Procurement of donor pancreases for islet isolation and transplantation is not yet widely practiced due to concerns about post-mortem ischemia upon functional islet yields. Perfusion/preservation technology can help to circumvent ischemic injury and is applied in this study to porcine pancreata (Px) prior to islet isolation. Px harvested from adult pigs were assigned to one of three preservation treatment groups:G1) Fresh controls - processed immediately with minimum cold ischemia(<1h) G2) Static Cold Storage-flushed with cold UW-Viaspan and stored at 2–4ºC for 24h, and G3) Hypothermic Machine Perfusion (HMP)-perfused on a pulsatile LifePort® machine with KPS1 solution at 4–7ºC and low pressure(10mmHg) for 24h. Islet isolation was then accomplished using conventional methods and standard accepted product release criteria were used to assess islet yield and function. Islet yield was markedly different between the treatment groups and the increased yield in the HMP group over the static cold storage in UW-Viaspan was statistically significant (p<0.05). Functionally, the islets from each experimental group were equivalent and not significantly different to fresh controls (G1). Dithizone staining for islets showed a consistently more uniform digestion of the Px from G3 compared with G1 and G2, with greater separation of the tissue and less entrapped islets. HMP for 24h is well tolerated leading to moderate edema but no loss of function of the harvested islets. The edema appears to aid in enzymatic digestion producing a greater yield and purity of islets compared with Px subjected to 24h of static cold storage.
Machine perfusion (MP) has potential benefits for marginal organs such as from deceased from cardiac death donors (DCD). However, there is still no consensus on MP benefits. We aimed to determine machine perfusion benefits on kidney grafts.
We evaluated kidney grafts preserved in ViaspanUW or KPS solutions either by CS or MP, in a DCD pig model (60 min warm ischemia + 24 h hypothermic preservation). Endpoints were: function recovery, quality of function during follow up (3 month), inflammation, fibrosis, animal survival.
ViaspanUW-CS animals did not recover function, while in other groups early follow up showed similar values for kidney function. Alanine peptidase and β-NAG activities in the urine were higher in CS than in MP groups. Oxydative stress was lower in KPS-MP animals. Histology was improved by MP over CS. Survival was 0% in ViaspanUW-CS and 60% in other groups. Chronic inflammation, epithelial-to-mesenchymal transition and fibrosis were lowest in KPS-MP, followed by KPS-CS and ViaspanUW-MP.
With ViaspanUW, effects of MP are obvious as only MP kidney recovered function and allowed survival. With KPS, the benefits of MP over CS are not directly obvious in the early follow up period and only histological analysis, urinary tubular enzymes and red/ox status was discriminating. Chronic follow-up was more conclusive, with a clear superiority of MP over CS, independently of the solution used. KPS was proven superior to ViaspanUW in each preservation method in terms of function and outcome. In our pre-clinical animal model of DCD transplantation, MP offers critical benefits.
We previously demonstrated that adding pyruvate to Perfadex® increased graft metabolism during 24 hours storage and improved reperfusion lung function. This increased metabolism was associated with progressively lower storage solution pH over the preservation interval. We hypothesized that more effective pH regulation would result in further improvements in lung survival after hypothermic storage. Rat lungs were stored for 24 hours in Perfadex, Perfadex with N-2-hydroxyethylpiperazine-propanesulfonic acid buffer (HEPES), pyruvate-modified Perfadex, and pyruvate-modified Perfadex with HEPES. pH change in the storage solution was measured. Structural lung injury was evaluated on hematoxylin-eosin stained tissue sections. Cell death was quantified by measuring necrotic cells by trypan blue exclusion and apoptotic cells by TUNEL assay. Lungs stored in Perfadex® demonstrated the greatest degree of cell death. Lungs from the Pyruvate group exhibited decreased cell death despite greater acidosis. The addition of HEPES reduced cell death and preservation solution acidosis in both Perfadex and pyruvate-modified Perfadex (p<.05). Almost all cell death resulted from necrosis. Adding pyruvate to the preservation solution increases acid formation during storage but reduces cell death. HEPES ameliorates this acidosis and reduces allograft cell destruction. Increasing the preservation solution buffering capacity may be a simple strategy to improve lung preservation for transplantation.
