Factors influencing myeloid stem cell (CFU-C) survival after cryopreservation of human marrow and chronic granulocytic leukemia cells

Human marrow stem cells obtained from 20 patients (9 with nonhematological malignancy, 11 with acute leukemia in remission) and peripheral blood stem cells from 27 patients with chronic granulocytic leukemia were cryopreserved in 10% dimethyl sulfoxide (Me₂SO). It was found that the optimal cooling rate for the human myeloid stem cells (CFU-C) ranged from 1 to 3 °C per minute. The myeloid stem cells (CFU-C) maintained their viability for up to one year of storage in liquid nitrogen, after an initial 20% reduction due to the freezing procedure. Myeldoid stem cells survived better when thawed and diluted at room temperature (RT) than at 4 °C. However, the viability of thawed stem cells decreased when stored at RT for more than 1 hr. The viability of stem cells cryopreserved in bags and ampoules was similar. No differences were noted in the surivial of normal human marrow stem cells and cells from patients with chronic granulocytic leukemia when cryopreserved under similar conditions.     

Effects of cryoprotective agents and freezing on abdominal ganglia of aplysia.

Isolated Aplysia depilans abdominal ganglia were exposed to 10 and 20% dimethylsulphoxide (Me₂SO) or glycerol at room temperature. Results indicate that Me₂SO induced an irreversible depression of extracellularly recorded ganglionic spontaneous spike generation while glycerol proved to be non-toxic. Intracellular recordings of individual nerve cell spontaneous activity during exposure to the cryoprotective agents were obtained in a few preliminary experiments. Both Me₂SO and glycerol induced a decrement in the nerve cell membrane potential. The main difference between the action of the two cryoprotectants was in the rate and the amount of depolarization, both being higher in the case of Me₂SO exposure.The Aplysia abdominal ganglia were frozen to ?20 °C and to ?196 °C. In all but one ganglia frozen to ?20 °C, including the preparations frozen in the absence of any cryoprotective agent, functional recovery was obtained after thawing. However, only the application of 20% glycerol improved the recovery of the preparations to a significant extent. In ganglia protected with 20% glycerol a full recovery of the action potential amplitude and frequency was obtained. In ganglia protected with 20% glycerol intracellular recordings of individual nerve cells demonstrated spontaneous spike activities before freezing and after thawing.No functional recovery was observed in ganglia frozen to ?196 °C in the absence of a cryoprotective agent. While in most preparations frozen with a cryoprotectant spontaneously generated spikes were recorded after thawing. However, the action potential frequency and amplitude were significantly depressed. It is concluded that further investigation is required to improve the freezing technique so that Aplysia ganglia may be preserved at low temperatures. It is suggested that intracellular exploration of the effects of cryoprotectants and freezing on identified nerve cell membrane may prove to be useful in future investigations.     

Analysis of “solution effects” injury: Rabbit renal cortex frozen in the presence of dimethyl sulfoxide

Slices of rabbit renal cortex were frozen in 0.64 or 1.92 M dimethyl sulfoxide (Me₂SO) to various subzero temperatures, thawed, and assayed for viability. Salt and Me₂SO concentrations were calculated and correlated with the injury taking place during freezing. In separate experiments, slices were treated with NaCl or Me₂SO in concentrations sufficient to simulate the exposure brought about as a result of freezing. The effects of these treatments on cortical viability were compared with the results of freezing to equivalent concentrations of either NaCl or Me₂SO. The results show that whereas slices will tolerate exposure to at least six times the isotonic concentration of NaCl at 0 °C, they are unable to tolerate even three times the isotonic salt concentration when frozen in 1.92 M Me₂SO. They can, however, tolerate 3 × NaCl when frozen in 0.64 M Me₂SO. Freezing damage did not depend upon the amount of ice formed per se, since slices frozen in the low concentration of Me₂SO tolerated removal of about 75% of the initial fluid content of the system, whereas slices frozen in 1.92 M Me₂SO did not tolerate an identical removal of unfrozen solution. It was found that treatment of slices with high concentrations of Me₂SO at subzero temperatures in accordance with Elford's application (14) of Farrant's method (20) produced damage which correlated approximately with the damage observed when the same concentrations of Me₂SO were produced by freezing. It is concluded that most of the damage caused by freezing in 1.92 M Me₂SO is produced either directly or indirectly by Me₂SO. Possible mechanisms for this injury are discussed.     

