Fourier analysis of isolated rat heart electrocardiographs as a measure of viability after cooling to −22°C with the aid of a cryoprotective agent

The viability of isolated rat hearts cooled to −22°C with the aid of a cryoprotective agent, ethylene glycol, has been assessed by a novel technique of comparing the Fourier analysed spectrum of the electrocardiogram before and after cooling. The frequency spectrum of the electrocardiogram of a good isolated heart being retrograde perfused at 37°C, contains 3 peaks with the second harmonic dominant in power. A damaged heart contains higher harmonics and additional components.     

The contribution of the cryoprotectant to total injury in rabbit hearts frozen with ethylene glycol.

Following the failure of hearts to recover function after freezing at ?20 ° in the presence of 3 m ethylene glycol, a variety of experimental treatments was devised to determine the relative harmfulness of ice, high concentrations of electrolytes and high ethylene glycol concentration. Neither cooling to ?20 °C without freezing in a Ca²⁺-free solution containing twice the normal salt concentration and 6 m ethylene glycol (freezing 3 m ethylene glycol at ?20 °C doubles the solute concentration in the liquid phase), nor perfusion at ?1 °C with this solution were conducive to the recovery of hearts. However, perfusion with Ca²⁺-free 3 m ethylene glycol solution with twice the normal concentration of salts did allow full recovery of function, whereas perfusion with Ca²⁺-free 6 m ethylene glycol solution with normal salt concentrations did not. Therefore, the high ethylene glycol concentration encountered during freezing was the main cause of damage.     

Isolated sheep heart hypothermic (5–13 °C) perfusion with fresh blood: Successful preservation for 24–72 hours with continuous strong ventricular activity

Isolated lamb hearts perfused with fresh whole blood at 10 and 13 °C in an ex vivo perfusion circuit continuously contracted at a rate of 15 to 20 times/min with a peak left ventricular systolic pressure (LVPSP) up to 70 mm Hg. These contractions persisted for the duration of the hypothermic study, up to three days with no change in vascular resistance. On rewarming to 38 °C, the hearts resumed regular and efficient contractions. Hearts perfused at 5 °C, however, exhibited no electrical or mechanical activity during hypothermic preservation and were uniformly poorly preserved.Quality of heart preservation was improved if, prior to final cooling, hearts were first rewarmed to 38 °C, followed by cooling. Change of the support animal, or interruption of flow of fresh blood into the perfusion circuit, resulted in cessation of ventricular contractions, ventricular fibrillation, and poor organ preservation.     

Effect of two cooling protocols on the post-thaw characteristics of Iberian ibex sperms

The rate at which lethal intracellular ice forms during sperm cryopreservation is highly dependent on the cooling protocol. The present work compares two cooling protocols for use with Iberian ibex (Capra pyrenaica) sperm by assessing the effects on the motility, viability, and size of frozen-thawed sperm cells. Ejaculates, obtained from six adult ibex males via transrectal, ultrasound-guided massage of the accessory sex glands plus electroejaculation if necessary, were cooled via either 1) Protocol 1 (decelerating cooling), involving cooling in liquid nitrogen vapor from 5 °C to −35 °C (40 °C/min), from −35 °C to −65 °C (17 °C/min), and then from −65 °C to −85 °C (3 °C/min); or 2) Protocol 2 (accelerating cooling) involving cooling in a biological freezer from 5 °C to −5 °C (4 °C/min), from −5 °C to −110 °C (25 °C/min), and then from −110 °C to −140 °C (35 °C/min). Compared to fresh ejaculates, sperm quality at thawing was found to be reduced by both protocols (p < .05), but especially by Protocol 1. Sperm head size was also significantly reduced by both protocols, although the Protocol 1 sperm heads were also significantly smaller than those of Protocol 2 sperms heads (p < .05). In fresh sperm samples, clustering analyses revealed two subpopulations of sperms with different morphometric characteristics, SP1 with larger cells, and SP2 with smaller cells. Both cooling protocols caused reduction in the proportion of SP1 cells, and an increase in the proportion of SP2 cells. In conclusion, the decelerating cooling protocol (Protocol 1) caused greater cryodamage to the sperm cells than the accelerating protocol (Protocol 2).     