Most of the tissue used for penetrating keratoplasty is issued through eye banks that store the corneoscleral button either in hypothermic storage at 2–6°C or in organ culture at 31–37°C.
These two preservation techniques differ in technical aspects, tissue evaluation possibilities, storage time and microbiological safety. Hypothermic storage is simple and requires little expensive equipment. In general a pre-storage evaluation of the endothelium is performed by specular microscopy and storage time is usually around 7–10 days. Organ culture is a relatively complicated technique requiring more expertise and well-equipped facilities. Evaluation of the endothelium is not only performed before storage, but is routinely performed after storage through the use of light microscopy. With organ culture the allowed storage period is longer, up to four weeks. The vulnerability of organ culture to microbial contamination can be turned into an advantage because it allows the detection of residual micro-organisms on the cornea before surgery. Both preservation techniques seem to result in similar graft survival.
The method of choice for preservation of the donor cornea is dictated by a number of factors mentioned in this review and this helps to explain the geographical differences in the use of the different techniques.
Organ culture; Hypothermic storage; Eye banking
Osteochondral allografts are an increasingly popular treatment for the repair of articular cartilage lesions. Current tissue bank protocols require bacteriological testing that takes from 21 to 28 days to process. During this time, tumor necrosis factor-alpha TNF-α (a pro-apoptotic cytokine) is upregulated resulting in loss of chondrocyte viability. To date, etanercept (a cytokine inhibitor) has not been studied in the current storage paradigm with the intention of preserving cell viability.
To assess whether or not the addition of Etanercept can improve the chondrocytic viability of osteochondral allograft during storage.
Controlled, randomized and blinded in vitro laboratory study.
Osteochondral allografts were harvested from eight Boer goat femurs and placed into storage media and stored at 4°C for 28 days. The experimental group was supplemented with 10 µg/mL of Etanercept. After storage, cell viability was assessed by live/dead staining and confocal microscopy. Specimens were also analyzed histologically and underwent histomorphological analysis. TNF-α expression was measured with semi-quantitative PCR.
At 28 days, the percent viability of the superficial zone in etanercept-treated allografts was maintained at significantly higher levels than those measured in the untreated group (69.3 ± 9.4 compared to 47.8 ± 19.1, p=0.01). No difference was found histologically between the etanercept and the untreated group (i.e. safranin-O staining for GAG expression). Histomorphologic assessment showed no difference in indentation stiffness or roughness between groups. TNF-α expression was significantly decreased in the etanercept group compared to the untreated group.
Etanercept was able to maintain cell viability of osteochondral allografts significantly better than the current storage paradigm after 28 days storage.
Maintaining the viability of the superficial zone will benefit outcomes by facilitating joint articulation via improved lubrication. Additionally, maintaining the cellular viability for increased periods of time may allow a greater window of time in which a suitable recipient may be found.
Osteochondral; Allograft; Cartilage; Etanercept
BACKGROUND/AIMS—The use of "fresh" (hypothermically stored) and frozen amniotic membrane (AM) was compared in a patient with cicatricial pemphigoid with stem cell failure. The viability of both "fresh" and frozen AM epithelial cells was assessed after storage.
METHODS—AM was stored at either +4°C ("fresh") or at −80°C (frozen). A "fresh" graft was applied to the cornea following superficial keratectomy. Subsequently, a further frozen graft was applied to the same eye. Viability of the stored AM epithelium was assessed by investigating membrane integrity and mitochondrial activity.
RESULTS—In both cases the cornea re-epithelialised and visual acuity improved. Improvement, however, was not sustained.
CONCLUSION—Although both procedures led to an improvement in visual acuity, "fresh" tissue performed no better than frozen in promoting re-epithelialisation. The authors suggest that logistical, safety, and cost considerations outweigh any benefits of using "fresh" as opposed to frozen graft material.
