The cold chain delivery of organs for transplantation: from research laboratories and individual enthusiasts to pan- global networks in 50 years.

It is some 50 years since the first published reports appeared of ex vivo preservation of organs for transplantation. Over the intervening decades, organ preservation strategies have become one essential component of world-wide clinical transplant services. In the formative years, translational research in organ hypothermic preservation was grappling with the questions about whether static or dynamic storage was preferable, and the practical implications of those choices. Those studies were also informing the newly expanding clinical transplant services. During the middle years, both preservation modalities were practiced by individual group choices. By the 2000s, the shift in donor demographics demanded a re-evaluation of organ preservation strategies, and now a new era of research and development is promoting adoption of new technologies. In this review we outline many important academic studies which have contributed to this successful history, and give profile to the increasing innovative approaches which are being evaluated for the future. Doi.org/10.54680/fr24310110112.

Biopreservation of hepatocytes: current concepts on hypothermic preservation, cryopreservation, and vitrification.

Isolated liver cells (primarily isolated hepatocytes) have found important applications in science and medicine over the past 40 years in a wide range of areas, including physiological studies, investigations on liver metabolism, organ preservation and drug de-toxification, experimental and clinical transplantation. An integral component of many of these works is the need to store the isolated cells, either for short or long-term periods. This review covers the biopreservation of liver cells, with a focus on the history of liver cell biopreservation, the application of hypothermia for short-term storage, standard cryopreservation methods for isolated hepatocytes, the biopreservation of other types of liver cells, and recent developments such as vitrification of hepatocytes. By understanding the basis for the different approaches, it will be possible to select the best options for liver cell biopreservation in different applications, and identify ways to improve preservation protocols for the future.

Translating cryobiology principles into trans-disciplinary storage guidelines for biorepositories and biobanks: a concept paper.

Low temperatures are used routinely to preserve diverse biospecimens, genetic resources and non-viable or viable biosamples for medical and clinical research in hospital-based biobanks and non-medical biorepositories, such as genebanks and culture, scientific, museum, and environmental collections. However, the basic knowledge underpinning preservation can sometimes be overlooked by practitioners who are unfamiliar with fundamental cryobiological principles which are more usually described in research literature rather than in quality and risk management documents. Whilst procedures vary, low temperature storage is a common requirement and reaching consensus as to how best it is applied could facilitate the entire biopreservation sector. This may be achieved by encouraging an understanding of cryoprotection theory and emphasizing the criticality of thermal events (glass transitions, ice nucleation, thawing) for sample integrity, functionality and stability. The objective of this paper is to inspire diverse biopreservation sectors to communicate more clearly about low temperature storage and, raise awareness of the importance of cryobiology principles to field newcomers and biopreservation practitioners, by considering how the principles may be translated into evidence-based guidelines for biobank and biorepository operations.

Influence of zinc and zinc chelator on HT-29 colorectal cell line.

Trace elements are involved in many key pathways involving cell cycle control. The influence of zinc and zinc chelator (TPEN) on transcription levels of the main zinc transporters (ZnT1 and ZIP1) in the HT-29 colorectal cell line has not been reported. Proliferation of HT-29 cells was measured using the methylene blue assay after exposure to zinc (two concentrations), TPEN (two concentrations), or a combination of zinc and TPEN (simultaneously and sequentially) for 4 h, 8 h, and 24 h. The transcription levels of ZnT1, ZIP1, vascular endothelial growth factor (VEGF), and caspase-3 were determined using reverse transcriptase real-time polymerase chain reaction (RT-PCR) after exposure of cells to zinc and TPEN. The zinc content in the substrate (medium used for culture) was determined using atomic absorption spectrometry. TPEN decreased cellular proliferation causing complete cell death by 8 h. Zinc had a protective effect against short periods of exposure to TPEN. There was no correlation between the transcripts of main zinc transporters and the zinc content in the substrate. The zinc content in the substrate remained constant after varying periods of cell culture. TPEN decreased the transcript levels of caspase-3 and VEGF, which are surrogate markers for apoptosis and angiogenesis. Zinc chelation of HT-29 cells causes cell death. Zinc appears to be protective for short periods of exposure to TPEN but has no protective effect on prolonged exposure. HT-29 cells are not able to counteract the effect of intracellular chelation of zinc by altering zinc transport. Further research into the mechanisms of these findings is necessary and may lead to novel therapeutic options.

