Intracellular ice formation in yeast cells vs. cooling rate: Predictions from modeling vs. experimental observations by differential scanning calorimetry

To survive freezing, cells must not undergo internal ice formation during cooling. One vital factor is the cooling rate. The faster cells are cooled, the more their contents supercool, and at some subzero temperature that supercooled cytoplasm will freeze. The question is at what temperature? The relation between cooling rate and cell supercooling can be computed. Two important parameters are the water permeability (Lp) and its temperature dependence. To avoid intracellular ice formation (IIF), the supercooling must be eliminated by dehydration before the cell cools to its ice nucleation temperature. With an observed nucleation temperature of −25 °C, the modeling predicts that IIF should not occur in yeast cooled at <20 °C/min and it should occur with near certainty in cells cooled at ?30 °C/min. Experiments with differential scanning calorimetry (DSC) confirmed these predictions closely. The premise with the DSC is that if there is no IIF, one should see only a single exotherm representing the freezing of the external water. If IIF occurs, one should see a second, lower temperature exotherm. A further test of whether this second exotherm is IIF is whether it disappears on repeated freezing. IIF disrupts the plasma membrane; consequently, in a subsequent freeze cycle, the cell can no longer supercool and will not exhibit a second exotherm. This proved to be the case at cooling rates >20 °C/min.     

Comparative cryopreservation study of trochophore larvae from two species of bivalves: Pacific oyster (Crassostrea gigas) and Blue mussel (Mytilus galloprovincialis)

Oysters and mussels are among the most farmed species in aquaculture industry around the world. The aim of this study was to test the toxicity of cryoprotective agents to trochophore larvae from two different species of bivalves and develop an improved cryopreservation protocol to ensure greater efficiency in the development of cryopreserved trochophores (14 h old oyster larvae and 20 h old mussel larvae) to normal D-larvae for future developments of hatchery spat production. The cryopreservation protocol producing the best results for oyster trochophores (60.0 ± 6.7% normal D-larvae) was obtained by holding at 0 °C for 5 min then cooling at 1 °C min⁻¹ to −10 °C and holding for 5 min before cooling at 0.5 °C to −35 °C, holding 5 min and then plunging into liquid nitrogen (LN), using 10% ethylene glycol. For mussel experiments, no significant differences were found when cooling at 0.5 °C min⁻¹ or at 1 °C min⁻¹ for CPA combinations with 10% ethylene glycol and at 0.5 °C min⁻¹. Using these combinations, around half of trochophores were able to develop to normal D-larvae post-thawing (48.9 ± 7.6% normal D-larvae).     

Cooling rate optimization for zebrafish sperm cryopreservation using a cryomicroscope coupled with SYBR14/PI dual staining

The Zebrafish has gained increased popularity as an aquatic model species in various research fields, and its widespread use has led to numerous mutant strains and transgenic lines. This creates the need to store these important genetic materials as frozen gametes. Sperm cryopreservation in zebrafish has been shown to yield very low post-thaw survival and many protocols suffer from great variability and poor reproducibility. The present study was intended to develop a freezing protocol that can be reliably used to cryopreserve zebrafish sperm with high post-thaw survival. In particular, our study focused on cooling protocol optimization with the aid of cryomicroscopy. Specifically, sperm suspended in 8% DMSO or 4% MeOH were first incubated with live/dead fluorescent dyes (SYBR14/PI) and then cooled at various rates from 4 °C to different intermediate stopping temperatures such as −10, −20, −30 and −80 °C before rewarming to 35 °C at the rate of 100 °C/min. %PI-positive (dead) cells were monitored throughout the cooling process and this screening yielded an optimal rate of 25 °C/min for this initial phase of freezing. We then tested the optimal cooling rate for the second phase of freezing from various intermediate stopping temperatures to −80 °C before plunging into liquid nitrogen. Our finding yielded an optimal intermediate stopping temperature of −30 °C and an optimal rate of 5 °C/min for this second phase of freezing. When we further applied this two-step cooling protocol to the conventional controlled-rate freezer, the average post-thaw motility measured by CASA was 46.8 ± 6.40% across 11 males, indicating high post-thaw survival and consistent results among different individuals. Our study indicates that cryomiscroscopy is a powerful tool to devise the optimal cooling conditions for species with sperm that are very sensitive to cryodamage.     

