membrane integrity and fluidity were evaluated by flow cytometry with dual labeling (M540/YO-PRO®-1). Results demonstrated that the lack of exposure to a HT (T5) results in reduced sample motility compared to those having a HT (T6), with sperm exposed to HT exhibiting less plasma membrane fluidity. Collectively, these results provide empirical evidence that incorporation of a HT in semen processing protects boar sperm against cold injury through maintenance of lipid architecture of the plasma membrane. [Copyright &y& Elsevier]

Abstract: The exchange of genetically engineered mouse strains between research facilities requires transporting fresh mouse sperm under refrigerated temperatures. Although sperm generally maintains fertility for 48h at cold temperatures, in vitro fertilization rates of C57BL/6 mouse sperm are low after 48-h cold storage. Furthermore, 48h is often not sufficient for the specimens to reach their destinations. To increase the availability of this technology, we aimed to extend the cold storage period while maintaining sperm fertility. In this study, we determined the optimal medium for sperm preservation and evaluated the effect of reduced glutathione in the fertilization medium on sperm fertility after cold storage. We found that higher fertility levels were maintained after 72-h cold storage in the preservation medium Lifor compared with storage in paraffin oil, M2 medium, or CPS-1 medium. In addition, 1.0mM glutathione enhanced sperm fertility. After transporting sperm from Asahikawa Medical University to our laboratory, embryos were efficiently produced from the cold-stored sperm. After transfer, these embryos developed normally into live pups. Finally, we tested the transport system using genetically engineered mouse strains and obtained similar high fertilization rates with all specimens. In summary, we demonstrated that cold storage of sperm in Lifor maintains fertility, and glutathione supplementation increased the in vitro fertilization rates of sperm after up to 96h of cold storage. This improved protocol provides a simple alternative to transporting live animals or cryopreserved samples for the exchange of genetically engineered mouse strains among research facilities. [Copyright &y& Elsevier]

Abstract: Background: Delayed graft function still represents a major complication in clinical kidney transplantation. Here we tested the possibility to improve functional outcome of cold stored kidneys a posteriori by hypothermic reconditioning using retrograde oxygen persufflation (ROP) immediately prior to reperfusion. Methods: Kidneys from female German Landrace pigs were flushed with Histidine–Tryptophan–Ketoglutarate (HTK) solution and cold-stored for 18h (control). Some grafts were subsequently subjected to 90min of retrograde oxygen persufflation (ROP) via the renal vein during cold preservation. Early graft function of all kidneys was assessed thereafter by warm reperfusion in vitro (n =6, resp.). Results: Renal function upon reperfusion was significantly enhanced by ROP with an approximately twofold increase in renal clearances of creatinine and urea. ROP also led to higher renal vascular flow rates, enhanced urine output and mitigated histological alterations. Conclusion: It is concluded that initial graft function can be improved by 90min of hypothermic gaseous oxygenation after arrival of the preserved organ in the transplantation clinic. [Copyright &y& Elsevier]

Abstract: We previously reported that machine perfusion (MP) performed at 20°C enhanced the preservation of steatotic rat livers. Here, we tested whether rat livers retrieved 30min after cardiac arrest (NHBDs) were better protected by MP at 20°C than with cold storage. We compared the recovery of livers from NHBDs with organs obtained from heart beating donors (HBDs) preserved by cold storage. MP technique: livers were perfused for 6h with UW-G modified at 20°C. Cold storage: livers were perfused in situ and preserved with UW solution at 4°C for 6h. Both MP and cold storage preserved livers were reperfused with Krebs-Heinselet buffer (2h at 37°C). AST and LDH release and mitochondrial glutamate dehydrogenase (GDH) levels were evaluated. Parameters assessed included: bile production and biliary enzymes; tissue ATP; reduced and oxidized glutathione (GSH/GSSG); protein–SH group concentration. Livers preserved by MP at 20°C showed significantly lower hepatic damage at the end of reperfusion compared with cold storage. GDH release was significantly reduced and bile production, ATP levels, GSH/GSSG and protein–SH groups were higher in livers preserved by MP at 20°C than with cold storage. The best preserved morphology and high glycogen content was obtained with livers submitted to MP at 20°C. Liver recovery using MP at 20°C was comparable to recovery with HBDs. MP at 20°C improves cell survival and gives a better-quality of preservation for livers obtained from NHBDs and may provide a new method for the successful utilization of marginal livers. [Copyright &y& Elsevier]