Protective effects of a carbon monoxide-releasing molecule (CORM-3) during hepatic cold preservation.

UNLABELLED: There is increasing evidence that carbon monoxide (CO), a signaling molecule generated during the degradation of heme by heme oxygenase-1 (HO-1) in biological systems, has a variety of cytoprotective actions, including anti-hypoxic effects at low temperatures. However, during liver cold preservation, a direct effect needs to be established. Here, we designed a study to analyze the role of CO, delivered via a carbon monoxide-releasing molecule (CO-RM) in the maintenance of liver function, and integrity in rats during cold ischemia/reperfusion (CI/R) injury. We used an isolated normothermic perfused liver system (INPL) following a clinically relevant model of ex vivo 48 h cold ischemia stored in a modified University of Wisconsin (UW) solution, to determine the specific effects of CO in a rat model. CO was generated from 50 microM tricarbonylchloro ruthenium-glycinato (CORM-3), a water-soluble transition metal carbonyl that exerts pharmacological activities via the liberation of controlled amounts of CO in biological systems. The physiological effects of CORM-3 were confirmed by the parallel use of a specific inactive compound (iCORM-3), which does not liberate CO in the cellular environment. CORM-3 addition was found to prevent the injury caused by cold storage by improving significantly the perfusion flow during reperfusion (by almost 90%), and by decreasing the intrahepatic resistance (by 88%) when compared with livers cold preserved in UW alone. Also, CORM-3 supplementation preserved good metabolic capacity as indicated by hepatic oxygen consumption, glycogen content, and release of lactate dehydrogenase. Liver histology was also partially preserved by CORM-3 treatment. CONCLUSIONS: These findings suggest that CO-RM could be utilized as adjuvant therapeutics in UW solutions to limit the injury sustained by donor livers during cold storage prior to transplantation, as has been similarly proposed for the heart, and kidney.

Mitochondrial function after liver preservation in high or low ionic-strength solutions: a comparison between UW-based and sucrose-based solution.

In this study we evaluated mitochondrial function after liver cold storage and normothermic reperfusion. The preservation solutions were: modified University of Wisconsin (mod UW) and sucrose-based solution (SBS). After cold preservation liver was re-perfused for 1 hour in vitro with Krebs-Ringer buffer at 37 degree C. Samples of tissue were taken for ATP determination. Mitochondrial respiratory parameters, succinate oxidase complex activity, mitochondrial H+- ATPase and intramitochondrial potassium concentration were assayed. It was shown, that brief (1 hour) cold storage and subsequent normothermic reperfusion revealed no difference in liver ATP content between mod UW and SBS groups but resulted in a gradual decrease of 50 percent after 24-hour storage and reperfusion. Mitochondrial potassium ion concentration increased by 40 percent after 1-hour cold storage in the mod UW as compared to control (P value less than 0.05) and SBS. After brief cold storage ADP and uncoupler-stimulated respiration increased by 120 percent in SBS group, unlike mod UW, when succinate was used as substrate, and was more pronounced after 24 hour. Succinate oxidase complex activity did not change over either cold storage or warm reperfusion. Mitochondrial H+-ATPase activities in SBS and mod UW did not differ and both were inhibited after 24-hour cold storage. Our data demonstrate that low ionic strength preservation solution can substantially modulate mitochondrial energy turnover due to substrate oxidation increase. Many of the changes in mitochondrial function follow brief exposure to low temperatures.