Kinetics and activation energy of recrystallization of intracellular ice in mouse oocytes subjected to interrupted rapid cooling

Intracellular ice formation (IIF) is almost invariably lethal. In most cases, it results from the too rapid cooling of cells to below −40 °C, but in some cases it is manifested, not during cooling, but during warming when cell water that vitrified during cooling first devitrifies and then recrystallizes during warming. Recently, Mazur et al. [P. Mazur, I.L. Pinn, F.W. Kleinhans, Intracellular ice formation in mouse oocytes subjected to interrupted rapid cooling, Cryobiology 55 (2007) 158–166] dealt with one such case in mouse oocytes. It involved rapidly cooling the oocytes to −25 °C, holding them 10 min, rapidly cooling them to −70 °C, and warming them slowly until thawed. No IIF occurred during cooling but intracellular freezing, as evidenced by blackening of the cells, became detectable at −56 °C during warming and was complete by −46 °C. The present study differs in that the oocytes were warmed rapidly from −70 °C to temperatures between −65 and −50 °C and held for 3–60 min. This permitted us to determine the rate of blackening as function of temperature. That in turn allowed us to calculate the activation energy (E_a) for the blackening process; namely, 27.5 kcal/mol. This translates to about a quadrupling of the blackening rate for every 5 °C rise in temperature. These data then allowed us to compute the degree of blackening as a function of temperature for oocytes warmed at rates ranging from 10 to 10,000 °C/min. A 10-fold increase in warming rate increased the temperature at which a given degree of blackening occurred by 8 °C. These findings have significant implications both for cryobiology and cryo-electron microscopy.     

Calorimetric measurement of water transport and intracellular ice formation during freezing in cell suspensions

The current study presents a new and novel analysis of heat release signatures measured by a differential scanning calorimeter (DSC) associated with water transport (WT), intracellular ice formation (IIF) and extracellular ice formation (EIF). Correlative cryomicroscopy experiments were also performed to validate the DSC data. The DSC and cryomicroscopy experiments were performed on human dermal fibroblast cells (HDFs) at various cytocrit values (0–0.8) at various cooling rates (0.5–250 °C/min). A comparison of the cryomicroscopy experiments with the DSC analysis show reasonable agreement in the water transport (cellular dehydration) and IIF characteristics between both the techniques with the caveat that IIF measured by DSC lagged that measured by cryomicroscopy. This was ascribed to differences in the techniques (i.e. cell vs. bulk measurement) and the possibility that not all IIF is associated with visual darkening. High and low rates of 0.5 °C/min and 250 °C/min were chosen as HDFs did not exhibit significant IIF or WT at each of these extremes respectively. Analysis of post-thaw viability data suggested that 10 °C/min was the presumptive optimal cooling rate for HDFs and was independent of the cytocrit value. The ratio of measured heat values associated with IIF (q_IIF) to the total heat released from both IIF and water transport or from the total cell water content in the sample (q_CW) was also found to increase as the cooling rate was increased from 10 to 250 °C/min and was independent of the sample cytocrit value. Taken together, these observations suggest that the proposed analysis is capable of deconvolving water transport and IIF data from the measured DSC latent heat thermograms in cell suspensions during freezing.     

Membrane hydration correlates to cellular biophysics during freezing in mammalian cells

