Freezing tolerance in embryos and spermatozoa of the sea urchin

Late embryos of the sea urchin survive freezing, at least for a short period of time, at ?196 °C in the presence of a cryoprotectant. The freezing tolerance in glycolated embryos is greater in advanced developmental stages. High rates of both cooling and warming during a freezethaw sequence were more dangerous in one-cell embryos than in late ones. Both ethylene glycol and DMSO exerted a significant protection against freezing injury on embryos in all the stages after fertilization, but not on unfertilized egg cells. Cryopreservation of sea urchin sperm at ?196 °C in the presence of 1.5 m ethylene glycol has been achieved for 3 days. The fertilizability of eggs inseminated by frozen-thawed sperm was more than two-thirds. Nearly all the eggs thus fertilized developed to normal gastrulae.     

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.     

Histological studies on cultured canine heart valves recovered from −196 °C

Adult canine heart valves have been frozen to ?196 °C, (0.5 to 0.7 °C/min from 0 to ?100 °C) with 10% DMSO ($\text{v}\text{v}$), stored, thawed at ~150 °C/min, and then cultured for 9 to 12 days. A histological analysis of sections derived from several valves indicates viability, but with a not inconsiderable loss of stromal fibrocytes and some damage to the endothelial lining. The practicality of freezing valve tissue for banking will have to be looked at critically, before valve transplants can be considered as a possible alternative to the well established use of mechanical valve prosthesis. However, demonstrating viability of heart valve tissue extends the range of tissues that are amenable to cryopreservation.     

Regeneration of Plants from Cell Suspensions Frozen at −20, −70 and −196°C

Cell suspensions of carrot, Datura, tobacco and soybean subjected to ?20°C, ?70°C and ?196°C in the presence of a suitable cryoprotective agent, and stored for various lengths of time have been revived. After revival these cells divided to form callus masses. Direct immersion in liquid nitrogen invariably killed the cells, whereas cooling at the rate of 1 or 2°C/min, or pre-freezing briefly at ?20 and ?70°C, followed by freezing at ?196°C retained the viability. Depending on the plant species up to 70% of the cell clumps could withstand ultra-cooling. Tobacco and Datura cell suspensions were more sensitive to cold treatment than were those of carrot. Actively growing cell suspensions containing small cell-clumps revived rapidly, while filtered cell-suspensions of free cells only occasionally survived. Calli of tobacco and carrot obtained from frozen suspensions have been regenerated into plants.     

Cooling of embryonic cells,isolated blastoderms,and intact embryos of the zebra fish Brachydanio rerio to −196 °c

Single cells from the developing embryo of the zebra fish survive freezing when protected with 1 M DMSO and cooled to ?196 °C in two steps. Cell survival drops from 85 to 26% when clumps of 5–10 cells are similarly frozen, and to 2% when isolated blastoderms are treated in the same way. This drastic decrease in survival is interpreted as an example of the “scale-up problem,” in which diffusional barriers prevent cryoprotectant equilibration and osmotic dehydration in large cell assemblanges.Isolated blastoderms develop considerably in culture, and retain some of this ability following cooling to ?25 °C after protection with DMSO or glycerol.Intact embryos protected with high concentrations of glycerol (2.8 M) tolerate slow cooling to ?196 °C surprisingly well, with most of the embryonic cells morphologically intact and actively extruding lobopodia. Glycerol could, however, only be removed from cells by disrupting the embryo so that diffusional barriers were removed. DMSO (2.8 M) was ineffective in preserving embryos or cells cooled to ?196 °C.     

DNA replication in eucaryotic nuclei. Evidence suggesting a specific model of replication

Preimplantation-stage mouse embryos suspended in dimethyl sulfoxide (DMSO) have been used as a model to study details of the response of a simple multicellular system to freezing and thawing. Rapid freezing to ?196 °C kills the embryos unless they have first been cooled very slowly to at least below ?50 °C. The survival of both 2-cell and 8-cell embryos has been found to depend as critically on the rate at which the frozen embryos were thawed as on the rate at which they were first frozen. The damaging consequences of thawing frozen embryos too rapidly have been shown to occur between ?70 and ?20 °C. Finally, the survival of embryos as a function of the time in DMSO prior to freezing and thawing has been compared with their volume changes as a function of time in DMSO. This comparison leads to the tentative conclusion that dimethyl sulfoxide need not permeate the embryos to protect them against freezing damage. Overall, the embryos' response to freezing and thawing is qualitatively similar to that displayed by many other cell types.     