Positive inotropic effects of carbon monoxide-releasing molecules (CO-RMs) in the isolated perfused rat heart.

BACKGROUND AND PURPOSE: Carbon monoxide (CO) generated by the enzyme haeme oxygenase-1 (HO-1) during the breakdown of haeme is known to mediate a number of biological effects. Here, we investigated whether CO liberated from two water soluble carbon monoxide-releasing molecules (CO-RMs) exerts inotropic effects on the myocardium. EXPERIMENTAL APPROACH: Rat isolated hearts perfused either at constant flow or constant pressure were used to test the effect of CO-RMs. KEY RESULTS: CORM-3, a fast CO releaser, produced a direct positive inotropic effect when cumulative doses (3, 10 and 30 microg min(-1)) or a single dose (5 microM) were infused at either constant coronary pressure (CCP) or constant coronary flow (CCF). The inotropic effect mediated by CORM-3 was abolished by blockade of soluble guanylate cyclase or Na(+)/H(+) exchanger, but not by inhibitors of L-type Ca(2+) channels and protein kinase C. CORM-3 also caused a slight reduction in heart rate but did not alter coronary flow. In contrast, the slow CO releaser CORM-A1 produced significant coronary vasodilatation when given at the highest concentration (30 mug min(-1)) but exerted no effect on myocardial contractility or heart rate. CONCLUSION AND IMPLICATIONS: A rapid CO release from CORM-3 exerts a direct positive inotropic effect on rat isolated perfused hearts, whereas CO slowly released by CORM-A1 had no effect on myocardial contractility but caused significant coronary vasodilatation. Both cGMP and Na(+)/H(+) exchange appear to be involved in this effect but further work is needed to determine the relative contribution of each pathway in CO-mediated inotropic effect.

Membrane integrity and development of immature murine cumulus-oocyte complexes following slow cooling to -60 degrees C: the effect of immediate rewarming, plunging into LN2 and two-controlled-rate-stage cooling.

Cryopreservation of murine germinal vesicle (GV) stage cumulus-oocyte complexes (COCs) has been shown to result in poor development and cumulus cell damage. In an attempt to determine the stage of the cryopreservation protocol at which damage occurs, three cooling profiles were compared: slow-cooling (0.3 degrees C/min) to -60 degrees C (protocol A); slow-cooling to -60 degrees C and plunging to -196 degrees C (protocol B); or slow-cooling to -60 degrees C followed by further cooling at 10 degrees C/min to -150 degrees C, then plunging to -196 degrees C (protocol C). GV-stage COCs were collected from hormone-primed mice by repeated puncturing of ovarian follicles. COCs were exposed to 1.5 M Me(2)SO prior to cooling to -60 or -196 degrees C. Membrane integrity was assessed immediately after thawing using carboxy fluorescein and propidium iodide. A greater proportion of cumulus cells were damaged following protocol B than protocol A. Damage was less extensive following protocol C than following protocol B. For assessment of development, COCs were matured and fertilised in vitro. Morphological normality was significantly reduced following cooling to -60 or -196 degrees C compared with non-cryopreserved controls. Fertilisation of oocytes assessed as normal post-treatment was not significantly different between any of the groups. Development to blastocyst was least from oocytes exposed to protocol B, being significantly worse than for oocytes exposed to protocol A, but not significantly different to protocol C. A protocol comprising two stages of controlled-rate cooling decreased damage to the membranes of cumulus cells but did not significantly improve embryo development.

Treatment with CO-RMs during cold storage improves renal function at reperfusion.