Little is known about the long-term properties of fresh cold-stored osteochondral allograft tissue. We hypothesized fresh cold-stored tissue would yield superior material properties in an in vivo ovine model compared to those using freeze-thawed acellular grafts. In addition, we speculated that a long storage time would yield less successful grafts. We created 10-mm defects in medial femoral condyles of 20 sheep. Defects were reconstructed with allograft plugs stored at 4°C for 1, 14, and 42 days; control specimens were freeze-thawed or defect-only. At 52 weeks, animals were euthanized and retrieved grafts were analyzed for cell viability, gross morphology, histologic grade, and biomechanical and biochemical analysis. Explanted cold-stored tissue had superior histologic scores over freeze-thawed and defect-only grafts. Specimens stored for 1 and 42 days had higher equilibrium moduli and proteoglycan content than freeze-thawed specimens. We observed no difference among any of the cold-stored specimens for chondrocyte viability, histology, equilibrium aggregate modulus, proteoglycan content, or hypotonic swelling. Reconstructing cartilage defects with cold-stored allograft resulted in superior histologic and biomechanical properties compared with acellular freeze-thawed specimens; however, storage time did not appear to be a critical factor in the success of the transplanted allograft.
OBJECTIVES—It has been suggested that the humoral immune system plays a part in the pathogenesis of pulmonary fibrosis. Although circulating autoantibodies to lung protein(s) have been suggested, few lung proteins have been characterised. The purpose of this study is to determine the antigen recognised by serum of a patient with pulmonary fibrosis associated with dermatomyositis.
METHODS—To accomplish this, anti-small airway epithelial cell (SAEC) antibody in a patient's serum was evaluated using a western immunoblot.
RESULTS—An autoantibody against SAEC was found, and the antigen had a molecular weight of 62 kDa. Using the patient's serum, clones from the normal lung cDNA library were screened and demonstrated that anti-SAEC antibody in the patient's serum was against ADAM (A disintegrin and metalloprotease) 10.
CONCLUSION—This is the first report that demonstrates the existence of anti-ADAM 10 antibody in a patient with pulmonary fibrosis associated with dermatomyositis.
The study of small airway diseases such as post-transplant bronchiolitis obliterans syndrome (BOS) is hampered by the difficulty in assessing peripheral airway function either physiologically or directly. Our aims were to develop robust methods for sampling small airway epithelial cells (SAEC) and to establish submerged SAEC cultures for downstream experimentation.
SAEC were obtained at 62 post-transplant bronchoscopies in 26 patients using radiologically guided bronchial brushings. Submerged cell cultures were established and SAEC lineage was confirmed using expression of clara cell secretory protein (CCSP).
The cell yield for SAEC (0.956 ± 0.063 × 106) was lower than for large airway cells (1.306 ± 0.077 × 106) but did not significantly impact on the culture establishment rate (79.0 ± 5.2% vs. 83.8 ± 4.7% p = 0.49). The presence of BOS significantly compromised culture success (independent of cell yield) for SAEC (odds ratio (95%CI) 0.067 (0.01-0.40)) but not LAEC (0.3 (0.05-1.9)). Established cultures were successfully passaged and expanded.
Primary SAEC can be successfully obtained from human lung transplant recipients and maintained in culture for downstream experimentation. This technique will facilitate the development of primary in vitro models for BOS and other diseases with a small airway component such as asthma, cystic fibrosis and COPD.
Background. L-asparaginase is effective in treating canine and feline lymphoma, however chemotherapy poses a significant financial cost to veterinary clients, limiting therapy for many pets. Single dose vials result in significant drug wastage, and drug shortages limit consistent availability for pets. Hypothesis. E. coli-derived asparaginase retains enzymatic and antineoplastic activity in canine and feline lymphoma cells after cold storage. Methods. E. coli-derived asparaginase was cold-stored: refrigeration (7–14 days) and freezing (14 days–six months, one to three freeze/thaw cycles). Enzymatic activity of asparaginase was measured via a modified asparagine assay. Effects of cold-stored asparaginase on cell proliferation and cytotoxicity were measured in feline (MYA-1, F1B) and canine (17–71, OSW) lymphoma cells. Results. Cold-stored E. coli-derived asparaginase retains antineoplastic activity in all four cell lines tested. Cold-stored E. coli-derived L-asparaginase depletes asparagine and retains enzymatic activity. Duration of refrigeration, duration of freezing, and number of freeze-thaw cycles have minimal effect on asparaginase enzyme activity. Conclusions and Clinical Importance. This study establishes a scientific basis for long-term cold storage of reconstituted E. coli-derived asparaginase that may result in better utilization of limited drug resources and improve financial feasibility of E. coli-derived asparaginase as a therapeutic option for pets with lymphoma.
