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In this study, mature female mice of the ICR strain were induced to superovultate, mated, and collected at either zygote or early morula stages. Embryos suspended in 1 M ethylene glycol in PBS containing 10 mg/L Snomax for 15 min, then transferred in sample holder to Linkam cryostage, cooled to and seeded at 7 °C, and then observed and photographed while being cooled to −70 °C at 0.5–20 °C/min. Intracellular ice formation (IIF) was observed as abrupt ‘‘flashing’’. Two types of flashing or IIF were observed in this study. Extracellular freezing occurred at a mean of −7.7 °C. In morulae, about 25% turned dark within ±1 °C of extracellular ice formation (EIF). These we refer to as “high temperature’’ flashers. In zygotes, there were no high temperature flashers. All the zygotes flashed at temperatures well below the temperature for EIF. Presumably high temperature flashers were a consequence of membrane damage prior to EIF or damage from EIF. We shall not discuss them further. In the majority of cases, IIF occurred well below −7.7 °C; these we call ‘‘low temperature’’ flashers. None flashed with cooling rate (CR) of 0.5 °C/min in either zygotes or morulae. Nearly all flashed with CR of 4 °C/min or higher, but the distribution of temperatures is much broader with morulae than with zygotes. Also, the mean flashing temperature is much higher with morulae (−20.9 °C) than with zygotes (−40.3 °C). We computed the kinetics of water loss with respect to CR and temperature in both mouse zygotes and in morulae based on published estimates of Lp and it is Ea. The resulting dehydration curves combined with knowledge of the embryo nucleation temperature permits an estimate of the likelihood of IIF as a function of CR and subzero temperature. The agreement between these computed probabilities and the observed values are good.

We report additional details of the thermal modeling, selection of the laser, and construction of the Cryo Jig used for our ultra-rapid warming studies of mouse oocytes (B Jin, FW Kleinhans, Peter Mazur, Cryobiology 68 (2014) 419–430). A Nd:YAG laser operating at 1064 nm was selected to deliver short 1 msec pulses of sufficient power to produce a warming rate of 1 × 107 °C/min from –190°C to 0°C. A special Cryo Jig was designed and built to rapidly remove the sample from LN2 and expose it to the laser pulse. India ink carbon black particles were required to increase the laser energy absorption of the sample. The thermal model reported here is more general than that previously reported. The modeling reveals that the maximum warming rate achievable via external warming across the cell membrane is proportional to (1/R2) where R is the cell radius.

Vitrification is the most sought after route to the cryopreservation of animal embryos and oocytes and other cells of medical, genetic, and agricultural importance. Current thinking is that successful vitrification requires that cells be suspended in and permeated by high concentrations of protective solutes and that they be cooled at very high rates to below − 100°C. We report here that neither of these beliefs holds for mouse oocytes. Rather, we find that if mouse oocytes are suspended in media that produce considerable osmotic dehydration before vitrification and are subsequently warmed at ultra high rates (10,000,000°C/min) achieved by a laser pulse, nearly 100% will survive even when cooled rather slowly and when the concentration of solutes in the medium is only 1/3rd of standard.

To date, traditional cryopreservation techniques have not been amenable to zebrafish (Danio rerio) embryos, due in part to their large yolky eggs, which have a low surface to volume ratio and limited permeability to water and cryoprotectants. However, recent vitrification and ultra-rapid warming studies in mice have demonstrated successful preservation by dehydrating 85% or more of their total water content. We hypothesized that this approach may help overcome the barriers to embryo cryopreservation among D. rerio. The purpose of this study was to determine the osmotic tolerance limit of D. rerio embryos under conditions relevant to cryopreservation. We found that embryos undergoing gastrulation (30%–70% epiboly) were particularly sensitive to osmotic dehydration/rehydration. By contrast, a subset of embryos dehydrated during or after segmentation (20–22 somite, prim 5) survived 3 h in a 2 M sucrose solution but exhibit developmental delay, edema and trunk necrosis 2–4 days post-treatment.