Low concentrations of carbon monoxide (CO) can protect tissues against ischemia-reperfusion (I-R) injury. We have recently identified a novel class of compounds, CO-releasing molecules (CO-RMs), which exert important pharmacological activities by carrying and delivering CO to biological systems. Here, we examined the possible beneficial effects of CO liberated from CO-RMs on the damage inflicted by cold storage and I-R in isolated perfused kidneys. Hemodynamic and biochemical parameters as well as mitochondrial respiration were measured in isolated perfused rabbit kidneys that were previously flushed with CO-RMs and stored at 4 degrees C for 24 h. Two water-soluble CO-RMs were tested: (1) sodium boranocarbonate (CORM-A1), a boron-containing carbonate that releases CO at a slow rate, and (2) tricarbonylchloro(glycinato)ruthenium(II) (CORM-3), a transition metal carbonyl that liberates CO very rapidly in solution. Kidneys flushed with Celsior solution supplemented with CO-RMs (50 microM) and stored at 4 degrees C for 24 h displayed at reperfusion a significantly higher perfusion flow rate (PFR), glomerular filtration rate, and sodium and glucose reabsorption rates compared to control kidneys flushed with Celsior solution alone. Addition of 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ), a guanylate cyclase inhibitor, prevented the increase in PFR mediated by CO-RMs. The respiratory control index from kidney mitochondria treated with CO-RMs was also markedly increased. Notably, renal protection was lost when kidneys were flushed with Celsior containing an inactive compound (iCO-RM), which had been deliberately depleted of CO. CO-RMs are effective therapeutic agents that deliver CO during kidney cold preservation and can be used to ameliorate vascular activity, energy metabolism and renal function at reperfusion.

Engineering of bypass conduits to improve patency.

For patients with severe coronary artery and distal peripheral vascular disease not amenable to angioplasty and lacking sufficient autologous vessels there is a pressing need for improvements to current surgical bypass options. It has been decades since any real progress in bypass material has reached mainstream surgical practice. This review looks at possible remedies to this situation. Options considered are methods to reduce prosthetic graft thrombogenicity, including endothelial cell seeding and developments of new prosthetic materials. The promise of tissue-engineered blood vessels is examined with a specific look at how peptides can improve cell adhesion to scaffolds.

Identification of cytochrome P450 enzymes in human colorectal metastases and the surrounding liver: a proteomic approach.

We describe the direct identification of multiple cytochrome P450 (CYP) enzymes in healthy and cancerous tissue. CYPs in human liver colorectal metastases were compared with those in the surrounding liver using a mass spectrometry-based proteomic approach. Nano-scale reversed phase liquid chromatography combined with electrospray ionisation tandem mass spectrometry has been used to identify CYPs with no pre-selection of the proteins anticipated. Fourteen distinct CYP enzymes from the subfamilies 1A, 2A, 2B, 2C, 2D, 2E, 3A, 4A, 4F, 8B and 27A were positively identified; 13 in the liver samples and 12 in the tumour tissue. It was found that three of the colorectal metastases expressed essentially the same drug-metabolising pattern of CYPs as the surrounding liver, whilst three tumour samples from different individuals showed no CYP expression. This was likely in at least one case to be due to low sample mass. The CYP expression profile in an individual tumour is likely to be an important determinant in predicting the outcome of cancer chemotherapy.

Induction of adhesion molecule expression in liver ischaemia-reperfusion injury is associated with impaired hepatic parenchymal microcirculation.

BACKGROUND: Activated neutrophils may be important mediators in liver ischaemia-reperfusion injury (I/R). Adhesion of leucocytes to the endothelial cell surface is a result of activation of cell adhesion molecules. The aim of this study was to investigate the effect of I/R on the hepatic microcirculation (HM) and intercellular adhesion molecule (ICAM) 1 expression. METHODS: Four groups of six Sprague-Dawley rats underwent laparotomy for liver exposure. Group 1 acted as controls, and groups 2-4 underwent partial liver ischaemia for 30, 45 and 60 min respectively followed by reperfusion for 60 min. Flow in the HM was measured by laser Doppler flowmetry. Liver biopsies were taken at the end of the reperfusion period. ICAM-1 expression was assessed by immunohistochemistry (graded 0-3). RESULTS: Mean flow in the HM was significantly reduced with I/R (mean(s.e.m.) red cell flux 140(21), 52(3) and 43(2) with 30, 45 and 60 min ischaemia compared with control 230(17); all P < 0.001). ICAM-1 expression was significantly induced (mean(s.e.m.) 1.30(0.21), 2.50(0.22) and 2.80(0.17) with 30, 45 and 60 min ischaemia versus control 0.50(0.22); all P < 0.001). CONCLUSION: I/R produced a significant upregulation of ICAM-1 expression which correlated with impaired flow in the HM.