Microscopic observation of intracellular ice formation in unfertilized mouse ova as a function of cooling rate

A physical-chemical analysis of water loss from cells at subzero temperatures had shown that the likelihood of intracellular ice formation increased with increasing cooling rate (22). We have now used a modified version of a unique conductioncooled cryomicroscope stage (8) to observe the freezing of unfertilized mouse ova suspended in dimethyl sulfoxide. Survival measurements showed that the respective survivals of ova were about 65, 56, and 0% when they were cooled at rates of 0.2 to 1.5, 2.5, and 5.4 °C/min. Direct microscopic observation of mouse ova during freezing showed that the respective fractions of cells that froze intracellularly were 13, 72, and 100% when they were cooled at rates of 1.3, 2.9, and 4.8 °C/min or faster. These values agree with those predicted from the physical-chemical analysis for cells the size of mouse ova. The microscopic observations have also shown that intracellular freezing generally occurred at about ?40 to ?45 °C. We had previously observed that mouse embryos must be cooled slowly to ?50 °C or below if they are to survive subsequent rapid cooling to ?196 °C. The observation of intracellular ice formation at ?45 °C supports the interpretation that at temperatures above ?50 °C the embryos still contain water capable of freezing intracellularly.     

Survival of tissue culture cells frozen by a two-step procedure to -196 degrees C. I. Holding temperature and time.

A two-step freezing procedure has been examined in order to separate some of the causes of damage following freezing and thawing. Different holding temperatures and times have been studied during the freezing of Chinese hamster tissue culture cells in dimethyl sulphoxide (5%, $\text{v}\text{v}$). Damage following rapid cooling to, time at, and thawing from different holding temperatures was found to increase at lower holding temperatures and at longer times. Damage on subsequent cooling from the holding temperature to ?196 °C and thawing was found to diminish at lower holding temperatures and longer times. The net result was that optimal survival from ?196 °C was obtained after 10 min at ?25 °C. Protection against the second step of cooling to ?196 °C was acquired at the holding temperature itself and was absent at ?15 °C without freezing.It seems that this technique will allow the different phases of freezing injury to be separated. These phases may include thermal shock to the holding temperature, hypertonic damage at the holding temperature and dilution shock on thawing from ?196 °C.     

Variations in myocardial CPK and Na+-K+ ATPase following long-term freezing.

The activity of the soluble enzyme CPK and the membrane bound enzyme Na⁺-K⁺ ATPase as a function of storage temperature, time of storage and cryoprotectant type and concentration in canine myocardial tissue was invesigated. Activity of CPK is well preserved at ?196 °C and ?79 °C and falls off during one month storage at ?40 °, ?20 °, and 0 °C. Na⁺-K⁺ ATPase demonstrates a greater liability. After an initial cryoprotectant “activation,” activity drops. In all cases, however, addition of the cryoprotectant preserved activity better than in samples stored only in buffer.     

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.     

A simple method for the freeze-preservation of zoospores of the green macroalga Enteromorpha intestinalis

Settled zoospores of the green macroalga Enteromorpha intestinalis were subjected to several different freezing and storing treatments at both cryogenic and non-cryogenic temperatures after which their viability was assessed using a spore germination bioassay. Three different cooling rates were tested: slow cooling at –1°C min⁻¹ and –0.5°C min⁻¹ to end temperatures in the range –20°C to –40°C, and a two-step procedure whereby the spores were frozen to –30°C at a rate of –1°C min⁻¹ prior to immersion in liquid nitrogen at –196°C. Spore viability was also investigated using the cryoprotectants glycerol and dimethyl suphoxide (DMSO), a reduced saline medium and various storage times. In the majority of experiments, the use of a cryoprotectant during the freezing process significantly increased the viability of the spores, with DMSO affording slightly greater protection than glycerol. All treatments produced high viabilities (ranging from 75.3–100.0%) after 5-min storage at the different end temperatures. However, progressively longer storage up to 7 days generally resulted in a marked reduction in viability. This was with the exception of spores frozen in a reduced saline medium; a medium of 75% seawater and either 5 or 10% DMSO greatly increased spore viability, with values of > 40% recorded for spores stored at –20°C for up to 5 weeks. This revised version was published online in July 2006 with corrections to the Cover Date.     