Long-term (24h) cooling of ovarian fragments in the presence of permeable cryoprotectants prior to freezing: Two unsuccesful IVF-cycles and spontaneous pregnancy with baby born after re-transplantation

Cancer is the second major cause of death in the world. The problem of post-cancer infertility plays a significant role, because chemotherapy can be gonadotoxic. Cryopreservation of ovarian tissue before cancer therapy with re-implantation after convalescence is the potential key solution to this problem. The aim of this study was to test the viability of cryopreserved human ovarian cortex after long-term cooling in culture medium composed of permeable cryoprotectants. Ovarian fragments from sixteen patients were randomly divided into two groups. After the operation, tissue pieces assigned to both groups were cooled to 5 °C for 22–24 h, frozen and thawed. Group 1 pieces (n = 32) were cooled before cryopreservation in the standard culture medium, and Group 2 pieces (n = 32) were cooled in the freezing medium (culture medium+6% ethylene glycol+6% dimethyl sulfoxide+0.15 M sucrose). Freezing was performed in standard 5 ml cryo-vials with ice formation at −9 °C, cooling from −9 to −34 °C at a rate of −0.3 °C/min and plunging at −34 °C into liquid nitrogen. After thawing in a 100 °C (boiling) water bath, the removal of cryoprotectants was performed in 0.5 M sucrose with 20 min exposure in sucrose and 30 min stepping rehydration. The effectiveness of the pre-freezing cooling of tissue was evaluated by the development of follicles (histology). Six months after the autotransplantation, oocytes from the twenty-seven-year old, hormonally stimulated patient were retrieved and fertilized with her partner sperm through the intracytoplasmic spermatozoa injection (ICSI). For groups 1 and 2, 93.5 ± 1.9% and 96.4 ± 2.0% of the preantral follicles, respectively, were morphologically normal (P > 0.1) (with a tendency toward increasing in quality in Group 2). Six months after the auto-transplantation, two ICSI cycles resulted in the gathering and transplantation of high quality embryos, but no pregnancy had been established. Thirteen months after the auto-transplantation, the patient became spontaneously pregnant and delivered a healthy baby girl at term. Long-term (24 h) cooling of ovarian tissue to 5 °C before cryopreservation in the presence of permeable cryoprotectants simplifies the protocol of cryopreservation and has a tendency of increasing of the cells viability after thawing.     

In situ cryopreservation of human embryonic stem cells in gas-permeable membrane culture cassettes for high post-thaw yield and good manufacturing practice

Current strategies for marine pollution monitoring are based on the integration of chemical and biological techniques. The sea urchin embryo-larval bioassays are among the biological methods most widely used worldwide. Cryopreservation of early embryos of sea urchins could provide a useful tool to overcome one of the main limitations of such bioassays, the availability of high quality biological material all year round. The present study aimed to determine the suitability of several permeant (dimethyl sulfoxide, Me₂SO; propylene glycol, PG; and ethylene glycol, EG) and non-permeant (trehalose, TRE; polyvinylpyrrolidone, PVP) cryoprotectant agents (CPAs) and their combination, for the cryopreservation of eggs and embryos of the sea urchin Paracentrotus lividus. On the basis of the CPAs toxicity, PG and EG, in combination with PVP, seem to be most suitable for the cryopreservation of P. lividus eggs and embryos. Several freezing procedures were also assayed. The most successful freezing regime consisted on cooling from 4 to −12 °C at 1 °C/min, holding for 2 min for seeding, cooling to −20 °C at 0.5 °C/min, and then cooling to −35 °C at 1 °C/min. Maximum normal larvae percentages of 41.5% and 68.5%, and maximum larval growth values of 42.9% and 60.5%, were obtained for frozen fertilized eggs and frozen blastulae, respectively.     