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 degrees 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 degrees 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 1.5M ethylene glycol in PBS and included a transient hold of 2 min for equilibration at -10 degrees C, just below the extracellar ice formation temperature. We obtain an Lp=1.8 x 10(-3)microm min(-1)atm(-1) at -25 degrees 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 degrees 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.

Intracellular ice formation (IIF) is almost invariably lethal. In most cases, it results from the too rapid cooling of cells to below -40 degrees 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 degrees C, holding them 10 min, rapidly cooling them to -70 degrees 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 degrees C during warming and was complete by -46 degrees C. The present study differs in that the oocytes were warmed rapidly from -70 degrees C to temperatures between -65 and -50 degrees 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 degrees 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 degrees C/min. A 10-fold increase in warming rate increased the temperature at which a given degree of blackening occurred by 8 degrees C. These findings have significant implications both for cryobiology and cryo-electron microscopy.

We have previously reported that intracellular ice formation (IIF) in mouse oocytes suspended in glycerol/PBS solutions or ethylene glycol (EG)/PBS solutions and rapidly cooled to −50 °C or below occurs at temperatures where a critical fraction of the external water remains unfrozen [P. Mazur, S. Seki, I.L. Pinn, F.W. Kleinhans, K. Edashige, Extra- and intracellular ice formation in mouse oocytes, Cryobiology 51 (2005) 29–53; P. Mazur, I.L. Pinn, F.W. Kleinhans, The temperature of intracellular ice formation in mouse oocytes vs. the unfrozen fraction at that temperature, Cryobiology 54 (2007) 223–233]. For mouse oocytes in PBS or glycerol/PBS that fraction is 0.06; for oocytes in EG that fraction was calculated to be 0.13, more than double. The fractions unfrozen are computed from ternary phase diagrams. In the previous publication, we used the EG data of Woods et al. [E.J. Woods, M.A.J. Zieger, D.Y. Gao, J.K. Critser, Equations for obtaining melting points for the ternary system ethylene glycol/sodium chloride/Water and their application to cryopreservation., Cryobiology 38 (1999) 403–407]. Since then, we have determined that ternary phase diagrams for EG/NaCl/water synthesized by summing binary phase data for EG/water NaCl/water gives substantially different curves, which seem more realistic [F.W. Kleinhans, P. Mazur, Comparison of actual vs. synthesized ternary phase diagrams for solutes of cryobiological interest, Cryobiology 54 (2007) 212–222]. Unfrozen fractions at the temperatures of IIF computed from these synthesized phase diagrams are about half of those calculated from the Woods et al. data, and are in close agreement with the computations for glycerol; i.e., IIF occurs when about 92–94% of the external water is frozen. A parallel paper was published by Guenther et al. [J.F. Guenther, S. Seki, F.W. Kleinhans, K. Edashige, D.M. Roberts, P. Mazur, Extra-and intra-cellular ice formation in Stage I and II Xenopus laevis oocytes, Cryobiology 52 (2006) 401–416] on IIF in oocytes of the frog Xenopus . It too examined whether the temperatures of IIF were related to the unfrozen fractions at those temperatures. It also used the Woods et al. ternary phase data to calculate the unfrozen fractions for EG solutions. As reported here, once again the values of these unfrozen fractions are substantially different from those calculated using synthesized phase diagrams. With the latter, the unfrozen fractions at IIF become very similar for EG and glycerol.