Purpose. Hypothermic machine perfusion systems seem more effective than the current static storage to prevent cold ischemic liver injury. Thus, we test an innovative hyperbaric hypothermic machine perfusion (HHMP), which combines hyperbaric oxygenation of the preservation solution and continuous perfusion of the graft. Methods. Rat livers were preserved with Celsior solution according to 4 different modalities: normobaric static preservation; hyperbaric static preservation at 2 atmosphere absolute (ATA); normobaric dynamic preservation, with continuous perfusion; hyperbaric dynamic preservation, with continuous perfusion at 2 ATA. After 24 h cold preservation, we assessed different parameters. Results. Compared to baseline, livers preserved with the current static storage showed severe ultrastructural damage, glycogen depletion and an increased oxidative stress. Normobaric perfused livers showed improved hepatocyte ultrastructure and ameliorated glycogen stores, but they still suffered a significant oxidative damage. The addition of hyperbaric oxygen produces an extra benefit by improving oxidative injury and by inducing endothelial NO synthase (eNOS) gene expression. Conclusions. Preservation by means of the present innovative HHMP reduced the liver injury occurring after the current static cold storage by lowering glycogen depletion and oxidative damage. Interestingly, only the use of hyperbaric oxygen was associated to a blunted oxidative stress and an increased eNOS gene expression.
Several studies have reported that pancreatic ductal preservation greatly improved the islet yield and function after cold storage. However, these studies were devoid of appropriate controls, such as vascular perfusion, which is routinely performed to preserve organs in the clinical setting. In this study, we created a vascular perfusion model using inbred rats, and investigated the effect of ductal injection on the islet yield and function after cold storage. Rat pancreases after 10 h cold ischemia were classified as follows: without ductal/vascular perfusion; with ductal injection; with vascular perfusion; and with ductal/vascular perfusion. The islet yield, function, viability, release of inflammatory mediators, and pathological changes in the exocrine tissues were assessed in the Hanks' Balanced Salt Solution (HBSS) model. The islet yield was also assesed by introducing University of Wisconsin Solution (UWS) and Histidine-Tryptophan-Ketoglutarate solution (HTK), which are the standard clinical preservation solutions. In the HBSS model, ductal injection and vascular perfusion significantly improved the islet yield compared with the control group. However, ductal injection showed no additional effects on the islet yield, function, viability and suppressing the release of inflammatory mediators when vascular perfusion was performed. Although ductal injection significantly decreased the apoptosis of exocrine cells, no beneficial effect on vacuolation was observed. In contrast, vascular perfusion significantly suppressed vacuolation in the exocrine tissues. Likewise, in the UWS and HTK model, ductal injection and vascular perfusion improved the islet yield compared with the control group. Nevertheless, the combination group showed no additional effects. These data suggest that ductal injection has no additional effect on islet yield and function after cold storage in a vascular perfusion model. We propose that ductal injection can be an effective and simple alternative for vascular perfusion prior to pancreas harvest, but is not necessary in most cases, since vascular perfusion is routinely performed.
Improved kidney preservation methods are needed to reduce ischemia-reperfusion (IR) injury in kidney allografts. Lifor is an artificial preservation solution comprised of nutrients, growth factors, and a non-protein oxygen and nutrient carrier. The current study compared the effectiveness of Lifor to University of Wisconsin solution (UW) in protecting rat kidneys from warm IR and cold storage injury.
Materials and Methods
In a warm IR model, rat kidneys were perfused in situ with either saline, UW, or Lifor for 45 minutes. Renal function and histology were assessed 24 hours later. In a cold IR model, kidney slices were cold stored in saline, UW, or Lifor at 4°C. Kidney injury was assessed by the release of lactate dehydrogenase (LDH) and immunoblot analysis for cleaved caspase-3.
Lifor perfusion significantly mitigated renal dysfunction and tubular injury at 24 hours compared to saline or UW. Lifor and UW prevented LDH release in hypoxic kidney slices in vitro, however activation of caspase-3 following hypoxia-reoxygenation was attenuated only with Lifor. Cold storage with Lifor or UW significantly decreased LDH release from kidney slices or normal rat kidney cells in comparison to storage in saline or culture media. After 24 hours of cold storage there was a significant decrease in cleaved caspase-3 in Lifor stored slices compared that seen following cold storage in saline or UW solution.