Optimizing conditions for cryopreservation of an insect cell line

A cell line (UM-BGE-2) derived from embryos of the cockroach Blattella germanica was frozen to ?196 °C under a variety of conditions and cell viability was assayed after warming. It was found that cell viability was affected by the cooling rate, the warming rate, the controlled cooling endpoint temperature, and the type and concentration of cryoprotectant. The best survival for cells suspended in Grace's tissue culture medium containing 1 M Me₂SO was obtained when cells were cooled at 1 °C/ min to at least ?90 °C before being placed in liquid nitrogen and warmed at more than 900 °C/min. Cultures initiated from these frozen cells produce typical growth curves and appear normal after several passages.     

Cryopreservation of some useful microalgae species for biotechnological exploitation

Culture collections of microalgae represent a biological resource for scientific research and biotechnological applications. When compared to the current methods of maintenance and sub-culturing, cryopreservation minimizes labor costs and is an effective method for maintaining a large range of species over long periods with high stability. In order to determine the best cryopreservation method for microalgae species with great biotechnological potential, three freezing protocols were employed using different cryoprotectants (dimethyl sulfoxide—Me₂SO; methanol—MeOH). Three marine microalgae species (Thalassiosira weissflogii; Nannochloropsis oculata, and Skeletonema sp.) were cooled by directly plunging into liquid nitrogen (?196°C) and with two-step controlled cooling protocols (?18°C and ?80°C pre-treatments). After storage periods ranging from 10 to 120 days, viability was determined by the ability of cells to actively grow again. Results obtained for T. weissflogii showed that this species could be preserved at ultra-low temperature (?196°C) for 10 and 30 days with 10?% Me₂SO and 5?% MeOH when employed a controlled cooling protocol (?80°C). N. oculata was successfully cryopreserved either by direct freezing or with controlled cooling protocols. N. oculata samples presented good responses when treated with 5?% Me₂SO, 10?% Me₂SO, 5?% MeOH and even without any cryoprotectant. Skeletonema sp. did not survive cryopreservation in any of the tested conditions. The results indicate the difficulty in establishing common protocols for different microalgae species, being necessary further studies for a better understanding of cell damages during freezing and thawing conditions for each species.     

A novel strategy for conservation of European eel (Anguilla anguilla) genetic resources: Cryopreservation of ovarian stem cells

The aim of this study was to develop short- and long-term preservation protocols for European eel ovarian stem cells (OSCs) through hypothermic storage and cryopreservation of ovarian fragments that will assist in current conservation programs of this critically endangered species. Firstly, a freezing procedure was developed by testing different cryomedia and technical aspects of freezing. Utilization of 1.5 M of dimethyl sulfoxide (Me₂SO), 0.1 M glucose and 1.5% BSA yielded optimal OSCs survival. Additionally, equilibration of 50-mg ovarian fragments for 30 min and plunging into lN₂ at −80 °C displayed the highest OSC viability. Different cooling rates ranging from −1 to −40 °C/min did not significantly affect OSC viability when thawing in a 10 °C water bath. In addition, application of needle-immersed vitrification (NIV), combining ES3 (1.5 M PG and 1.5 M Me₂SO) with VS3 (3 M PG and 3 M Me₂SO) yielded the highest viability rates. Finally, hypothermic storage (4 °C) of ovarian fragments and ovarian cell suspensions displayed favorable viability of ~90% after 48 h of storage and ~65% after 72 h of storage. The development of OSC preservation methods presents an onset of further development of germline stem cell (GSC) manipulation techniques in this species. Cryopreservation of OSCs can enable a continuous supply of cells for either transplantation or in vitro cell culture thus enabling new and improved management and conservation strategies for this endangered species.     