II: Effect of Cooling Rate and Duration of Freezing Point Plateau on Boar Semen Frozen in Mini- and Maxi-Straws and Plastic Bags

The post-thaw motility and the acrosome integrity of semen from 4 boars frozen with a programmable freezing machine, in mini (0.25 ml) and maxi (5 ml) plastic straws and in 10 × 5 cm TeflonR FEP-plastic bags (0.12 mm thick, 5 ml), were compared. The freezing of the semen was monitored by way of thermocouples placed in the straws and the bags. Three freezing programmes were used, namely A: from + 5° C, at a rate of 3° C/min, to −6° C, held for 1 min at –6° C, and followed by a cooling rate of 20° C/min to −100° C; B: a similar curve except that there was no holding time at −6° C and that the cooling rate was 30° C/min, and C: from +5°C to −100° C, with a cooling rate of 35° C/min, followed by storage in liquid N2. Despite the treezing curve assayed, both the mini-straws and the bags depicted much shorter freezing point plateaus as compared to the maxi-straws. Post-thaw sperm motility as well as the amount of normal apical ridges were equally significantly higher when semen was frozen in mini-straws or in bags than in maxi-straws. Significant differences in these post-thawing parameters were obtained between the freezing curves used. The stepwise freezing procedure A appeared as the best alternative for boar semen, considering this in vitro evaluation.     

Two-step accelerating freezing protocol yields a better motility,membranes and DNA integrities of thawed ram sperm than three-steps freezing protocols

The present study compares a protocol that mimics freezing of ram semen in static nitrogen vapor with two protocols with an initial low cooling rate in the first step, followed by higher cooling rates where ice nucleation occurs. Semen ejaculates, obtained from twelve adults rams, were diluted with TEST-based extender and frozen with either Protocol 1 (three-step decelerating cooling): from +5 °C to −35 °C (40 °C/min), from −35 °C to −65 °C (17 °C/min), and then from −65 °C to −85 °C (3 °C/min); or Protocol 2 (three-step accelerating cooling): from +5 °C to −5 °C (4 °C/min), from −5 °C to −110 °C (25 °C/min), and then from −110 °C to −140 °C (35 °C/min); or Protocol 3 (two-step accelerating cooling), from +5 °C to −10 °C (5 °C/min), and then from −10 °C to −130 °C (60 °C/min). Post-thaw sperm quality was reduced for all protocols (p < .05) compared with fresh semen. Post-thaw percentages of sperm motility characteristics and sperm with intact plasma membrane, intact acrosome, and intact mitochondrial membrane were greater using Protocol 3 than Protocol 2 (p < .05) and Protocol 1 (p < .01). In addition, the post-thaw percentage of sperm with fragmented DNA was lower (p < .05) using Protocol 3 compared with Protocol 1. The present results indicate that a cooling rate of 60 °C/min around and after the time point of ice nucleation provided better post thaw survival and function of ram sperm than lower (and/or decelerating) cooling rates.     

Increased cold hardiness in eggs of Arcynopteryx compacta (Plecoptera) by dehydration

Eggs of the stonefly, Arcynopteryx compacta, that overwinter in the alpine region of Norwegian mountains, increase their cold-hardiness by dehydration. Eggs enclosed in ice at −22°C survive the loss of about two-thirds of their total water content by shrinkage due to passive diffusion of body water along the concentration gradient. Fully hydrated eggs are killed by freezing at their supercooling point of −26°C, and by direct cooling to −30°C. Dehydrated eggs have a mean supercooling point of −31°C, and survive exposure at −27 and −29°C in ice. Judged from their melting points the eggs do not accumulate low-molecular-weight cryoprotective substances. The difference between freezing and melting points corresponds to a thermal hysteresis of up to 1.8°C. The presence of thermal hysteresis antifreezes may stabilize their supercooled state when enclosed by ice during overwintering. The eggs enter diapause in the autumn, and diapause completion is enhanced both by temperature and time during enclosure in ice.     

Cooling of rabbit kidneys permeated with glycerol to sub-zero temperatures.

The aims of this study were to investigate if kidney preservation could be enhanced by cooling of the organs to high sub-zero temperatures after depression of their freezing points by addition of glycerol, and to study whether the added amounts of this compound would confer protection to the organs during freezing and thawing at slow rates.Glycerol was added and removed gradually by continuous, hypothermic perfusion, and the post-preservation viability was assessed by autotransplantation.Brief cooling to ?5 °C of kidneys perfused with 3 m glycerol was found to be compatible with life-sustaining posttransplant function, whereas no kidneys stored at that temperature for 5 days survived.Slow cooling af kidneys glycerolized to 3 m to ?80 °C was associated with a marked increase in vascular resistance after thawing, and none of such frozen kidneys functioned after transplantation. They showed immediately after revascularization severe impairment of the circulation, and vascular damage was observed by light microscopy. The use of 5 m glycerol for cryoprotection attenuated this rise in vascular resistance and reduced the release of the endocellular enzyme, lactate dehydrogenase after thawing, indicating less cellular damage although no kidneys functioned after grafting.It is suggested that the mechanical effect of interstitial and intravascular ice formation is a major factor in damage to intact organs during freezing, and that further injury is produced by incomplete removal of the cryoprotectant before transplantation.     