Lifor solution mitigates both warm and cold renal IR and appears to provide greater protection from apoptosis as compared to UW solution.
renal ischemia-reperfusion; cold preservation; UW solution; Lifor; apoptosis
Background & Aims
Primary hepatocytes are of great importance for basic research as well as cell transplantation. However, their stability, especially in suspension, is very low. This feature severely compromises storage and shipment. Based on previous studies with adherent cells, we here assessed cold storage injury in rat hepatocyte suspensions and aimed to find a cold storage solution that preserves viability, attachment ability and functionality of these cells.
Rat hepatocyte suspensions were stored in cell culture medium, organ preservation solutions and modified TiProtec solutions at 4°C for one week. Viability and cell volume were determined by flow cytometry. Thereafter, cells were seeded and density and metabolic capacity (reductive metabolism, forskolin-induced glucose release, urea production) of adherent cells were assessed.
Cold storage injury in hepatocyte suspensions became evident as cell death occurring during cold storage or rewarming or as loss of attachment ability. Cell death during cold storage was not dependent on cell swelling and was almost completely inhibited in the presence of glycine and L-alanine. Cell attachment could be greatly improved by use of chloride-poor solutions and addition of iron chelators. Using a chloride-poor, potassium-rich storage solution containing glycine, alanine and iron chelators, cultures with 75% of the density of control cultures and with practically normal cell metabolism could be obtained after one week of cold storage.
In the solution presented here, cold storage injury of hepatocyte suspensions, differing from that of adherent hepatocytes, was effectively inhibited. The components which acted on the different injurious processes were identified.
A study was undertaken to determine the survival patterns of Salmonella montevideo G4639 on and in tomatoes during storage and the efficacy of chlorine treatment on inactivation of the pathogen. The population of S. montevideo on the surfaces of inoculated tomatoes stored at 10 degrees C did not change significantly (P < 0.05) throughout an 18-day storage period. Significant increases in population occurred within 7 days and within 1 day when tomatoes were stored at 20 and 30 degrees C, respectively. A significantly higher number of cells was taken up by the core tissue of tomatoes tempered at 25 degrees C when the tomatoes were dipped in a suspension at 10 degrees C compared with the number taken up when the tomatoes were dipped in cell suspensions tempered at 25 or 37 degrees C. Populations remained constant throughout subsequent storage for 8 days at 10 degrees C, regardless of the temperature differential between tomatoes and the dip suspension. Storage of tomatoes at 20 degrees C, however, resulted in significant increases in populations of S. montevideo. Populations of the pathogen on the surfaces and in the core tissues of tomatoes were significantly reduced by dipping for 2 min in a solution containing 60 or 110 ppm (60 or 110 micrograms/ml) chlorine, respectively; however, treatment in solution containing 320 ppm chlorine did not result in complete inactivation. Populations of S. montevideo remained unchanged in chopped tomatoes stored at 5 degrees C for 216 h (9 days) but increased significantly after storage for 96 or 22 h at 20 or 30 degrees C, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
To compare UW-solution (UW) and Euro-Collins (EC) for long-term liver preservation we investigated
the morphology and metabolic capacity of rat liver after 18 and 42-hours cold-storage in either UW or
After harvesting the rat liver was transferred to a perfusion chamber where it was perfused for 10 min
with UW or EC at 4°C. Thereafter livers were stored at 4°C in UW or EC for 18 hours (both groups
n = 6) or for 42 hours (both groups n = 8). After 18-hr or 42-hr cold-storage a 2-hr warm perfusion
(37°C) was started with Krebs-Ringer solution with carbogen to which 125Iodine-triiodothyronine
was added. Control livers (n = 8) were immediately perfused with Krebs-Ringer without cold-storage.
The following parameters were assessed: ASAT-levels in the perfusate, T3-metabolites in the bile and
the perfusate, the perfusion pressure, the volume of bile secreted and light-microscopical morphology at
the end of the warm perfusion period.