We are currently investigating factors that influence intracellular ice formation (IIF) in mouse oocytes and oocytes of the frog Xenopus. A major reason for choosing these two species is that while their eggs normally do not possess aquaporin channels in their plasma membranes, these channels can be made to express. We wish to see whether IIF is affected by the presence of these channels. The present Xenopus study deals with control eggs not expressing aquaporins. The main factor studied has been the effect of a cryoprotective agent [ethylene glycol (EG) or glycerol] and its concentration. The general procedure was to (a) cool the oocytes on a cryostage to slightly below the temperatures at which extracellular ice formation occurs, (b) warm them to just below the melting point, and (c) then re-cool them to -50 degrees C at 10 degrees C/min. In the majority of cases, IIF occurs well into step (c), but a sizeable minority undergo IIF in steps (a) or (b). The former group we refer to as low-temperature flashers; the latter as high-temperature flashers. IIF is manifested as abrupt blackening of the egg, which we refer to as "flashing." Observations on the Linkam cryostage are restricted to Stage I and II oocytes, which have diameters of 200 300 microm. In the absence of a cryoprotective agent, that is in frog Ringers, the mean flash temperature for the low-temperature freezers is -11.4 degrees C, although a sizeable percentage flash at temperatures much closer to that of the EIF (-3.9 degrees C). When EG is present, the flash temperature for the low-temperatures freezers drops significantly to approximately -20 degrees C for EG concentrations ranging from 0.5 to 1.5 M. The presence of 1.5 M glycerol also substantially reduces the IIF temperature of the low-temperature freezers; namely, to -29 degrees C, but 0.5 and 1 M glycerol exert little or no effect. The IIF temperatures observed using the Linkam cryostage agree well with those estimated by calorimetry [F.W. Kleinhans, J.F. Guenther, D.M. Roberts, P. Mazur, Analysis of intracellular ice nucleation in Xenopus oocytes by differential scanning calorimetry, Cryobiology 52 (2006) 128-138]. The IIF temperatures in Xenopus are substantially higher than those observed in mouse oocytes [P. Mazur, S. Seki, I.L. Pinn, F.W. Kleinhans, K. Edashige, Extra- and intracellular ice formation in mouse oocytes, Cryobiology 51 (2005) 29-53]. Perhaps that is a reflection of their much larger size.

Intracellular ice formation (IIF) plays a central role in cell damage during cryopreservation. We are investigating the factors which trigger IIF in Xenopus oocytes, with and without aquaporin water channels. Here, we report differential scanning calorimeter studies of Xenopus control oocytes which do not express aquaporins. Stage I to VI oocytes (which increase progressively in size) were investigated with emphasis on stage I and II because they are translucent and can also be studied under the cryomicroscope. Measurements were made in 1, 1.5, and 2M ethylene glycol (EG) in frog Ringers plus SnoMax. A multistep freezing protocol was used in which the samples were cooled until extracellular ice formation (EIF) occurred, partially remelted, slowly recooled through the EIF temperature, and then rapidly (10 degrees C/min) cooled. EIF in the 1, 1.5, and 2M EG occurred at -6.4, -7.8, and -8.9 degrees C, respectively. Freezing exotherms of individual stage I-VI oocytes were readily visible. A general trend was observed in which the IIF temperature of the early stage oocytes (I-III) was well below T(EIF) while the later stages (IV-VI) froze at temperatures much closer to T(EIF). Thus, in 1.5M EG, T(IIF) was -21.1, -25, and -26.6 degrees C in stages I-III, but was -17 and -8.5 degrees C for stage IV and V-VI. Concurrently, the percentage of oocytes in which IIF was observed fell dramatically from a high of 40 to 72% in early stages (I-III) to a low of only 7% in stage V-VI because, particularly in the later stages, IIF was hidden in the EIF exotherm. We conclude that early stage oocytes are a good model system in which to investigate modulators of IIF, but that late stage oocytes are damaged during EIF and infrequently supercool.

MII mouse oocytes in 1 and 1.5M ethylene glycol(EG)/phosphate buffered saline have been subjected to rapid freezing at 50 degrees C/min to -70 degrees C. When this rapid freezing is preceded by a variable hold time of 0-3 min after the initial extracellular ice formation (EIF), the duration of the hold time has a substantial effect on the temperature at which the oocytes subsequently undergo intracellular ice formation (IIF). For example, in 1M EG, the IIF temperatures are -23.7 and -39.2 degrees C with 0 and 2 min hold times; in 1.5M EG, the corresponding IIF temperatures are -29.1 and -40.8 degrees C.