Near-infrared spectroscopic assessment of mitochondrial oxygenation status--comparison during normothermic extracorporeal liver perfusion by buffer only or buffer fortified with washed red blood cells: an experimental study.

BACKGROUND: Use of marginal and non-heart-beating donors leads to an increased incidence of complications after clinical liver transplantation. Normothermic extracorporeal liver perfusion (NELP) may allow resuscitation and evaluation of such organs. Despite recent success in long-term liver preservation by NELP, no methods of organ evaluation have been defined. Mitochondrial cytochrome oxidase (Cyt Ox) levels reflect oxygen and substrate delivery, and hence ATP production at the cellular level. This study used near-infrared spectroscopy (NIRS) to measure Cyt Ox levels during NELP. METHODS: Livers retrieved from New Zealand white rabbits were immediately perfused in an extracorporeal circuit with oxygenated buffer (group A, n = 4) or red blood cell (RBC)-fortified buffer (group B, n = 4). Perfusion was continued for 3 hours at 37 degrees C pH 7.4, and perfusate was gassed with 95%O2/5%CO2 at 1 liter per minute. Cyt Ox levels were monitored continuously by NIRS and bile output was measured. RESULTS: Cyt Ox was reduced at the start of perfusion in both groups, but even more rapidly in the buffer-perfused group. After initial deterioration, Cyt Ox levels improved significantly (P < .05) with perfusion in the RBG-perfused group, but remained impaired in the buffer group 5.74 +/- 1.51 Deltamicromol/L and -25.77 +/- 21.94 Deltamicromol/L for groups B and A, respectively, at 180 minutes. Differences in bile output were not significant (19.33 +/- 9.50 and 25.00 +/- 16.81 micromol/min/100 g for groups B and A respectively). CONCLUSIONS: Cyt Ox levels may offer better viability markers than bile output. NIRS is a practical method to measure tissue oxygenation, and RBC-based perfusion provided better oxygenation during NELP.

A comparison of bile composition from heart-beating and non-heart-beating rabbit organ donors during normothermic extracorporeal liver perfusion: experimental evaluation using proton magnetic resonance spectroscopy.

BACKGROUND: Organs retrieved from marginal and non-heart-beating donors (NHBDs) have sustained variable degrees of preretrieval damage that result in an increased incidence of complications. Normothermic extracorporeal liver perfusion (NELP) provides an opportunity to evaluate and resuscitate such organs. The aim of this study was to identify markers of ischemic injury in bile during perfusion of livers from heart-beating donors (HBDs) and NHBDs. METHODS: Livers were retrieved from New Zealand white rabbits. The HBD group (n=4) had no in situ warm ischemia before retrieval and the NHBD group (n=4), 45 minutes of in situ warm ischemia before liver retrieval. After 40 minutes of postretrieval cold ischemia, all livers were dual vessel reperfused, normothermically with oxygenated buffer solution supplemented with rabbit red blood cells, for 6 hours. Bile was collected and examined with 1HMRS. RESULTS: Perfusion bile from HBD group showed an increased concentration of bile acids, lactate, glucose, and phosphatidylcholine, but a decreased concentration of acetate as compared to retrieval bile. This trend was further enhanced in NHBD group. The mean +/- SD (in micromol/L) were bile acids (10.48 +/- 2.8 vs 26.05 +/- 12.1 vs 44.5 +/- 44.5), lactate (10.66 +/- 4.5 vs 14.66 +/- 5.2 vs 13.22 +/- 1.8), glucose (5.37 +/- 2 vs 21.2 +/- 5.0 vs 29.09 +/- 15.3), phosphatidylcholine (0.21 +/- 0.02 vs 5.57 +/- 1.7 vs 6.42 +/- 0.3), and acetate (1.8 +/- 0.5 vs 0.39 +/- 0.1 vs 0.38 +/- 0.09) for retrieval bile, HBD perfusion bile, and NHBD perfusion bile, respectively. One animal from each group did not produce any bile during perfusion. CONCLUSIONS: 1HMRS of biliary constituents revealed differences with the type of ischemia. These indices may be potential markers of the extent of warm ischemic injury and the functional activity of an extracorporeally perfused liver.