After cold storage in UW-solution the ASAT-levels in the perfusate were lower than after storage in
EC as well as the perfusion pressures. These livers demonstrated a better T3-metabolism and secreted
more bile than EC-stored livers. Histological examination showed more tissue damage in the EC-stored
livers than in the UW stored livers.
We conclude that cold-storage of rat liver in UW-solution resulted in a better morphology and
metabolic capacity as compared with EC-solution.
In the current study, the mechanical and hypothermic damage induced by vibration and cold storage on human mesenchymal stem cells (hMSCs) stored at 2–8°C was quantified by measuring the total cell number and cell viability after exposure to vibration at 50 Hz (peak acceleration 140 m s−2 and peak displacement 1.4 mm), 25 Hz (peak acceleration 140 m s−2, peak displacement 5.7 mm), 10 Hz (peak acceleration 20 m s−2, peak displacement 5.1 mm) and cold storage for several durations. To quantify the viability of the cells, in addition to the trypan blue exclusion method, the combination of annexin V-FITC and propidium iodide was applied to understand the mode of cell death. Cell granularity and a panel of cell surface markers for stemness, including CD29, CD44, CD105 and CD166, were also evaluated for each condition. It was found that hMSCs were sensitive to vibration at 25 Hz, with moderate effects at 50 Hz and no effects at 10 Hz. Vibration at 25 Hz also increased CD29 and CD44 expression. The study further showed that cold storage alone caused a decrease in cell viability, especially after 48 h, and also increased CD29 and CD44 and attenuated CD105 expressions. Cell death would most likely be the consequence of membrane rupture, owing to necrosis induced by cold storage. The sensitivity of cells to different vibrations within the mechanical system is due to a combined effect of displacement and acceleration, and hMSCs with a longer cold storage duration were more susceptible to vibration damage, indicating a coupling between the effects of vibration and cold storage.
stem cells; mechanical stress; vibration; regenerative medicine; hypothermia; viability
Early graft dysfunction due to preservation/reperfusion injury represents a dramatic event after liver transplantation. Enhancement of donor organ criteria, in order to cope with the ever increasing donor shortage, further increases graft susceptibility to ischemic alterations.
Major parts of post-preservation injury, however, occur at the time of warm reperfusion but not during ischemic storage; successful reperfusion of ischemic tissue in turn depends on an adequate redox and intracellular signal homeostasis. The latter has been shown experimentally to be favorably influenced by oxygen persufflation within short time spans. Thus viability of marginally preserved liver grafts could still be augmented by transient hypothermic reconditioning even after normal procurement and static cold storage. The present study is aimed to confirm the conceptual expectations, that hypothermic reconditioning by gaseous oxygen persufflation is a useful method to suppress injurious cellular activation cascades and to improve post-ischemic recovery of marginally preserved liver grafts.
OPAL is a prospective single center randomized proof of concept study, including two parallel groups in a total of 116 liver transplant patients. The effect of an in hospital treatment of the isolated liver graft by 2 hours of oxygen persufflation immediately prior to transplantation will be assesses as compared to standard procedure (cold storage without further intervention). The primary endpoint is the peak transaminase serum level (AST) during the first three days after transplantation as a surrogate readout for parenchymal liver injury. Other outcomes comprise patient and graft survival, time of intensive care requirement, hepatic tissue perfusion 1h after revascularisation, early onset of graft dysfunction based on coagulation parameters, as well as the use of a refined scoring-system for initial graft function based on a multi-parameter (AST, ALT, Quick and bilirubin) score. Furthermore, the effect of OPAL on molecular pathways of autophagy and inflammatory cell activation will be evaluated. Final analysis will be based on all participants as randomized (intention to treat).
Current Controlled Trials ISRCTN00167887
Understanding how human cells in tissue culture adapt to hypothermia may aid in developing new clinical procedures for improved ischemic and hypothermic protection. Human coronary artery endothelial cells grown to confluence at 37°C and then transferred to 25°C become resistant over time to oxidative stress and injury induced by 0°C storage and rewarming. This protection correlates with an increase in intracellular glutathione at 25°C. To help understand the molecular basis of endothelial cold-adaptation, isolated proteins from cold-adapted (25°C/72 h) and pre-adapted cells were analyzed by quantitative proteomic methods and differentially expressed proteins were categorized using the DAVID Bioinformatics Resource.