Behavioral responses of hatchling painted turtles (Chrysemys picta) and snapping turtles (Chelydra serpentina) at subzero temperatures

We monitored behavioral responses of cold-acclimated hatchling painted turtles (Chrysemys picta) indigenous to Nebraska and hatchling snapping turtles (Chelydra serpentina) indigenous to Nebraska and Arkansas during cooling (0.1°C/min) to temperatures as low as −19°C. All turtles made exploratory movements during cooling and locomotion occurred at temperatures as low as −2 to −4°C, but C. picta maintained relatively higher levels of locomotor activity than C. serpentina, and no differences in motility occurred between northern and southern groups of C. serpentina. Slow movements of the head and limbs were observed in supercooled hatchling C. picta at temperatures as low as −10°C, whereas at about −5°C, C. serpentina exhibited an increase in spontaneous motor activity followed by muscle contracture, immobility, and spontaneous freezing. C. picta spontaneously froze at about −16°C without exhibiting cold contracture, suggesting that they are better adapted to survive exposure to extreme cold.     

Mild hypothermia protects against postischemic hepatic endothelial injury and decreases the formation of reactive oxygen species

Abstract

The ability of mild hypothermia (MH; 34°C) to protect against postischemic endothelial injury and decrease reactive oxygen species' (ROS) formation was studied using lucigenin and luminol enhanced chemiluminescence (CL). Lucigenin CL is largely specific for superoxide, while luminol reacts with many ROS.

Isolated rat livers perfused under constant flow in a non-recirculating system were exposed to 2.5 h of ischemia after 0.5 h perfusion with Krebs-Henseleit buffer at either normothermia (38°C) or mild hypothermia (34°C) (n = 5, all groups). CL (cps), vascular resistance (Woods units), O₂ consumption, and potassium efflux were measured at the end of perfusion, and at 0 min reperfusion, and every 30 min during reperfusion.

For both the lucigenin and luminol groups, CL and vascular resistance increased significantly (repeat measures ANOVA, P <0.05) for normothermia (NT, 38°C) but not mild hypothermia. Potassium efflux did not change significantly for the mild hypothermia groups. In the luminol enhanced group, oxygen consumption was greater in the mildly hypothermic group at 1 h and 1.5 h of reperfusion.

Mild hypothermia decreased postischemic ROS production. Increased vascular resistance in the normothermia group may indicate an endothelial injury. Mild hypothermia appears to protect against this injury.     

Viability and Morphology of rat heart cells after freezing and thawing of the whole heart

Intact adult rat hearts were cooled in the presence of 10% DMSO according to an external cooling program which approximated the optimal external three-step cooling program for the isolated adult heart cells: 20 min at ?20 °C, 0.2 °C/min from ?20 to ?25, ?30, or ?50 °C, and rapid cooling to ?196 °C. Following rapid thawing, cells were isolated after perfusion with a 0.1% collagenase solution. Only cells which originated from the free wall of the right ventricle could be isolated, even after cooling to ?20 °C. Most cells from hearts cooled to ?196 °C did not survive. When the third cooling step was omitted and the end temperature of the second cooling step was ?30 °C, 38% of the cells excluded trypan blue, 29% were morphologically intact, and 30% showed spontaneous contractions after thawing, expressed as percentages of the control, A much lower survival was found after cooling to ?50 °C.Histological and electron microscopical study of the heart immediately after thawing revealed no differences between hearts cooled to ?20, ?30, or ?196 °C. Also no marked differences were observed between the morphological integrity after freezing and thawing of the atrium, the left and right ventricle walls, and the ventricular septum. The survival data suggest the presence of nonmorphologically detectable alterations in cells frozen to ?196 °C, compared to cells frozen to ?30 °C. The morphological investigations indicate no essential differences in resistance of atrial and ventricular cells to the freezing process.Experiments involving neonatal rat hearts cooled to ?196 °C, according to the method which gave optimal preservation of the isolated cells, revealed that after thawing cells are present from which growing and contracting cultures can be derived. It appears that cells in the neonatal rat heart are more resistant to freezing to ?196 °C than cells in the adult rat heart.     