Abstract: Background: The benefit of carbon monoxide as applied by controlled, continuous gaseous persufflation during liver preservation on postischemic graft recovery was investigated in an isolated rat liver model. Methods: Livers from male Wistar rats were retrieved 30min after cardiac arrest of the donor and subjected to 18h of cold storage. Some grafts were subjected to gaseous persufflation with carbon monoxide (CO, dissolved in nitrogen) during static cold storage at a concentration of 50ppm or 250ppm. Graft viability was assessed thereafter upon warm reperfusion in vitro. Results: CO-persufflation significantly reduced cellular enzyme loss (maximal at 50ppm) and functional recovery (bile production and energy charge) upon reperfusion by about 50%. The effect was associated with a reduction of free radical-induced lipid peroxidation, lower vascular perfusion resistance, and improved mitochondrial ultrastructure. Conclusion: Viability of cold stored liver grafts can be notably augmented by gaseous ex vivo application of low dose CO to the isolated organ. [ABSTRACT FROM AUTHOR]

Abstract: Objectives: Especially for preservation of marginal donor organs, machine perfusion (MP) and retrograde oxygen persufflation (ROP) are alternatives to cold storage (CS). Using a porcine kidney autotransplantation model we compared metabolic and morphologic effects of CS, ROP, and MP on kidneys exposed to warm ischemia. Methods: Kidneys of 21 pigs were exposed in situ to warm ischemia for 60min. The kidneys were randomly allocated to three experimental groups, each receiving a 4-h treatment of either cold storage, machine perfusion, or retrograde oxygen persufflation. Tissue samples were examined for malondialdehyde and histological changes. Daily blood samples were examined for creatinine levels. Results: Seven days after transplantation, the plasma creatinine levels in the CS and MP groups were still significantly elevated above the baseline values. In the ROP group, all animals exhibited nearly normal creatinine levels. Malondialdehyde, an indicator of lipidperoxidation, was dramatically increased in the machine perfused kidneys on day 7, whereas the malondialdehyde levels in the other two groups were near normal values. The MP kidneys exhibited the most striking histological changes. Conclusion: Though MP has been well introduced in organ transplantation, in our opinion, it must still be optimized and standardized. It is necessary to clarify questions such as whether there is a need for oxygenation during perfusion, the length of perfusion, the impact of pressure, and the effects of additional scavengers. The results of the present study suggest the reconsideration of the ROP-technique for the preservation of predamaged donor grafts especially of NHBD and further studies, comparing MP and ROP upon long term preservation are strongly encouraged. [Copyright &y& Elsevier]

Abstract: Cryopreservation of 2-cell embryos is an effective technology for storage of genetically engineered mouse strains. Transport of genetically engineered mice between laboratories has frequently been performed using such cryopreserved 2-cell embryos. However, the receiving laboratory requires proficient skills and special instruments to obtain live young from cryopreserved and transported embryos. Therefore, in this study, we tried to address the storage and transport of vitrified/warmed 2-cell embryos at a cold temperature. In cold storage experiments, the development rates of 2-cell embryos stored in M2 medium for 24, 48 and 72h into blastocysts were relatively high (83%, 63% and 43%, respectively). Although, 2-cell embryos stored in PB1 and mWM maintained the developmental potency for 24h, the rates were markedly decreased to low levels after 48h (PB1: 0%; mWM: 5%). In transport experiments, many pups were obtained from vitrified/warmed 2-cell embryos transported at a cold temperature in all receiving laboratories (incidence of successful development: 49%; 249/511). In summary, short-term storage and transport of vitrified/warmed 2-cell embryos in M2 medium at a cold temperature can maintain their ability to develop into live young. [Copyright &y& Elsevier]