Cells adapted to 25°C expressed changes in the abundance of 219 unique proteins representing a broad range of categories such as translation, glycolysis, biosynthetic (anabolic) processes, NAD, cytoskeletal organization, RNA processing, oxidoreductase activity, response-to-stress and cell redox homeostasis. The number of proteins that decreased significantly with cold-adaptation exceeded the number that increased by 2:1. Almost half of the decreases were associated with protein metabolic processes and a third were related to anabolic processes including protein, DNA and fatty acid synthesis. Changes consistent with the suppression of cytoskeletal dynamics provided further evidence that cold-adapted cells are in an energy conserving state. Among the specific changes were increases in the abundance and activity of redox proteins glutathione S-transferase, thioredoxin and thioredoxin reductase, which correlated with a decrease in oxidative stress, an increase in protein glutathionylation, and a recovery of reduced protein thiols during rewarming from 0°C. Increases in S-adenosylhomocysteine hydrolase and nicotinamide phosphoribosyltransferase implicate a central role for the methionine-cysteine transulfuration pathway in increasing glutathione levels and the NAD salvage pathway in increasing the reducing capacity of cold-adapted cells.
Endothelial adaptation to mild-moderate hypothermia down-regulates anabolic processes and increases the reducing capacity of cells to enhance their resistance to oxidation and injury associated with 0°C storage and rewarming. Inducing these characteristics in a clinical setting could potentially limit the damaging effects of energy insufficiency due to ischemia and prevent the disruption of integrated metabolism at low temperatures.
Peach (Prunus persica) fruits of cv. ‘Earli Grande’ were treated with CaCl2 (4 and 6%) and stored at 0–2 °C and 85–90% RH for 21 days followed by storage at ambient conditions (28–30 °C, 65–70% RH) for 72 h. CaCl2 at 6% effectively in reduced spoilage, physiological loss in weight (PLW) effectively reduced and maintained fruit firmness, palatability rating, acidity, vitamin A content and pectin methyl estrase (PME) activity during storage. Results revealed that peach fruits harvested at optimum stage followed by post-harvest dip in 6% CaCl2 solution for 10 min can be stored for 3 weeks in cold storage (0–2 °C, 85–90% RH) with post-storage shelf-life of 3 days at ambient conditions (28–30 °C, 65–70% RH) with acceptable edible quality of fruits.
Peach; Quality; Calcium; Storage
Machine perfusion at subnormothermic temperature (20°C), MP20, was developed by Vairetti et al. and showed to afford a better preservation of fatty livers respect to traditional cold storage (CS) in terms of enzyme release into the perfusate, bile production, glycogen stores, energy charge and oxidative stress. Here we investigated whether it also caused decreased cell death by apoptosis. Fatty and lean Zucker rats were submitted to MP20 or CS for 6 h and reperfused normothermically for 2 h. Apoptotic cells were revealed by immunohistochemistry of activated caspase-3 and M30 (new epitope on CK18 degraded by caspase-3) and by the TUNEL assay. Portal pressure was also determined. A statistically significant reduction of hepatocyte apoptosis, but especially of sinusoidal cells was determined for fatty livers submitted to MP20 respect to CS. Portal pressure was significantly lower after MP20 respect to CS. The reduction of sinusoidal cell death by apoptosis without need for anti-apoptotic therapies appears particularly positive since apoptotic sinusoidal cells hinder microcirculation in the sinusoids and are thrombogenic. These results further confirm the potential of MP20 for preserving fatty livers that would be otherwise discarded as grafts, and thus for increasing the donor pool for liver transplantation.
fatty liver; transplantation; apoptosis; sinusoidal cells; sub-normothermic machine perfusion; cold storage.