Biochemical functionality and recovery of hepatocytes after deep freezing storage

Summary The present study was undertaken to define the conditions for optimal cryopreservation of hepatocytes. Two different freezing procedures were analyzed: a slow freezing rate (SFR) (−2° C/min down to −30°C and then quick freezing to −196° C) and a fast freezing rate (FFR) (direct freezing of tubes to −196° C: −39° C/min). Cells were frozen in fetal bovine serum containing 10% Dimethyl sulfoxide (DMSO). After rapid thawing at 37° C, followed by dilution and removal of the cryoprotectant, cells were plated and several parameters were followed as criteria for optimal cryopreservation of cells. The FFR cells showed no apparent ultrastructural damage after 24 h of culture. Plating efficiency and spreading were similar as controls. Gluconeogenesis from pyruvate and fructose, tyrosine amino transferase induction by glucagon and dexamethasone, urea production, and plasma protein synthesis of FFR cells were similar to those found in control cultures. The FFR procedure, in comparison to the SFR method, seemed to render the best preserved hepatocytes. The financial support for this work was from Fondo de Investigaciones Sanitarias de la Seguridad Social, Grants 41/82 and 48/82.     

The effect of two cryopreservation methods on human sperm DNA damage

Several methods are currently available for selection when conducting sperm cryopreservation, however, these methods might cause different degrees of damage on sperm DNA. The aim of the this study is to compare the effects of storage at −80 °C (in ultra-low temperature refrigerator) and at −196 °C (in liquid nitrogen) on sperm DNA damage, thus to provide a reference for choosing the right method according to different aims. We randomly collected 28 semen samples from college students of Chongqing city. The samples stored at −80 °C were neat semen samples and the samples stored at −196 °C were mixed with additional cryoprotectants. Each sample was subjected to two freezing-thawing cycles, and the sperm DNA damage levels of fresh and thawed samples were measured by single cell gel electrophoresis (SCGE) and sperm chromatin structure assay (SCSA). Both SCGE and SCSA assays showed cryopreservation induced significant damage to sperm DNA. However, storage at −196 °C lead to more severe damage to sperm DNA than storage at −80 °C measured by SCSA. Sperm DNA damage increased simultaneously with the higher frequency of freezing-thawing cycles. We concluded that storage of neat semen samples at −80 °C had milder damage to sperm DNA than storage at −196 °C mixed with cryoprotectants. To avoid additional sperm DNA damage, repeated freezing and thawing should be prevented.     

Development of a spermatogonia cryopreservation protocol for blue catfish,Ictalurus furcatus

Sustainability of channel catfish, Ictalurus punctatus ♀ × blue catfish, Ictalurus furcatus ♂ hybrid aquaculture relies on new innovative technologies to maximize fry output. Transplanting spermatogonial stem cells (SSCs) from blue catfish into channel catfish hosts has the potential to greatly increase gamete availability and improve hybrid catfish fry outputs. Cryopreservation would make these cells readily accessible for xenogenesis, but a freezing protocol for blue catfish testicular tissues has not yet been fully developed. Therefore, the objectives of this experiment were to identify the best permeating [dimethyl sulfoxide (DMSO), ethylene glycol (EG), glycerol, methanol] and non-permeating (lactose or trehalose with egg yolk or BSA) cryoprotectants, their optimal concentrations, and the best freezing rates (−0.5, −1.0, −5.0, −10 °C/min until −80 °C) that yield the highest number of viable type A spermatogonia cells. Results showed that all of these factors had significant impacts on post-thaw cell production and viability. DMSO was the most efficient permeating cryoprotectant at a concentration of 1.0 M. The optimal concentration of each cryoprotectant depended on the specific cryoprotectant due to interactions between the two factors. Of the non-permeating cryoprotectants, 0.2 M lactose with egg yolk consistently improved type A spermatogonia production and viability beyond that of the 1.0 M DMSO control. The overall best freezing rate was consistent at −1 °C/min, but similar results were obtained using −0.5 °C/min. Overall, we recommend cryopreserving blue catfish testicular tissues in 1.0 M DMSO with 0.2 M lactose and egg yolk at a rate of either -0.5 or −1 °C/min to achieve the best cryopreservation outcomes. Continued development of cryopreservation protocols for blue catfish and other species will make spermatogonia available for xenogenic applications and genetic improvement programs.     