Developmental potential of murine germinal vesicle stage cumulus-oocyte complexes following exposure to dimethylsulphoxide or cryopreservation: loss of membrane integrity of cumulus cells after thawing.

BACKGROUND: Cumulus cells of the cumulus-oocyte complex (COC) are important in oocyte maturation. Thus, in preserving immature oocytes it is prudent to also preserve their associated cumulus cells. The survival and function of oocytes and their associated cumulus cells was assessed following cryopreservation or exposure to cryoprotectant without freezing. METHODS: Immature COCs were collected from mice primed with pregnant mare's serum. COCs were either slow-cooled or exposed to 1.5 mol/l dimethylsulphoxide without freezing. Treated and fresh COCs were stained for membrane integrity or, after in-vitro maturation and IVF, were assessed for developmental capability. Development of cumulus-denuded fresh oocytes, as well as denuded and frozen-thawed oocytes co-cultured with fresh cumulus cells, was assessed. RESULTS: Slow-cooled oocytes had significantly reduced coverage by intact cumulus cells compared with fresh COCs. Cumulus cell association and developmental capability were not substantially affected by exposure to cryoprotectant without freezing. Denuded fresh oocytes and cryopreserved COCs had decreased developmental potential that was not overcome by co-culture with fresh cumulus cells. CONCLUSIONS: Loss of association between oocyte and cumulus cells was induced by cryopreservation, but not by treatment with cryoprotectant alone. The data indicate that direct physical contact between cumulus cells and the oocyte, throughout maturation, improves subsequent embryo development.

Dynamics of the changes in homogenate respiratory activity after rat whole liver storage at 4 degrees C.

Homogenate respiratory activity was studied after different storage terms of the whole rat liver at 4 degrees C in sucrose-based solution and following normothermic reperfusion. Preservation of homogenate respiratory activity in all metabolic states after normothermic reperfusion of the control liver (60 min, 4 degrees C) is shown. Further storage (6 and 24 hrs) of isolated liver under the mentioned above conditions strengthens the substrate respiration of homogenate both after storage and after normothermic reperfusion. At the same time oxidative phosphorylation does not practically change. No change was noted in respiratory activity in the states 3, 4ADP and 3DNP after 24 hrs of liver storage in respect of a previous term. Following normothermic liver reperfusion contributes to a statistically true reduction of mentioned parameters of respiration, that correlates with a decrease in the degree of respiration and phosphorylation coupled of the studied system.

Hepatic uptake of solutes from the preservation solution during hypothermic storage: a (1)H NMR study in rat liver.

The hepatic uptake of histidine and carnosine (histidyl-alanine), used as buffer agents in four preservation solutions, was studied during 24-h hypothermic storage of rat livers by use of (1)H nuclear magnetic resonance (NMR) spectroscopy. Results demonstrated that there was a progressive, concentration-linked passive diffusion of histidine into liver tissues throughout the storage period. A similar inward diffusion of carnosine was also noted. Of the carbohydrate osmotic buffers in the preservation solutions, mannitol permeated the liver tissues to a greater degree and more rapidly than raffinose after the flushing with equivalent concentrations and storage at hypothermia. In general, many solutes from preservation solutions will increasingly penetrate the hepatic inter- and intracellular spaces during extended hypothermic preservation and (1)H NMR spectroscopy is one technique that can assist in the identification of these changes.