Cardiac transplantation remains the first choice for the surgical treatment of end stage heart failure. An inadequate supply of donor grafts that meet existing criteria has limited the application of this therapy to suitable candidates and increased interest in extended criteria donors. Although cold storage (CS) is a time-tested method for the preservation of hearts during the ex vivo transport interval, its disadvantages are highlighted in hearts from the extended criteria donor. In contrast, transport of high-risk hearts using hypothermic machine perfusion (MP) provides continuous support of aerobic metabolism and ongoing washout of metabolic byproducts. Perhaps more importantly, monitoring the organ’s response to this intervention provides insight into the viability of a heart initially deemed as extended criteria. Obviously, ex vivo MP introduces challenges, such as ensuring homogeneous tissue perfusion and avoiding myocardial edema. Though numerous groups have experimented with this technology, the best perfusate and perfusion parameters needed to achieve optimal results remain unclear. In the present review, we outline the benefits of ex vivo MP with particular attention to how the challenges can be addressed in order to achieve the most consistent results in a large animal model of the ideal heart donor. We provide evidence that MP can be used to resuscitate and evaluate hearts from animal and human extended criteria donors, including the non-heart beating donor, which we feel is the most compelling argument for why this technology is likely to impact the donor pool.
Non-heart beating donor; Myocardial viability; Machine perfusion; Heart transplantation; Myocardial preservation
Fresh allograft valves stored in a nutrient medium at +4 degrees C have a limited storage time of eight weeks. Dura mater has been stored in glycerol for longer periods, and this paper presents work on the glycerol storage of allograft heart valves. The elastic properties of the valve cusps showed a fall during storage in glycerol that was associated with an altered histological appearance of the cusp tissue. The loss in nuclearity in the histological sections of stored tissue was partially responsible for the observed decrease in viability during storage. All these changes during storage in glycerol, or glycerol and subsequent antibiotic treatment, were similar to the changes seen in valves stored in a nutrient medium. Glycerol therefore offers an alternative storage system to the cold nutrient medium but has no practical advantages. Glycerol alone will not sterilise the allograft tissue, and a post-storage treatment with antibiotics is essential.
Storage duration of red cells has been associated with increased morbidity and mortality following transfusion. This association has been attributed to the loss of deformability of stored red cells leading to deterioration of microvascular perfusion. ATP content is considered a critical determinant of the deformability of stored red cells.
ATP content and deformability were determined after storage for up to 49 days in 40 leukocyte-depleted whole blood units. Red cell deformability was determined using a laser-assisted optical rotational cell analyzer (LORCA®) employing shear stress (SS) ranging from 0.3 to 30 Pa. Deformability was expressed as the elongation index (EI). EI was correlated with ATP content.
ATP content decreased from 3.5 to 1.7 ?mol/g hemoglobin. EI increased from 0.03 to 0.05 at an SS of 0.3 Pa, and decreased from 0.62 to 0.59 at an SS of 30 Pa. Correlation coefficient (r) of ATP vs. EI at 0.3 Pa ranged from –0.17 to +0.15 during storage. At 30 Pa, r ranged from –0.03 to +0.45. Correlation increased with storage irrespective of SS, and increased with SS irrespective of storage.
ATP content is not a valid surrogate marker for red cell deformability and may not reflect in vivo survival of stored red cells.
ATP content; Deformability; Quality; Red cell storage; Storage lesion; In vivo survival; Whole blood
Following kidney transplantation, ischemia-reperfusion injury contributes to adverse outcomes. The purpose of this study was to determine whether a cold-storage solution saturated with noble gas (xenon or argon) could limit ischemia-reperfusion injury following cold ischemia.
Sixty Wistar rats were randomly allocated to 4 experimental groups. Kidneys were harvested and then stored for 6 h before transplantation in cold-storage solution (Celsior®) saturated with either air, nitrogen, xenon or argon. A syngenic orthotopic transplantation was performed. Renal function was determined on days 7 and 14 after transplantation. Transplanted kidneys were removed on day 14 for histological and immunohistochemical analyses.
Creatinine clearance was significantly higher and urinary albumin significantly lower in the argon and xenon groups than in the other groups at days 7 and 14. These effects were considerably more pronounced for argon than for xenon. In addition, kidneys stored with argon, and to a lesser extent those stored with xenon, displayed preserved renal architecture as well as higher CD-10 and little active caspase-3 expression compared to other groups.
Argon- or xenon-satured cold-storage solution preserved renal architecture and function following transplantation by reducing ischemia-reperfusion injury.
Argon; Xenon; Transplantation; Kidney; Organ-protection; Ischemia-reperfusion injury; Storage