Effects of freeze-thaw on the biomechanical and structural properties of the rat Achilles tendon

Rodent models are commonly used to investigate tendon healing, with the biomechanical and structural properties of the healed tendons being important outcome measures. Tendon storage for later testing becomes necessary when performing large experiments with multiple time-points. However, it is unclear whether freezing rodent tendons affects their material properties. Thus the aim of this study was to determine whether freezing rat Achilles tendons affects their biomechanical or structural properties. Tendons were frozen at either −20 °C or −80 °C directly after harvesting, or tested when freshly harvested. Groups of tendons were subjected to several freeze-thaw cycles (1, 2, and 5) within 3 months, or frozen for 9 months, after which the tendons were subjected to biomechanical testing. Additionally, fresh and thawed tendons were compared morphologically, histologically and by transmission electron microscopy. No major differences in biomechanical properties were found between fresh tendons and those frozen once or twice at −20 °C or −80 °C. However, deterioration of tendon properties was found for 5-cycle groups and both long-term freezing groups; after 9 months of freezing at −80 °C the tear resistance of the tendon was reduced from 125.4 ± 16.4N to 74.3 ± 18.4N (p = 0.0132). Moreover, tendons stored under these conditions showed major disruption of collagen fibrils when examined by transmission electron microscopy. When examined histologically, fresh samples exhibited the best cellularity and proteoglycan content of the enthesis. These properties were preserved better after freezing at −80 °C than after freezing at −20 °C, which resulted in markedly smaller chondrocytes and less proteoglycan content. Overall, the best preservation of histological integrity was seen with tendons frozen once at −80 °C. In conclusion, rat Achilles tendons can be frozen once or twice for short periods of time (up to 3 months) at −20 °C or −80 °C for later testing. However, freezing for 9 months at either −20 °C or −80 °C leads to deterioration of certain parameters.     

The efficacy of ice recrystallization inhibitors in rat lung cryopreservation using a low cost technique for ex vivo subnormothermic lung perfusion

Although lung transplant remains the only option for patients with end-stage lung failure, short preservation times result in an inability to meet patient demand. Successful cryopreservation may ameliorate this problem; however, very little research has been performed on lung cryopreservation due to the inability to prevent ice nucleation or growth. Therefore, this research sought to characterize the efficacy of a small-molecule ice recrystallization inhibitor (IRI) for lung cryopreservation given its well-documented ability to control ice growth.Sprague-Dawley heart-lung blocks were perfused at room temperature using a syringe-pump. Cytotoxicity of the IRI was assessed through the subsequent perfusion with 0.4% (w/v) trypan blue followed by formalin-fixation. Ice control was assessed by freezing at a chamber rate of −5 °C/min to −20 °C and cryofixation using a low-temperature fixative. Post-thaw cell survival was determined by freezing at a chamber rate of −5 °C/min to −20 °C and thawing in a 37 °C water bath before formalin-fixation. In all cases, samples were paraffin-embedded, sliced, and stained with eosin.The IRI studied was found to be non-toxic, as cell membrane integrity following perfusion was not significantly different than controls (p = 0.9292). Alveolar ice grain size was significantly reduced by the addition of this IRI (p = 0.0096), and the addition of the IRI to DMSO significantly improved post-thaw cell membrane integrity when compared to controls treated with DMSO alone (p = 0.0034).The techniques described here provide a low-cost solution for rat ex vivo lung perfusion which demonstrated that the ice control and improved post-thaw cell survival afforded by IRI-use warrants further study.     