Cell survival during freezing applications in biomedicine is highly correlated to the temperature history and its dependent cellular biophysical events of dehydration and intracellular ice formation (IIF). Although cell membranes are known to play a significant role in cell injury, a clear correlation between the membrane state and the surrounding intracellular and extracellular water is still lacking. We previously showed that lipid hydration in LNCaP tumor cells is related to cellular dehydration. The goal of this study is to build upon this work by correlating both the phase state of the membrane and the surrounding water to cellular biophysical events in three different mammalian cell types: human prostate tumor cells (LNCaP), human dermal fibroblasts (HDF), and porcine smooth muscle cells (SMC) using Fourier Transform Infrared spectroscopy (FTIR). Variable cooling rates were achieved by controlling the degree of supercooling prior to ice nucleation (− 3 °C and − 10 °C) while the sample was cooled at a set rate of 2 °C/min. Membranes displayed a highly cooperative phase transition under dehydrating conditions (i.e. NT = − 3 °C), which was not observed under IIF conditions (NT = − 10 °C). Spectral analysis showed a consistently greater amount of ice formation during dehydrating vs. IIF conditions in all cell types. This is hypothesized to be due to the extreme loss of membrane hydration in dehydrating cells that is manifested as excess water available for phase change. Interestingly, changes in residual membrane conformational disorder correlate strongly with cellular volumetric decreases as assessed by cryomicroscopy. A strong correlation was also found between the activation energies for freezing induced lyotropic membrane phase change determined using FTIR and the water transport measured by cryomicroscopy. Reduced lipid hydration under dehydration freezing conditions is suggested as one of the likely causes of what has been termed as “solution effects” injury in cryobiology.     

Determination of the water permeability (Lp) of mouse oocytes at −25 °C and its activation energy at subzero temperatures

Typically, subzero permeability measurements are experimentally difficult and infrequently reported. Here we report an approach we have applied to mouse oocytes. Interrupted cooling involves rapidly cooling oocytes (50 °C/min) to an intermediate temperature above the intracellular nucleation zone, holding them for up to 40 min while they dehydrate, and then rapidly cooling them to −70 °C or below. If the intermediate holding temperature and holding time are well chosen, high post thaw survival of the oocytes is possible because the freezable water is removed during the hold. The length of time required for the exit of the freezable water allows the water permeability at that temperature to be determined. These experiments used 1.5 M ethylene glycol in PBS and included a transient hold of 2 min for equilibration at −10 °C, just below the extracellar ice formation temperature. We obtain an Lp = 1.8 × 10⁻³ μm min⁻¹ atm⁻¹ at −25 °C based on a hold time of 30 min yielding 80% survival and the premise that most of the freezable water is removed during the 30 min hold. If we assume that the water permeability is a continuous function of temperature and that its Ea changes at 0 °C, we obtain a subzero Ea of 21 kcal/mol; higher than the suprazero value of 14 kcal/mol. A number of assumptions are required for these water loss calculations and the resulting value of Lp can vary by up to a factor of 2, depending on the choices make.     

Successful cryopreservation of Pacific oyster (Crassostrea gigas) oocytes

Protocols for cryopreservation of sperm and oocytes would provide the ultimate control over parental crosses in selective breeding programmes. Sperm freezing is routine for many species, but oocyte freezing remains problematic, with virtually zero success in aquatic species to date. This paper describes the development of a successful protocol for cryopreserving high concentrations of Pacific oyster (Crassostrea gigas) oocytes. Ethylene glycol (10%) and dimethyl sulfoxide (15%) were found to be the most effective cryoprotectants resulting in post-thaw fertilization rates of 51.0+/-8.0 and 45.1+/-8.3%, respectively. Propylene glycol was less effective and methanol resulted in zero fertilization post-thaw. The use of Milli-Q water rather than seawater as a base medium significantly improved fertilization (20.4+/-3.0 and 8.7+/-2.2%, respectively) as did the inclusion of a 5 min isothermal hold at -10 or -12 degrees C (35.9+/-5.0 and 31.9+/-4.6%, respectively). The optimal cooling rate post-hold was 0.3 degrees C min(-1), with virtually zero post-thaw fertilization with cooling rates of 3 and 6 degrees C min(-1). Using an optimized protocol, post-thaw fertilization rates for oocytes from eight individual females ranged from 0.8 to 74.5% and D-larval yields from 0.1 to 30.1%. For three individuals, larvae were reared through to spat. Development of D-larvae to eyed larvae and spat was similar for larvae produced from unfrozen (24.8+/-4.1% developed to eyed larvae and 16.5+/-3.2% to spat) and cryopreserved (28.4+/-0.6 and 18.7+/-0.5%, respectively) oocytes. The ability to cryopreserve large quantities of oyster oocytes represents a major advance in cryobiology and selective breeding.     