Abstract: Conventional cold storage and cryopreservation methods for hops (Humulus lupulus L.) are available but, to our knowledge, the genetic and epigenetic stability of the recovered plants have not been tested. This study analyzed 51 accessions of hop using the molecular techniques, Random Amplified DNA Polymorphism (RAPD) and Amplified Fragment Length Polymorphism (AFLP), revealing no genetic variation among greenhouse-grown controls and cold stored or cryopreserved plants. Epigenetic stability was evaluated using Methylation Sensitive Amplified Polymorphism (MSAP). Over 36% of the loci were polymorphic when the cold and cryo-treated plants were compared to greenhouse plants. The main changes were demethylation events and they were common to the cryopreserved and cold stored plants indicating the possible effect of the in vitro establishment process, an essential step in both protocols. Protocol-specific methylation patterns were also detected indicating that both methods produced epigenetic changes in plants following cold storage and cryopreservation. [Copyright &y& Elsevier]

Abstract: 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 (12–14h) 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 24h at 4°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. [Copyright &y& Elsevier]

Abstract: Background: Cold ischemic injury plays an important role in short- and long-term function of kidneys after transplant. Antimicrobial peptides have not previously been studied for their impact on cold ischemia in transplanted kidneys. Methods: Bactenecin (l- and d-forms) was added to University of Wisconsin (UW) preservation solution for 3-day cold storage of dog kidneys. Effects on membrane permeability were studied in synthetic liposomes and in kidney cortex tissue slices. The role of bactenecin as a tissue mitogen and direct cytoskeletal stabilizer were studied with cultured cells and in vitro. Results: Bactenecin (both l- and d- forms) resulted in significant decreases in postoperative serum creatinine and time required for return of creatinine to the normal range showing the effect was independent of chirality. Bactenecin permeabilized synthetic liposomes and altered kidney cortex tissue slice membrane permeability characteristics, irrespective of chirality. Neither did bactenecin act as a mitogen for either primary renal tubule or Madin–Darby canine kidney (MDCK) cells stored in UW solution, nor did it appear to directly affect cytoskeletal dynamics. Conclusions: These results show that the antimicrobial peptide bactenecin can improve the quality of static cold storage of kidneys. The mechanism of its action is independent of receptor binding and does not appear to involve either an effect on the cytoskeleton or via activity as a mitogen. Current evidence best supports the hypothesis that bactenecin protects against cold ischemic injury by a controlled permeabilization of the membranes of the kidney during cold storage. [Copyright &y& Elsevier]

In cultured renal tubular cells hypothermia results in cell damage caused by iron-dependent formation of reactive oxygen species. It is unknown whether cold preservation affects function of renal vessels. Rat renal arcuate arteries were stored in a physiological salt solution at 4 °C for 24 h and compared to control arteries (not stored). To some of the stored arteries the iron chelator 2,2′-dipyridyl was added. Endothelium-independent vasoconstriction was assessed by cumulative concentration–response curves for potassium and phenylephrine in a small vessel myograph. Endothelium-independent vasodilation was assessed with sodium nitroprusside and endothelium-dependent vasodilation with histamine. Cold storage for 24 h did not affect vascular reactivity of renal small arteries and no influence of the iron chelator was seen. Since 24 h of cold storage considerable damages renal tubular cells both in vitro and after kidney transplantation, these results suggest that renal arteries are less sensitive to cold-induced damage than tubular cells. [Copyright &y& Elsevier]

One mode of action of protectants in the storage of biological materials is by promoting the formation of a vitrified state on cooling or drying. In the case of preservation by drying, the glassy material comprises a low water content mixture of protectant and organic material. The protectant must on drying form a glassy state of glass transition temperature (Tg) above the desired storage temperature. However, in some applications it must also be easily transported through cell membranes and this restricts the choice to a relatively limited number of small molecules, which typically exhibit very low glass transition temperatures. In this work we describe a self-polymerizing protectant comprising an inorganic salt and a small hydroxy functional molecule such as glycerol. This forms co-ordinate polymer chains of high Tg on drying but rapidly depolymerizes into the original components on rehydration. The polymerization process is general for polyhydroxy compounds including glucose and related compounds. [Copyright &y& Elsevier]