Respiratory activity of isolated rat hepatocytes following cold storage and subsequent rewarming: a comparison of sucrose-based and University of Wisconsin solutions.

Investigations were carried out on the respiratory function of isolated rat hepatocytes after cold storage alone for periods up to 48 h in either sucrose-based solution (SBS) or University of Wisconsin (UW) solution and after subsequent normothermic preincubation. In both SBS and UW, cold storage for 24 h depressed respiratory function (to 21 +/- 3 and 23 +/- 3 nmol O(2)/min/10(6) cells, respectively) compared to control cell values (31 +/- 3 and 33 +/- 5 nmol O(2)/min/10(6) cells; P < 0.01 in each case). However, normothermic preincubation for 60 min returned respiratory activity to control values (for SBS and UW storage: 41 +/- 6 and 40 +/- 5 nmol O(2)/min/10(6) cells; for control cells: 43 +/- 5 and 46 +/- 6 nmol O(2)/min/10(6) cells). Storage for 48 h in both SBS and UW allowed further depression of respiratory activity, with no recovery after preincubation. Stimulation of respiration by succinate in hepatocytes stored for longer periods was suggestive of increased membrane permeability. Both SBS and UW are effective storage solutions for isolated hepatocytes for up to 24 h as judged by aerobic metabolism, but significant damage was expressed in both solutions when preservation was extended.

Membrane permeability of human oocytes in the presence of the cryoprotectant propane-1,2-diol.

OBJECTIVE: To determine the permeability of unfertilized human oocytes to water and the cryoprotectant propane-1,2-diol over a range of temperatures and to use these data to predict osmotic responses under given conditions. DESIGN: Laboratory-based study. SETTING: Teaching hospital. PATIENT(S): Infertility patients donating unfertilized oocytes in excess of those required for treatment. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Water and cryoprotectant permeability were determined from measurements of oocyte volume excursions on exposure to 1.5 M propane-1,2-diol at 30 degrees C, 24 degrees C, and 10 degrees C. RESULT(S): Permeability of human oocytes to water and cryoprotectant increased as temperature increased. The predicted response of oocytes, based on these data, closely matched the measured response of an oocyte on exposure to a widely used method for addition of cryoprotectant before freezing. CONCLUSION(S): Commonly used cryopreservation protocols involving slow cooling in the presence of propane-1,2-diol cause potentially damaging excursions in cell volume on exposure to cryoprotectant. Modifications that can be expected to reduce cell volume excursions, based on oocyte permeability data, are suggested.

A comparison of the metabolic effects of continuous hypothermic perfusion or oxygenated persufflation during hypothermic storage of rat liver.

The metabolic consequences of supplying oxygen by two different modes were investigated. The effects of hypothermic liver preservation after cold hypoxic flush (Group I), oxygenated vascular persufflation (Group II), and continuous oxygenated perfusion (Group III) were compared. Adenine nucleotides were measured to assess energetics, and 1H nuclear magnetic resonance spectroscopy was employed to investigate other metabolic pathways. Energetics were maintained by both modes of oxygenation at 24 h. The mitochondrial redox state is indicated by the ratio of acetoacetate (Ace) and beta-hydroxybutyrate (betaHb). The detection of only betaHb or Ace in the hypoxic flush and perfused livers, respectively, suggested that the mitochondria of these livers were hyperreduced and hyperoxidized, respectively. In contrast, both components of the redox couple were detected in the persufflated livers, suggesting that persufflation may be a simple and effective method of maintaining hepatic energetics long-term while maintaining a more normal mitochondrial redox state.