Comparison between the temperatures of intracellular ice formation in fresh mouse oocytes and embryos and those previously subjected to a vitrification procedure

When cells that have been subjected to supposedly innocuous freezing or vitrification procedures are used as the source material for subsequent experiments, it is important that they possess or exhibit the same relevant properties as fresh cells. In this study, we compared the temperatures of intracellular ice formation (IIF) in previously vitrified mouse oocytes/embryos with those in fresh intact ones. In the case of MII oocytes, 2-cell embryos, 4-6-cell embryos, and morulae, there are no significant differences (p > 0.05); namely, -33.3 °C (fresh) vs. -35.4 °C (vitrified) with MII oocytes, -40.6 °C (fresh) vs. -38.7 °C (vitrified) with 2-cell embryos, -38.0 °C (fresh) vs. -39.4 °C (vitrified) with 4-6-cell embryos, -24.5 °C (fresh) vs. -24.2 °C (vitrified) with morulae. But, in 8-cell embryos, there is a significant difference (p < 0.05) between fresh (−37.9 °C) and vitrified (−32.9 °C). If we include this significant difference, the overall IIF temperature of fresh cells is 0.74 °C lower than that of previously vitrified cells. If we exclude it, the IIF temperature for fresh cells is 0.32 °C higher than that for previously vitrified cells. Our conclusion then is that there is no difference between the IIF temperatures of fresh and previously vitrified cells.     

Effect of cooling rate and cryoprotectant concentration on intracellular ice formation of small abalone (Haliotis diversicolor) eggs

The intracellular ice formation (IIF) behavior of Haliotis diversicolor (small abalone) eggs is investigated in this study, in relation to controlling the cooling rate and the concentration of dimethyl sulfoxide (DMSO). The IIF phenomena are monitored under a self-developed thermoelectric cooling (TEC) cryomicroscope system which can achieve accurate temperature control without the use of liquid nitrogen. The accuracy of the isothermal and ramp control is within ±0.5 °C. The IIF results indicate that the IIF of small abalone eggs is well suppressed at cooling rates of 1.5, 3, 7 and 12 °C/min with 2.0, 2.5, 3.0 and 4.0 M DMSO in sea water. As 2.0 M DMSO in sea water is the minimum concentration that has sufficient IIF suppression, it is selected as the suspension solution for the cryopreservation of small abalone eggs in order to consider the solution’s toxicity effect. Moreover, IIF characteristics of the cumulative probability of IIF temperature distribution are shown to be well fitted by the Weibull probabilistic distribution. According to our IIF results and the Weibull distribution parameters, we conclude that cooling at 1.5 °C/min from 20 to −50 °C with 2.0 M DMSO in sea water is more feasible than other combinations of cooling rates and DMSO concentrations in our experiments. Applying this protocol and observing the subsequent osmotic activity, 48.8% of small abalone eggs are osmotically active after thawing. In addition, the higher the cooling rate, the less chance of osmotically active eggs. A separate fertility test experiment, with a cryopreservation protocol of 1.5 °C/min cooling rate and 2.0 M DMSO in sea water, achieves a hatching rate of 23.7%. This study is the first to characterize the IIF behavior of small abalone eggs in regard to the cooling rate and the DMSO concentration. The Weibull probabilistic model fitting in this study is an approach that can be applied by other researchers for effective cryopreservation variability estimation and analysis.     

Cryopreservation of Greenshell™ mussel (Perna canaliculus) trochophore larvae

The Greenshell™ mussel (Perna canaliculus) is the main shellfish species farmed in New Zealand. The aim of this study was to evaluate the effects of cryoprotectant concentration, loading and unloading strategy as well as freezing and thawing method in order to develop a protocol for cryopreservation of trochophore larvae (16–20 h old). Toxicity tests showed that levels of 10–15% ethylene glycol (EG) were not toxic to larvae and could be loaded and unloaded in a single step. Through cryopreservation experiments, we designed a cryopreservation protocol that enabled 40–60% of trochophores to develop to D-larvae when normalized to controls. The protocol involved: holding at 0 °C for 5 min, then cooling at 1 °C min⁻¹ to −10 °C, holding for a further 5 min, then cooling at 0.5 °C min⁻¹ to −35 °C followed by a 5 min hold and then plunging into liquid nitrogen. A final larval rearing experiment of 18 days was conducted to assess the ability of these frozen larvae to develop further. Results showed that only 2.8% of the frozen trochophores were able to develop to competent pediveligers.     