Effects of temperature and doxorubicin exposure on keratinocyte damage in vitro

Cancer chemotherapy treatment often leads to hair loss, which may be prevented by cooling the scalp during drug administration. The current hypothesis for the hair preservative effect of scalp cooling is that cooling of the scalp skin reduces blood flow (perfusion) and chemical reaction rates. Reduced perfusion leads to less drugs available for uptake, whereas the reduced temperature decreases uptake of and damage by chemotherapy. Altogether, less damage is exerted to the hair cells, and the hair is preserved. However, the two mechanisms in the hypothesis have not been quantified yet. To quantify the effect of reduced drug damage caused by falling temperatures, we investigated the effect of local drug concentration and local tissue temperature on hair cell damage using in vitro experiments on keratinocytes. Cells were exposed for 4 h to a wide range of doxorubicin concentrations. During exposure, cells were kept at different temperatures. Cell viability was determined after 3 d using a viability test. Control samples were used to establish a concentration–viability curve. Results show that cell survival is significantly higher in cooled cells (T < 22° C) than in non-cooled cells (T = 37° C), but no significant differences are visible between T = 10° C and T = 22° C. Based on this result and previous work, we can conclude that there is an optimal temperature in scalp cooling. Further cooling will only result in unnecessary discomfort for the patient and should therefore be avoided.     

Low-temperature conditioning of the ice nucleation active bacterium,Erwinia herbicola

Rapid “low-temperature conditioning” and “solute conditioning” of the ice nucleation active bacterium Erwinia herbicola No. 26 are described. Conditioning is the process by which the ability to initiate ice at high temperatures is gained in these bacteria. The cumulative ice nucleator concentration, N[T], was used to measure the number of ice nucleators present in the bacterial systems. N[T] was determined at temperatures from −2 ° to −10 °C and was measured under varying conditioning temperature, time, and solute regimes. Values of N[T] increased rapidly on cooling samples from 30 to 5 °C. The optimum low temperature for conditioning was 5 °C. The conditioning process followed first-order reaction kinetics and time constants (1/rate constant) were between 43 and 62 min at 5 °C. Individual ice nucleators were isolated in droplets and were stable for at least 2 hr. Low-temperature conditioning did not occur when protein synthesis was inhibited by eliminating amino acids in the low-temperature conditioning media or by using the protein synthesis inhibitors chloramphenicol and streptomycin. Analysis of low-temperature conditioning, using heterogeneous ice nucleation theory predicted that ice nucleators are large and have diameters ranging from 80 Å (active at −8 °C) to 300 Å (active at −3 °C). In conclusion, it was predicted that conditioning resulted from growth of the nucleator from about 80 to 300 Å, from a change in the surface properties of 300 Å nucleator making it more similar to ice, or from a combination of these.     

Whole-body temperature gradients under surface,perfusion, and combined surface/perfusion hypothermia

Using various methods of hypothermia and halothane-diethyl ether azeotrope anesthesia whole-body temperature gradients were evaluated in 20 adult mongrel dogs. Simultaneous measurements were taken of brain, rectal, esophageal, pharyngeal, liver, jugular vein, shoulder muscle, thigh muscle, and subcutaneous temperatures during (i) surface, (ii) perfusion (slow and rapid cooling), and (iii) combined surface/perfusion methods of hypothermia. Throughout cooling and rewarming core temperature gradients averaged 1.2 °C and during circulatory arrest core temperatures decreased an average of 0.3 °C under pure surface hypothermia. Animals, thermoregulated by extracorporeal methods only, developed larger core temperature gradients during cooling and a significant increase (average = 3.1 °C) was noted in core temperatures during circulatory arrest. This pattern was particularly pronounced during rapid perfusion cooling. Hypothermia induction by combined surface/perfusion, in contrast to pure perfusion methods, resulted in smaller gradients without remarkable increase in core temperature (average = 1.3 °C) during the arrest period. These findings when correlated with the shorter total operating time and ease of operative management and resuscitation lead us to the conclusion that combined surface/ perfusion hypothermia techniques have certain advantages over either pure surface or pure perfusion techniques alone.     

Cryopreservation of cell line Paesun by a rapid cooling

The purpose of the present study was to clarify the possibility of a rapid cryopreservation for cell line Paesun by cooling in the range of 30–40 °C/min to vapor phase of −120 ∼-140 °C before immersion into liquid phase of liquid nitrogen using 10% Me₂SO. After thawing, these cells were examined with assaying viability by trypan blue exclusion staining and survival by cloning in monolayer; the percentages of cell and colony recovery obtained in rapid cooling had a tendency to be lower than that by slow cooling of 1 °C/min but there were no significant differences between them. In addition, post-thaw cells were examined by assaying proliferation and susceptibility to virus lines; there were no significant differences between before and after cryopreservation. In conclusion, these findings indicate that Paesun can be successfully cryopreserved by the rapid cooling rate of 30 °C–40 °C/min.