The developmental potential of frozen-thawed hamster preimplantation embryos following embryo transfer: Viability of slowly frozen embryos following slow and rapid thawing

Hamster preimplantation embryos were slowly frozen (0.33°C/min) and seeded above 10°C in TC-199 containing 1.5 M-DMSO. These embryos were thawed either slowly (1.5°C/min) or rapidly (90°C/min). The thawed embryos were examined by morphology, trypan blue exclusion and viability after embryo transfer. Slow thawing gave significantly higher viability compared to rapid thawing. The early preimplantation embryos demonstrate higher sensitivity to freezing. The three tests of viability (morphology, trypan blue exclusion and embryo transfer) were found to be positively correlated.     

The cryopreservation of Chlamydomonas.

A cryophilic strain of the unicellular green alga Chlamydomonas, C. nivalis was found to be more resistant to the stresses both of freezing and thawing and of shrinkage and rehydration than was a mesophilic strain C. reinhardii. C. nivalis was found to have a higher degree of unsaturation of phospholipid fatty acids. Following freezing and thawing of C. reinhardii there was a direct correlation between reduction in cell viability and loss of membrane selective permeability. Activation of intracellular phospholipases occurred at an early stage of freezing injury. Attempts to cold harden C. reinhardii were unsuccessful. For C. reinhardii methanol was the only effective cryoprotectant for freezing to and thawing from ?196 °C and the effects of cooling rate upon cellular survival are presented.     

A successful technique for the preservation of rabbit embryos

A technique for successfully freezing, thawing and transferring rabbit embryos has been developed. Morula stage embryos were collected from super-ovulated female rabbits by flushing both oviducts and uterine horns with a tissue culture medium. Well developed, viable embryos were then transferred to freezing vials and a cryoprotectant, dimethyl sulfoxide (DMSO) was added in several steps to bring its final concentration to 1.6 molar. To freeze the embryos the temperature was lowered slowly (either 0.5 degrees C/min or 1.0 degrees C/min) to -80 degrees C at which point the vials were transferred directly to liquid nitrogen (-196 degrees C). Thawing was done at 8 degrees C/min. After thawing, phosphate buffered saline was added in a stepwise manner to dilute the DMSO. The thawed embryos were then cultured at 37 degrees C. Transfer of the embryos was accomplished by laparotomizing a pseudopregnant doe and introducing the embryos into the fimbriated ends of the oviducts. The 101 positively transferred embryos resulted in 45 implantations and 34 live born young.     

A simplified method for freezing mouse embryos

Mouse oviducts containing eight-cell embryos were frozen to ?196 °C in 1.45 m DMSO. The cooling rate was 0.3 °C/min and thawing occurred at 3 °C/min. Dilution of DMSO took place either before or after flushing of the thawed oviducts. The yield of intact embryos was higher in the second group.In one particular series involving 21 donor mice (natural ovulation) 88 recovered embryos were transferred to the oviducts of recently mated pseudopregnant mice without prior in vitro culture to the blastocyst stage. Fifty-five live young were born.It is concluded that the freezing of embryos in the oviduct is a reliable method for establishing an embryo bank. Handling and collection of isolated embryos is not required and a large amount of material can be frozen at once. In vitro culturing of embryos is not required immediately after thawing in order to obtain a high yield of live young.     

Effects of freezing to −196 °C and thawing on Setaria lutescens seeds

The effects of single and repeated freezing and thawing of Setaria lutescens seeds in liquid nitrogen were investigated. One freeze to ?196 °C followed by a slow thaw, increased seed germination from 40 to 70%, but additional freeze-thaw cycles reduced germination to 30%. Using a scanning electron microscope, evidence was produced that seed coat cracking did not cause either initial increased, or subsequent reduced germination. Observations with a transmission electron microscope revealed that disruption of the integrity of lipid bodies accompanied increased damage from repeated freezing at ?196 °C and thawing. Repeated freezing and thawing of seeds stored in liquid nitrogen should be done with care to avoid loss of the germplasm.     

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.