Intracellular ice formation and growth in MCF-7 cancer cells

Direct cell injury in cryosurgery is highly related to intracellular ice formation (IIF) during tissue freezing and thawing. Mechanistic understanding of IIF in tumor cells is critical to the development of tumor cryo-ablation protocol. In aid of a high speed CMOS camera system, the events of IIF in MCF-7 cells have been studied using cryomicroscopy. Images of ‘darkening’ type IIF and recrystallization are compared between cells frozen with and without ice seeding. It is found that ice seeding has significant impact on the occurrence and growth of intracellular ice. Without ice seeding, IIF is observed to occur over a very small range of temperature (∼1 °C). The crystal dendrites are indistinguishable, which is independent of the cooling rate. Ice crystal grows much faster and covers the whole intracellular space in comparison to that with ice seeding, which ice stops growing near the cellular nucleus. Recrystallization is observed at the temperature from −13 °C to −9 °C during thawing. On the contrary, IIF occurs from −7 °C to −20 °C with ice seeding at a high subzero temperature (i.e., −2.5 °C). The morphology of intracellular ice frozen is greatly affected by the cooling rate, and no ‘darkening’ type ice formed inside cells during thawing. In addition, the intracellular ice formation is directional, which starts from the plasma membrane and grows toward the cellular nucleus with or without ice seeding. These results can be used to explain some findings of tumor cryosurgery in vivo, especially the causes of insufficient killing of tumor cells in the peripheral area near vessels.     

Cryopreservation of red snapper (Lutjanus argentimaculatus) sperm: Effect of cryoprotectants and cooling rates on sperm motility, sperm viability, and fertilization capacity

Sperm cryopreservation of red snapper (Lutjanus argentimaculatus) is essentially unexplored, although many species of the Lutjanidae family are considered to be high-value commercial species. The objective of this study was to develop a species-specific cryopreservation protocol for red snapper (L. argentimaculatus) sperm by optimizing cryoprotectants and cooling rates in the cryopreservation procedure. Ten cryoprotectants at four concentrations and two freezing protocols were examined in two separate experiments. In the first experiment, toxicity studies of dimethyl sulfoxide (DMSO), glycerol, propylene glycol (PG), ethylene glycol (EG), formamide, methanol, ethanol, sucrose, trehalose, and dimethylacetamide (DMA) on sperm motility were performed. Semen diluted 1:1 in Ringer solution were exposed to cryoprotectants at four final concentrations of 5%, 10%, 15%, or 20% for periods of 10, 20, 30, 40, 50, 60, 90, and 120 min at room temperature (25 °C). The cryoprotectants and concentrations that showed the least toxic effect on sperm motility were selected for cryopreservation trials. In the second experiment, selected cryoprotectants were then assessed for freezing capacity of sperm as follows: DMSO 5% and 10%, PG 5% and 10%, EG 5% and 10%, ethanol 5%, and methanol 5%. Semen was diluted 1:1 in Ringer solution and equilibrated with selected cryoprotectants for 10 min at room temperature. Sperm were frozen in a controlled-rate programmable freezer at four cooling rates of 3, 5, 10, and 12 °C/min from an initial temperature of 25 °C to final temperatures of −40 or −80 °C before plunging into liquid nitrogen. Sperm equilibrated in 10% DMSO and cooled at a rate of 10 °C/min to a final temperature of −80 °C had the highest motility (91.1 ± 2.2%) and viability (92.7 ± 2.3%) after thawing. The fertilization rate of frozen-thawed sperm (72.4 ± 2.4%) was not different (P > 0.05) from that of fresh sperm (75.5 ± 2.4%). This study apparently represents the first reported attempt for cryopreservation of L. argentimaculatus sperm.     