Preservation of polymorphonuclear neutrophils at cryogenic temperatures (−80 °C)

The effects of cryoprotectant alone and cryoprotectant plus additives on the preservation of polymorphonuclear neutrophils (PMNs) at cryogenic temperatures (?80 °C) were studied.A considerable difference among cryoprotective agents was observed in their protective effect against freeze injury of neutrophils. Based upon degree of chemotactic inhibition and impairment of trypan blue exclusion, Me₂SO proved to be superior to ethylene glycol and glycerol as a cryoprotectant and to exhibit the best protective effect at a concentration of 4.2%. When PMNs were frozen in Me₂SO alone, the ability of PMNs to exclude dye was retained after 3 days of cryopreservation, while chemotaxis was inhibited markedly. One-week preservation produced the death of 50% of the cells. To improve the protective effect of Me₂SO against chemotactic inhibition by cryopreservation, additives such as glucose, ATP, and albumin were included in the freezing medium. Addition of albumin displayed the most distinct improvement in the recovery of chemotaxis, although ATP also exhibited a protective effect, especially during short-term storage. Studies on the combined effect of these additives with ethylene glycol or glycerol showed that only albumin had a considerably better protective effect against dye exclusion injury but not against chemotactic inhibition. Phagocytosis and adhesion were less inhibited by freezing than was chemotaxis. A combination of Me₂SO and ATP markedly protected phagocytosis and adhesion from freeze injury. However, cyanide-insensitive oxygen uptake during phagocytosis, as well as chemotaxis, were considerably inhibited.     

Characterizing the ability of an ice recrystallization inhibitor to improve platelet cryopreservation

Improving aspects of platelet cryopreservation would help ease logistical challenges and potentially expand the utility of frozen platelets. Current cryopreservation procedures damage platelets, which may be caused by ice recrystallization. We hypothesized that the addition of a small molecule ice recrystallization inhibitor (IRI) to platelets prior to freezing may reduce cryopreservation-induced damage and/or improve the logistics of freezing and storage. Platelets were frozen using standard conditions of 5–6% dimethyl sulfoxide (Me₂SO) or with supplementation of an IRI, N-(2-fluorophenyl)-d-gluconamide (2FA), prior to storage at −80 °C. Alternatively, platelets were frozen with 5–6% Me₂SO at −30 °C or with 3% Me₂SO at −80 °C with or without 2FA supplementation. Supplementation of platelets with 2FA improved platelet recovery following storage under standard conditions (p = 0.0017) and with 3% Me₂SO (p = 0.0461) but not at −30 °C (p = 0.0835). 2FA supplementation was protective for GPVI expression under standard conditions (p = 0.0011) and with 3% Me₂SO (p = 0.0042). Markers of platelet activation, such as phosphatidylserine externalization and microparticle release, were increased following storage at −30 °C or with 3% Me₂SO, and 2FA showed no protective effect. Platelet function remained similar regardless of 2FA, although functionality was reduced following storage at −30 °C or with 3% Me₂SO compared to standard cryopreserved platelets. While the addition of 2FA to platelets provided a small level of protection for some quality parameters, it was unable to prevent alterations to the majority of in vitro parameters. Therefore, it is unlikely that ice recrystallization is the major cause of cryopreservation-induced damage.     

Cryopreservation of human umbilical vein and porcine corneal endothelial cell monolayers

Cryopreservation of endothelium is one of the major challenges in the cryopreservation of complex tissues. Human umbilical vein endothelial cells (HUVECs) in suspension are available commercially and recently their post-thaw cell membrane integrity was significantly improved by cryopreservation in 5% dimethyl sulfoxide (Me₂SO) and 6% hydroxyethyl starch (HES). However, cryopreservation of cells in monolayers has been elusive. The exact mechanisms of damage during cell monolayer cryopreservation are still under investigation. Here, we show that a combination of different factors contribute to significant progress in cryopreservation of endothelial monolayers. The addition of 2% chondroitin sulfate to 5% Me₂SO and 6% HES and cooling at 0.2 or 1 °C/min led to high membrane integrity (97.3 ± 3.2%) immediately after thaw when HUVECs were cultured on a substrate with a coefficient of thermal expansion similar to that of ice. The optimized cryopreservation protocol was applied to monolayers of primary porcine corneal endothelial cells, and resulted in high post-thaw viability (95.9 ± 3.7% membrane integrity) with metabolic activity 12 h post-thaw comparable to unfrozen control.     

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.     

Freeze-thaw survival of the free-living nematode Caenorhabditis briggsae.