Although fish embryos have been used in a number of slow-freezing cryopreservation experiments, they have never been successfully cryopreserved. In part this is because little is known about whether ice forms within the embryo during the slow-freezing dehydration process. Therefore, we examined the temperature of intraembryonic ice formation ( T IIF ) and the temperature of extraembryonic ice formation ( T EIF ), using a cryomicroscope. We used both unmodified zebrafish embryos and those with water channels (aquaporin-3 or AQP3) inserted into their membranes to increase permeability to water and cryoprotectants, examined at 100% epiboly to the 6-somite stage. In these experiments we examined: (1) the spontaneous freezing of (external) solutions; (2) the spontaneous freezing of solutions containing embryos; (3) the effect of preloading the embryos with cryoprotectants on T IIF ; (4) whether preloading the embryos with cryoprotectant helps in survival after nucleating events in the solution; and (5) the damaging effects of extracellular nucleation events versus solution toxicity on the embryos. The solutes alone (embryo medium—EM, sucrose culture medium, 1 M propylene glycol in EM, and 1 M propylene glycol in a sucrose culture medium) froze at −14.9 ± 1.1, −17.0 ± 0.3, −17.8 ± 1.0, and −17.7 ± 1.4, respectively. There was no difference amongst these means ( P > 0.05), thus adding cryoprotectant did not significantly lower the nucleation point. Adding embryos (preloaded with cryoprotectant or not) did not change the basic freezing characteristics of these solutes. In all these experiments, ( T EIF ) equaled ( T IIF ), and there was no difference in the freezing point of the solutions with or without the embryos ( P > 0.05). Additionally, there was no difference in the freezing characteristics of embryos with and without aquaporins ( P > 0.05). The formation of intraembryonic ice was lethal to the zebrafish embryos in all cases. But this lethal outcome was not related to solution injury effects, because 88–98% of embryos survived when exposed to a higher solute concentration with no ice present. Taken together, these data suggest that slow-freezing is not a suitable option for zebrafish embryos. The mechanism of this high temperature nucleation event in zebrafish embryos is still unknown.

Several factors have contributed to problems in mouse sperm cryopreservation, and we and others have found ways to ameliorate them. These include high sensitivity to several types of mechanical stresses and to oxygen-derived free radicals, low tolerance to osmotic cell volume changes, and rather rigorous requirements for cooling and warming rates. Another important factor is the cryoprotective agent. Mouse sperm are unusual in that our best results have been obtained in media containing the nonpermeating sugar raffinose (18% w/v) and lacking glycerol. This paper deals with questions about the basis of the protective action of sugars, and whether raffinose is unusual or unique in its ability to confer protection. More specifically, we investigated whether protection was more related to the total osmolality of the freezing solution, to the mass concentration of sugar, or to the molarity of the sugar, and we looked to see whether there are effects attributable to specific sugars. To investigate these questions, mouse sperm were frozen at the optimal rate of 25 degrees C/min in solutions prepared with different proportions of three sugars-raffinose, sucrose, and glucose-dissolved in 1/4x PBS. In the first experimental series, the total osmolality and the total sugar molarity were varied from 400 to 700 mOsm and from 300 to 530 mM, respectively, while holding the mass concentration of sugar constant at 18% (w/v). In the second experimental series, the mass concentration of sugars was varied from 10 to 18% while the sugar molarity and solution osmolality remained constant at 300 mM and 420 mOsm, respectively. The results suggest that protection against freezing and thawing depends more on the mass concentration of the sugar than on its molar concentration, a conclusion that has mechanistic implications.

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 (p0.05); namely, -33.3 degrees C (fresh) vs. -35.4 degrees C (vitrified) with MII oocytes, -40.6 degrees C (fresh) vs. -38.7 degrees C (vitrified) with 2-cell embryos, -38.0 degrees C (fresh) vs. -39.4 degrees C (vitrified) with 4-6-cell embryos, -24.5 degrees C (fresh) vs. -24.2 degrees C (vitrified) with morulae. But, in 8-cell embryos, there is a significant difference (p0.05) between fresh (-37.9 degrees C) and vitrified (-32.9 degrees C). If we include this significant difference, the overall IIF temperature of fresh cells is 0.74 degrees C lower than that of previously vitrified cells. If we exclude it, the IIF temperature for fresh cells is 0.32 degrees 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.