Evaluation of cryoprotectant and cooling rate for sperm cryopreservation in the euryhaline fish medaka Oryzias latipes

Medaka Oryzias latipes is a well-recognized biomedical fish model because of advantageous features such as small body size, transparency of embryos, and established techniques for gene knockout and modification. The goal of this study was to evaluate two critical factors, cryoprotectant and cooling rate, for sperm cryopreservation in 0.25-ml French straws. The objectives were to: (1) evaluate the acute toxicity of methanol, 2-methoxyethanol (ME), dimethyl sulfoxide (Me₂SO), N,N-dimethylacetamide (DMA), N,N-dimethyl formamide (DMF), and glycerol with concentrations of 5%, 10%, and 15% for 60 min of incubation at 4 °C; (2) evaluate cooling rates from 5 to 25 °C/min for freezing and their interaction with cryoprotectants, and (3) test fertility of thawed sperm cryopreserved with selected cryoprotectants and associated cooling rates. Evaluation of cryoprotectant toxicity showed that methanol and ME (5% and 10%) did not change the sperm motility after 30 min; Me₂SO, DMA, and DMF (10% and 15%) and glycerol (5%, 10% and 15%) significantly decreased the motility of sperm within 1 min after mixing. Based on these results, methanol and ME were selected as cryoprotectants (10%) to evaluate with different cooling rates (from 5 to 25 °C/min) and were compared to Me₂SO and DMF (10%) (based on their use as cryoprotectants in previous publications). Post-thaw motility was affected by cryoprotectant, cooling rate, and their interaction (P ? 0.000). The highest post-thaw motility (50 ± 10%) was observed at a cooling rate of 10 °C/min with methanol as cryoprotectant. Comparable post-thaw motility (37 ± 12%) was obtained at a cooling rate of 15 °C/min with ME as cryoprotectant. With DMF, post-thaw motility at all cooling rates was ?10% which was significantly lower than that of methanol and ME. With Me₂SO, post-thaw motilities were less than 1% at all cooling rates, and significantly lower compared to the other three cryoprotectants (P ? 0.000). When sperm from individual males were cryopreserved with 10% methanol at a cooling rate of 10 °C/min and 10% ME with a rate of 15 °C/min, no difference was found in post-thaw motility. Fertility testing of thawed sperm cryopreserved with 10% methanol at a rate of 10 °C/min showed average hatching of 70 ± 30% which was comparable to that of fresh sperm (86 ± 15%). Overall, this study established a baseline for high-throughput sperm cryopreservation of medaka provides an outline for protocol standardization and use of automated processing equipment in the future.     

Survival of mouse oocytes after being cooled in a vitrification solution to -196°C at 95° to 70,000°C/min and warmed at 610° to 118,000°C/min: A new paradigm for cryopreservation by vitrification

There is great interest in achieving reproducibly high survivals of mammalian oocytes (especially human) after cryopreservation, but the results to date have not matched the interest. A prime cause of cell death is the formation of more than trace amounts of intracellular ice, and one strategy to avoid it is vitrification. In vitrification procedures, cells are loaded with high concentrations of glass-inducing solutes and cooled to −196 °C at rates high enough to presumably induce the glassy state. In the last decade, several devices have been developed to achieve very high cooling rates. Nearly all in the field have assumed that the cooling rate is the critical factor. The purpose of our study was to test that assumption by examining the consequences of cooling mouse oocytes in a vitrification solution at four rates ranging from 95 to 69,250 °C/min to −196 °C and for each cooling rate, subjecting them to five warming rates back above 0 °C at rates ranging from 610 to 118,000 °C/min. In samples warmed at the highest rate (118,000 °C/min), survivals were 70% to 85% regardless of the prior cooling rate. In samples warmed at the lowest rate (610 °C/min), survivals were low regardless of the prior cooling rate, but decreased from 25% to 0% as the cooling rate was increased from 95 to 69,000 °C/min. Intermediate cooling and warming rates gave intermediate survivals. The especially high sensitivity of survival to warming rate suggests that either the crystallization of intracellular glass during warming or the growth by recrystallization of small intracellular ice crystals formed during cooling are responsible for the lethality of slow warming.     