Natural antifreeze proteins (AFPs) not only inhibit freezing at high subzero temperatures; they have the additional properties of inhibiting the recrystallization of ice during warming and of preventing devitrification. The natural AFP that occurs in the roots of cold-acclimated carrots can be extracted reasonably simply and is non-toxic: it was selected for study as a possible ingredient of the vitrification mixtures that are being developed for use in tissue cryopreservation. For this application, it would be essential for the AFP to remain active during prolonged storage at very low temperatures. For logistic reasons, it would also be essential to have an effective method of storage of the purified AFP itself. In this study, carrot AFP was isolated and purified, and its ability to inhibit recrystallization was monitored over 40 weeks of storage at −80 or −196 °C. The data revealed a progressive decrease in activity during storage, reaching half the original activity in 10–20 weeks and only 2–3% of the original activity at 40 week. These data suggest that carrot AFP will not be effective in tissue cryopreservation. [Copyright &y& Elsevier]

The hatching performance of embryos of the common carp (Cyprinus carpio L.) was examined after 1, 7, 14, 21, or 28 days of storage at −8, −6, −4, −2, 0, 2, or 4 °C with different concentrations of methanol (0.5–7.0 M in 0.5 M steps) or varying concentrations of methanol in 0.1 M sucrose or trehalose. Preserved embryos failed to hatch after storage at −8 and −6 °C, regardless of the duration of storage or the concentrations tested. Likewise, there was no hatching out above 5.0 M concentration of methanol, even with the addition of sucrose or trehalose. After storage at 2 or 4 °C, the hatching rate was higher with mixtures of methanol (1.5 M) and trehalose (0.1 M) than with methanol plus sucrose or methanol alone. At 4 °C, the solution containing 1.5 M methanol supplemented with trehalose gave the highest hatching response of embryos stored for 14 days. Comparison of hatching after 24 h of storage at the effective temperatures (−4, −2, 0, 2, and 4 °C) revealed that low concentrations of methanol were effective at high temperatures and high concentrations at sub-zero temperatures. The combination of 0.1 M trehalose with 1.5 M methanol gave the highest percentage hatching out both at 4 and 2 °C. At 0 °C, the highest percentage hatching occurred with 0.1 M trehalose plus 2.5 M methanol and at −2 and 4 °C, the best results were with 0.1 M trehalose plus 3.0 M methanol. [Copyright &y& Elsevier]

Post-transplantation thrombosis may occur in donor segments of iliac arteries and livers following surgical removal and storage in University of Wisconsin (UW) solution for transplantation. We have previously suggested that purine receptors are vulnerable to denaturation after UW storage. The aims of the present study were to determine what particular subtypes of purine P2Y receptors in rabbit thoracic aorta deteriorate after 8 days of UW storage by studying vascular reactivity to acetylcholine, ATP, 2MeSATP and UTP. Ring segments of aortae from male New Zealand White rabbits were mounted upon fine-wire myographs and vasodilatation to the above agents tested on fresh tissue, and after 8 days of UW storage. Vasodilatation to ATP was attenuated by 100 μM L-NAME in fresh tissue suggesting that the relaxant response was, in part, due to nitric oxide (NO). P2Y-mediated relaxation to ATP was significantly attenuated by UW storage and cholinergic responses were not. This attenuated relaxation to ATP was not further attenuated by L-NAME, suggesting a loss of the NO-dependent mechanism. De-endothelialisation indicated that UTP-mediated vasorelaxation, via P2Y2 receptors, was endothelium-dependent. Any residual endothelium-independent relaxation to UTP was abolished by UW storage and endothelium-dependent UTP relaxation was reduced to the same level as that seen in fresh, de-endothelialised tissue. In contrast responses to 2MeSATP, via P2Y1 receptors, were predominantly endothelium-independent and were only partially attenuated by UW storage. Responses to pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS) and L-NAME suggested that vasorelaxation to 2MeSATP and UTP was mediated by P2Y1 and P2Y2 receptors, respectively. It is therefore concluded that UW storage predominantly decreases P2Y2 receptor-mediated vascular reactivity. [Copyright &y& Elsevier]