To cryopreserve cells, it is essential to avoid intracellular ice formation during cooling and warming. One way to do so is to subject them to procedures that convert cell water into a non-crystalline glass. Current belief is that to achieve this vitrification, cells must be suspended in very high concentrations of glass-inducing solutes (i.e., ≥ 6 molal) and cooled at very high rates (i.e., >> 1,000°C/min). We report here that both these beliefs are incorrect with respect to the vitrification of 8-cell mouse embryos. In this study, precompaction 8-cell embryos were vitrified in several dilutions of EAFS10/10 using various cooling rates and warming rates. Survival was based on morphology, osmotic functionality, and on the ability to develop to expanded blastocysts. With a warming rate of 117,500°C/min, the percentages of embryos vitrified in 1×, 0.75×, and 0.5× EAFS that developed to blastocysts were 93%, 92%, and 86%, respectively. And the percentages of morphological survivors that developed to expanded blastocysts were 100%, 92%, and 97%, respectively. Even when the solute concentration of the EAFS was reduced to 33% of normal, we obtained 40% functional survival of these 8-cell embryos.

As survival of mouse oocytes subjected to vitrification depends far more on the warming rate than on the cooling rate, we wished to determine whether the lack of correlation between survival and cooling rate was mirrored by a lack of correlation between cooling rate and vitrification of the medium (EAFS), and between survival and the vitrification of the medium. The morphological and functional survival of the oocytes showed little or no relation to whether or not the EAFS medium vitrified or froze. We studied if the droplet size and the elapsed time (between placing the droplet on the Cryotop and the start of cooling) affects the result through modification of the cooling rate and solute concentration. Dehydration was rapid; consequently, the time between the placing the droplets into a Cryotop and cooling must be held to a minimum. The size of the EAFS droplet that is being cooled does not seem to affect vitrification. Finally, the degree to which samples of EAFS vitrify is firmly dependent on both its solute concentration and the cooling rate.

Although Anopheles gambiae embryos that have developed for 15 h at 17 degrees C are slightly permeable to water, they are impermeable to ethylene glycol, the cryoprotectant used in the cryopreservation of Drosophila melanogaster embryos. Success in cryopreservation requires that they be made permeable to protective solutes. Permeabilization of D. melanogaster was achieved by 1) dechorionation with 50% Clorox (household bleach) followed by 2) a water flush; 3) immersion in isopropyl alcohol to remove most extraembryonic water; 4) 2 min air drying to remove most isopropyl alcohol; 5) 90-s exposure to n-heptane containing 0.3% 1-butanol; and 6) 15-s exposure to pure n-heptane. The permeability of A. gambiae embryos was assessed by determining the times required for the initial dehydration of embryos in 1 M ethylene glycol in 260 mOsm Drosophila culture medium (permeability to water) and the times required for their return to normal volume (permeability to ethylene glycol). Based on these criteria, the above D. melanogaster procedure effectively permeabilizes 15 h/17 degrees C to 19 h/17 degrees C A. gambiae embryos. Nearly all collapsed in

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°C/min 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°C/min 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°C/min 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.