Approximately 75% or more of the L2 and L3 juvenile stages of the free-living nematode Caenorhabditis briggsae survived freezing and thawing without loss of fertility. Optimum survival depended upon a combination of conditions: (1) pretreatment with 5% DMSO at 0 °C for 10 min, (2) 0.2 °C per minute cooling rate from 0 to ?100 °C prior to immersion into liquid nitrogen, and (3) a 27.6 °C per minute warming rate from ?196 °C to ?10 °C. Storage at ?196 °C for more than 100 days was without effect on viability or fertility. Some of the L4 (about 50%) and adult (about 3%) stages survive the routine freeze-thaw treatment. However, there was no recovery of either embryonic stages or embryonated eggs from ?196 °C under these standard conditions. Either very fast cooling (about 545 °C/min) or fast warming (about 858 °C/min) rates diminished survival of the L2 and L3 stages drastically.Scanning electron microscopy revealed that freeze-thaw survivors with aberrant swimming behavior had cuticular defects. In juvenile forms, the altered swimming motion was lost after a molt whereas as abnormal adults grew, sinusoidal movement resumed. In the L4 and adult forms the cuticular abnormalities lowered viability and fertility. It is concluded that survival of nematodes from a freeze-thaw cycle is contingent upon establishing specific cryobiological conditions by varying aspects of the procedure that gave high recoveries of L2 and L3 stages.     

Cryopreservation of granulocytes for transfusion: Studies on human granulocyte isolation,the effect of glycerol on lysosomes,kinetics of glycerol uptake and cryopreservation with dimethyl sulfoxide and glycerol

Existing methods for the cryopreservation of granulocytes employ primarily dimethyl sulfoxide (Me₂SO) rather than glycerol as the cryoprotective additive of choice. Although Me₂SO has been demonstrated to be an effective cryoprotective additive for granulocyte preservation to yield viable cells (dye exclusion, phagocytosis, etc.), the inherent toxicity and clinical objections of Me₂SO as a cryoprotective additive for granulocyte preservation preclude its extensive and routine use in patients. Therefore, glycerol, with its important advantage of nontoxicity, has been investigated for its potential usefulness as a cryoprotective additive for preserving human granulocytes for transfusion.Granulocyte preparations were isolated from impure leukocyte concentrates obtained from the buffy coats of human whole blood. Studies on the isolation and purification of the granulocytes involved separation by sedimentation with dextran, removal of red cells by hypotonic shock with water, resuspension with Plasmatein and further purification by centrifugation. Intact viable granulocytes were obtained with a purity in excess of 90%.Lysosomes were studied as indicators of cryoinjury in granulocytes using β-glucuronidase as the key marker enzyme. This enzyme has been characterized as a sensitive indicator of damage to lysosomes and a direct linear relationship has been established between damage to granulocytes by freezing and amount of lysosomal enzyme released. Addition or presence of the cryoprotectant, glycerol, did not appear to have any adverse effect on lysosomes of intact granulocytes.Studies on the permeation kinetics of glycerol in granulocytes indicated that the additive was freely permeable and did not cause any potentially damaging osmotic changes in cell volume. Granulocytes in various concentrations of glycerol were then frozen at slow, moderate, and rapid cooling rates. Based on the small amount of β-glucuronidase released, good preservation of granulocyte lysosomes has been obtained with a slow cooling rate of 5 °C/min and a concentration of 15% glycerol. Further studies now are necessary to define those conditions of cooling rate and glycerol concentration required to develop a simple method for optimal preservation of granulocytes based on additional functional criteria of viability.     

Skin cryopreservation: I. Incorporation of radioactive sulfate as a criterion of pigskin graft viability after freezing to −196 °C in the presence of cryoprotectants

The incorporation of radioactive sulfate into glycosaminoglycans was used as a criterion of pigskin graft viability after surface treatment of the pig hide and after cooling and freezing of the graft. Complete surface treatment of the hide (soap, ethanolic iodine, antibiotics, and saline) diminished the incorporation of sulfate by about 40% compared with the control graft. During cooling and freezing the pigskin graft was submitted to 30-min exposures at 20, 4, ?18, ?50, ?150, and ?196 °C sequentially in a medium containing 0.65% NaCl, 3% sorbitol, and either 15% glycerol or 15% Me₂SO. Cooling to ?18 °C reduced the incorporation of sulfate only in the grafts protected by glycerol. A considerable decrease of incorporation was observed after freezing the graft to ?150 and ?196 °C in both cryoprotective solutions. The inclusion of a hold at ?50 °C was important, especially in the case of the Me₂SO medium when about 30% of ³⁵S radioactivity was recovered in the cryopreserved graft compared with the control sample.     