Directional freezing as an alternative method for cryopreserving rhesus macaque (Macaca mulatta) sperm

The objective was to develop a freezing protocol using a directional freezing (DF) technique for cryopreservation of rhesus macaque sperm and achieve a survival rate comparable to that achieved with a conventional freezing (CF) technique. Rhesus macaque sperm frozen with a DF technique, with cooling rates of 12 or 16 °C/min, had higher post-thaw motility (P < 0.05) than those cooled at 7 °C/min (59.3, 61.1, and 50.3%, respectively). Furthermore, sperm cryopreserved with 5% glycerol and a DF technique had similar frozen-thawed sperm motility to those cryopreserved by a CF technique (63.7 vs. 53.9%, P > 0.05). The function of sperm cryopreserved at the optimized cooling rate using a DF technique was evaluated by in vitro fertilization of oocytes collected from gonadotropin-stimulated rhesus macaques. Of the 38 mature oocytes collected, 78.9% were fertilized and 71.1, 47.4, and 42.1% of the oocytes developed to the 2-cell, morulae, and blastocyst stages, respectively. In conclusion, rhesus macaque sperm was effectively cryopreserved using a DF technique, providing a new and effective method for genetic preservation in this important species.     

Cryopreservation of sperm in farmed Australian greenlip abalone Haliotis laevigata

This study investigated factors important to the development of the liquid nitrogen (LN) vapor sperm cryopreservation technique in farmed greenlip abalone Haliotis laevigata, including (1) cryoprotectant agent (CPA) toxicity; (2) cooling temperature (height above LN surface); (3) thawing temperature; (4) sperm to egg ratio; and (5) sugar supplementation, using sperm motility, fertilization rate or integrity/potential of sperm components and organelles as quality assessment indicators. Results suggested that among the single CPAs evaluated 6% dimethyl sulfoxide (Me2SO) would be the most suitable for sperm cryopreservation in this species. The highest post-thaw sperm motility was achieved with the sperm that had been exposed to LN vapor for 10 min at 5.2 cm above the LN surface, thawed and recovered in 60 and 18 °C seawater bathes, respectively after at least 2 h storage in LN. The highest fertilization rates were achieved at a sperm to egg ratio of 10,000:1 or 15,000:1. Addition of 1% glucose or 2% sucrose produced significantly higher post-thaw sperm motility than 6% Me2SO alone. Among the three cryoprotectant solutions further trialled, 6% Me2SO + 1% glucose produced the highest fertilization rate of 83.6 ± 3.7%. Evaluation of sperm has shown that the addition of glucose could significantly improve the sperm plasma membrane integrity and mitochondrial membrane potential. These results demonstrated a positive role of glucose in the improvement of sperm cryopreservation in farmed greenlip abalone.     

Rationally optimized cryopreservation of multiple mouse embryonic stem cell lines: II—Mathematical prediction and experimental validation of optimal cryopreservation protocols

In Part I, we documented differences in cryopreservation success measured by membrane integrity in four mouse embryonic stem cell (mESC) lines from different genetic backgrounds (BALB/c, CBA, FVB, and 129R1), and we demonstrated a potential biophysical basis for these differences through a comparative study characterizing the membrane permeability characteristics and osmotic tolerance limits of each cell line. Here we use these values to predict optimal cryoprotectants, cooling rates, warming rates, and plunge temperatures. We subsequently verified these predictions experimentally for their effects on post-thaw recovery. From this study, we determined that a cryopreservation protocol utilizing 1 M propylene glycol, a cooling rate of 1 °C/minute, and plunging into liquid nitrogen at −41 °C, combined with subsequent warming in a 22 °C water bath with agitation, significantly improved post-thaw recovery for three of the four mESC lines, and did not diminish post-thaw recovery for our single exception. It is proposed that this protocol can be successfully applied to most mESC lines beyond those included within this study once the effect of propylene glycol on mESC gene expression, growth characteristics, and germ-line transmission has been determined. Mouse ESC lines with poor survival using current standard cryopreservation protocols or our proposed protocol can be optimized on a case-by-case basis using the method we have outlined over two papers. For our single exception, the CBA cell line, a cooling rate of 5 °C/minute in the presence of 1.0 M dimethyl sulfoxide or 1.0 M propylene glycol, combined with plunge temperature of −80 °C was optimal.     

Phase Transition Temperature and Chilling Sensitivity of Bovine Oocytes

A limiting factor for achieving cryopreservation of oocytes is direct chilling injury (DCI), which occurs during cooling. DCI, or cold shock, is defined as an irreversible damage expressed shortly after exposure to low, but not freezing, temperatures. The primary target of DCI is thought to be the plasma membrane. Recently, an association between DCI in sperm and the thermotropic phase transition of their membrane lipids was demonstrated. In the present study, we examined the phase transition of the membrane lipids of immature andin vitro-matured bovine oocytes during cooling, using Fourier transform infrared spectroscopy (FTIR). The phase transition of the membrane lipids of oocytes at the germinal vesicle (GV) stage occurred between 13 and 20°C, while a very broad phase transition, which centered around 10°C, was observed for mature oocytes (MII) stage. Thermotropic phase transitions were demonstrated to be related to the temperature at which DCI affected the integrity of the oocyte membranes. When immature oocytes were cooled to 13°C, fewer oocytes (40%) retained their membrane integrity than after exposure to 4°C (51%) or holding them at 38°C (78%), (as determined by the Fluorescein Diacetate-FDA test). This finding might suggest that holding immature oocytes at the phase transition temperature is more damaging to their membranes than exposure to lower temperatures. By contrast, no significant differences in membrane integrity were observed whenin vitro-matured oocytes were cooled to the same temperatures. Subsequently, GV oocytes were cooled to 4°C, and 26% underwent maturation and 19% underwent fertilizationin vitro. In vitro-matured oocytes that were cooled to 4°C displayed a slightly decreased rate of fertilization; the overall fertilization was 60% with 24% polyspermy, rather than the 76% fertilization rate with 12% polyspermy obtained with those not subjected to cooling. The high rate of polyspermy indicates that a site(s) other than the plasma membrane is affected during cooling of bovine oocytes. Nucleated bovine GV oocytes were electrofused within vitro-matured and enucleated oocytes, and then cooled to 4°C. Evaluation of the membrane integrity of the fused oocytes showed that these oocytes are chilling resistant, which strongly suggests that alteration of the membrane composition of an oocyte can change the cell's susceptibility to low temperatures. This finding led to an improvement in the survival of oocytes after cryopreservation.     

The effects of cooling rates and type of freezing extenders on cryosurvival of rat sperm

Cryopreservation of rat sperm is very challenging due to its sensitivity to various stress factors. The objective of this study was to determine the optimal cooling rate and extender for epididymal sperm of outbred Sprague Dawley (SD) and inbred Fischer 344 (F344) rat strains. The epididymal sperm from 10 to 12 weeks old sexually mature SD and F344 strains were suspended in five different freezing extenders, namely HEPES buffered Tyrode’s lactate (TL-HEPES), modified Kreb’s Ringer bicarbonate (mKRB), 3% dehydrated skim milk (SM), Salamon’s Tris-citrate (TRIS), and tes/tris (TES). All extenders contained 20% egg yolk, 0.75% Equex Paste and 0.1 M raffinose or 0.1 M sucrose. The sperm samples in each extender were cooled to 4 °C and held for 45 min for equilibration before freezing. The equilibrated sperm samples in each extender were placed onto a shallow quartz dish inserted into Linkam Cryostage (BCS 196). The samples were then cooled to a final temperature of −150 °C by using various cooling rates (10, 40, 70, and 100 °C/min). For thawing, the quartz dish containing the sperm samples were rapidly removed from the Linkam cryo-stage and placed on a 37 °C slide warmer and held for 1 min before motility analysis. Sperm membrane and acrosomal integrity and mitochondrial membrane potential (MMP) were assessed by SYBR-14/Propidium iodide, Alexa Fluor-488-PNA conjugate and JC-1, respectively. The total motility, acrosomal integrity, membrane integrity and MMP values were compared among cooling rates and extenders. Both cooling rate and type of extender had significant effect on cryosurvival (P < 0.05). Sperm motility increased as cooling rate was increased for both strains (P < 0.05). Highest cryosurvival was achieved when 100 °C/min cooling rate was used in combination with TES extender containing 20% egg yolk, 0.75% Equex paste and either 0.1 M sucrose or raffinose (P < 0.05). This study showed that TES extender containing 0.1 M raffinose or sucrose with 70 °C/min and 100 °C/min cooling rate improved post-thaw motility of rat sperm.