An experimental study on the freezing of red blood cells with and without hydroxyethyl starch

Red blood cells were frozen in small capillaries down to ?196 °C at different linear cooling rates with or without the cryoadditive HES; the thawing rate was 3000 or 6500 °C/min. Hematocrit and hydroxyethyl starch concentration varied independently. The hemolysis of red blood cells was determined photometrically after 250-fold dilution and compared to totally hemolyzed samples. The typical U-shaped curves for hemolysis as a function of the cooling rate were obtained for all cell suspensions investigated. Relative optimum cooling rates were determined for the respective combinations of HES and hct. The results show that increasing hct causes an increased hemolysis; increased HES concentration C_HES reduces the optimum cooling rate B_opt; increased hct results in higher optimal cooling rates. The findings allow one to establish a linear correlation of the HES concentration and the optimum cooling rates when the dilution of the extracellular medium by the cell water efflux during freezing is taken into account. A comparison with results from larger volumes frozen (25 ml) shows that the established relationship between hematocrit, HES concentration, and optimal cooling rate remains valid.     

Cell preservation in a programmed cooling machine: the effect of variations in supercooling

When cells are cryopreserved in programmed cooling machines, they supercool to a variable and uncontrolled extent. Experiments were carried out with three cell-types (human peripheral lymphocytes, Chinese hamster lung fibroblasts, and mouse lymphoma cells) to determine whether there was any effect of supercooling on cell survival. Samples were cooled at 1 °C min^?1 in the presence of 12% v/v dimethyl sulphoxide (Me₂SO) to ?100 °C, and then thawed rapidly in a 37 °C water bath. There was no correlation between the extent of supercooling or the maximum cooling rate after freezing and cell survival, but the time taken for the sample temperature to return to the temperature at which freezing occurred did influence the survival of the two tissue culture cell lines. These results are interpreted on the basis of current theories according to which cells require sufficient time to lose water as they cool in order to avoid subsquent intracellular freezing, but must be cooled sufficiently rapidly to minimise solution effects. It is concluded that the variations in supercooling that occur in programmed cooling machines present no particular difficulties, providing appropriate cooling rates are chosen.     

Effect of freezing extender composition and male line on semen traits and reproductive performance in rabbits

This study was conducted to elucidate the effect of different freezing extenders on two lines selected for hyperprolificacy and longevity (H and LP, respectively). In extender A, dimethyl sulphoxide (Me₂SO) and sucrose were used as cryoprotectants. In extenders B and C, the sucrose was replaced by 20% egg yolk, and in extender C the Me₂SO was substituted by acetamide. Semen was packaged in 0.25 ml plastic straws and cooled at 5°C for 45 min, and then was frozen in liquid nitrogen vapour for 10 min before being plunged into the liquid nitrogen. Thawing was carried out by immersing the straws in a water bath at 50°C for 10 s. Frozen-thawed semen characteristics and reproductive parameters were affected by freezing. Extender C showed significantly lower post-thawing quality traits than any of the three extenders. Acrosome integrity was significantly improved when Me₂SO was used as cryoprotectant. Sucrose replacement by 20% egg yolk had no effect on acrosome integrity but provided significantly lower sperm motility and viability. Freezing extender affected fertility rate, total born, number of implantation sites and gestational losses, obtaining better results when extender A was used. The acrosomal integrity after frozen-thawed process showed a significant correlation with fertility at 12^th day and also at birth, indicating that an increase in acrosomal integrity leads to an increase in both fertilities (12^th day and at birth). A positive correlation between motility of semen and implantation sites was found. The post-thawing quality traits of semen were not affected by the genetic line, although LP line showed higher total born and lower foetal and gestational losses. The findings of this study suggest that freezing extender composition has a significant effect on the success of rabbit sperm for preservation, and when Me₂SO was used as permeable cryoprotectant sucrose provided better protection compared with egg yolk and improved reproductive traits, and, on the other hand, the male genotypes used in the present study had no effect on frozen-thawed sperm parameters but negatively affected some of the reproductive parameters.     

Cryopreservation of embryos and larvae of the edible sea urchin loxechinus albus (Molina, 1782)

The natural population of the edible red sea urchin, Loxechinus albus, is decreasing due to overfishing. The embryos and larvae of the species are highly useful for monitoring marine pollution, which makes it necessary to conserve gametes, embryos and larvae to facilitate their use in diverse areas of aquaculture and environmental quality monitoring. This need can be met by cryopreserving individuals representing the different developmental stages to provide an ongoing supply of genetic material of the species. The present study establishes a reproducible protocol for cryopreserving red sea urchin blastula and larvae. Toxicity tests were conducted in the first stage of this study using two permeable cryoprotectors, dimethyl sulfoxide (Me₂SO) and propylene glycol (PG), at three concentrations (5%, 10% and 15%). The tests were repeated in the second stage, but mixing the cryoprotectors with 0.04 M of trehalose (TRE), a non-permeable cryoprotector. Cryopreservation tests were conducted in the third stage employing different freezing rates: 2 °C/min, 3 °C/min, 3.5 °C/min, 4 °C/min and 4.5 °C/min, using the cryoprotectors that yielded the highest post-incubation survival rates.The highest post-freezing survival rates for blastula (76 ± 7%) and larvae (79 ± 7%) were obtained with Me₂SO at 10% + 0.04 M of trehalose, with freezing rates of 3 °C/min and 4.5 °C/min, respectively.     

The effect of ice formation on the function of smooth muscle tissue stored at −21 or −60 °C

The mechanisms by which single cells are injured during freezing are relatively well understood, but it is likely that additional factors apply to tissues and organs, factors that may be responsible for the poor suecess of attempts to cryopreserve complex multicellular systems. One such factor may be the formation of extracellular ice.

This study was designed to discover whether ice formation as such is detrimental to the contractile recovery of pieces of mammalian smooth muscle after storage at subzero temperatures. Strips of taenia coli muscle were equilibrated with 2.56 M Me₂SO in a buffered solution, cooled at either 0.3 or 2 °C/min to ?21 °C and then held at this temperature in the frozen state. Other muscle strips were bathed in a solution the composition of which mimicked that of the unfrozen phase of the previous solution at ?21 °C; it contained 4.49 M Me₂SO and 1.75 times the normal concentration of salts, and muscles equilibrated with this solution were also cooled at either 0.3 or 2 °C/min to ?21 °C, and then held unfrozen for the same length of time.It was shown that exposure to ?21 °C and the increased concentration of solutes had little effect on the contractile recovery of the muscles, whereas ice formation was damaging. Furthermore, the rate of cooling had a marked effect upon functional recovery in the frozen muscles, and this could be correlated with the known effect of these cooling rates on the pattern of ice formation in the tissue. The effect was also seen in muscles frozen at ?60 °C. Improved buffering increased the functional recovery of all groups, but the effect of ice, and of cooling rate in the presence of ice, was confirmed. These findings may have significant implications for attempts to cryopreserve complex tissues and organs.     

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.     

The Effect of Freezing Rate on the Survival of Cryopreserved African Sharptooth Catfish (Clarias gariepinus) Spermatozoa

Cryoprotective agents were evaluated to find the optimal concentration of the cryoprotectant and most suitable combination of solution and cryoprotectant. A cryoprotective agent composed of 4% glucose and 9% glycerol yielded the best results. It was established that the optimal freezing rate is dependent on the composition of the cryoprotective agent. Maximal survival of catfish spermatozoa (60%) occurs at 5°C min^-1 and faster and slower freezing rates result in poor survival or no survival at all. Incorporation of an isothermal holding period into the freezing rate led to remarkable increase (20-30%) in sperm survival when Me₂SO was present in the cryoprotective agent. Cryoprotective agents containing glucose also showed improved survival when a three phase freezing rate was used. These results lead to the conclusion that the presence of an isothermal holding period in the freezing rate is beneficial for the cryoprotective action of Me₂SO and glucose.