Because of waxes in the vitelline membrane, the Drosophila egg is effectively impermeable to liquid water and to aqueous solutes, and consequently it cannot be cryopreserved unless it can be permeabilized. The more successful of the few published permeabilization procedures involve the removal of the chorion mechanically or by hypochlorite solution, the removal of all surrounding water by air drying or alcohol, the exposure of eggs to pure alkanes like octane or hexane for some 30 s, the removal of the alkane and the transfer of the eggs to aqueous culture medium without their desiccation, and lastly incubation of the permeabilized embryos under mineral oil. In following these procedures we opted for a somewhat different approach to applying hypochlorite, water, alcohol, and alkane; namely, eggs were placed between two Nucleopore filters, and the fluids drawn sequentially through the filters by vacuum. Extensive initial attempts were mystifying and discouraging in that although permeabilization was good, survivals were poor, and modifications that increased the latter reduced the former. The explanation turned out to be that permeabilization and survival depended critically on the amount of carry-over alcohol that contaminated the alkane. To determine the effects of alcohol concentration in the alkane, it was essential first to effectively eliminate carry-over contamination and then re-add precise amounts of alcohol (isopropanol) to the alkane (n-hexane, heptane, or octane). When the alcohol concentration is less than or equal to 0.2%, permeabilization is poor; when it is greater than or equal to 0.5%, permeabilization is good but survival (hatching) is poor. There are strong interactions between alcohol concentration and exposure time to alkane/alcohol mixtures with respect to the fraction of embryos that become permeabilized and the percentage that survive. There are also significant but less critical effects from the type of alcohol and alkane. The best results for 12-h embryos (greater than or equal to 90% permeabilization and 70-80% hatching) were achieved with eggs exposed to 0.3 or 0.4% 1-butanol in n-heptane for 90 s. High survivals of permeabilized 12-h embryos did not require incubation under mineral oil. Permeabilized embryos are permeable to water, ethylene glycol, glycerol, and the stain rhodamine B (which was used to assess permeabilization). They are effectively impermeable to sucrose. Embryo age is important. Between 14 and 16 h the above permeabilization procedures become dramatically less effective.(ABSTRACT TRUNCATED AT 400 WORDS)

The formation of more than trace amounts of ice in cells is lethal. The two contrasting routes to avoiding it are slow equilibrium freezing and vitrification. The cryopreservation of mammalian oocytes by either method continues to be difficult, but there seems a slowly emerging consensus that vitrification procedures are somewhat better for mouse and human oocytes. The approach in these latter procedures is to load cells with high concentrations of glass-inducing solutes and cool them at rates high enough to induce the glassy state. Several devices have been developed to achieve very high cooling rates. Our study has been concerned with the relative influences of warming rate and cooling rate on the survival of mouse oocytes subjected to a vitrification procedure. Oocytes suspended in an ethylene glycol-acetamide-Ficoll-sucrose solution were cooled to -196 degrees C at rates ranging from 37 to 1827 degrees C/min between 20 and -120 degrees C, and for each cooling rate, warmed at rates ranging from 139 to 2950 degrees C/min between -70 and -35 degrees C. The results are unambiguous. If the samples were warmed at the highest rate, survivals were80% over cooling rates of 187-1827 degrees C/min. If the samples were warmed at the lowest rate, survivals were near 0% regardless of the cooling rate. We interpret the lethality of slow warming to be a consequence of it allowing time for the growth of small intracellular ice crystals by recrystallization.

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 degrees C, the modeling predicts that IIF should not occur in yeast cooled at20 degrees C/min and it should occur with near certainty in cells cooled ator=30 degrees 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 rates20 degrees C/min.

We have previously reported that intracellular ice formation (IIF) in mouse oocytes suspended in glycerol/PBS solutions or ethylene glycol (EG)/PBS solutions and rapidly cooled to -50 degrees C or below occurs at temperatures where a critical fraction of the external water remains unfrozen [P. Mazur, S. Seki, I.L. Pinn, F.W. Kleinhans, K. Edashige, Extra- and intracellular ice formation in mouse oocytes, Cryobiology 51 (2005) 29-53; P. Mazur, I.L. Pinn, F.W. Kleinhans, The temperature of intracellular ice formation in mouse oocytes vs. the unfrozen fraction at that temperature, Cryobiology 54 (2007) 223-233]. For mouse oocytes in PBS or glycerol/PBS that fraction is 0.06; for oocytes in EG that fraction was calculated to be 0.13, more than double. The fractions unfrozen are computed from ternary phase diagrams. In the previous publication, we used the EG data of Woods et al. [E.J. Woods, M.A.J. Zieger, D.Y. Gao, J.K. Critser, Equations for obtaining melting points for the ternary system ethylene glycol/sodium chloride/Water and their application to cryopreservation., Cryobiology 38 (1999) 403-407]. Since then, we have determined that ternary phase diagrams for EG/NaCl/water synthesized by summing binary phase data for EG/water NaCl/water gives substantially different curves, which seem more realistic [F.W. Kleinhans, P. Mazur, Comparison of actual vs. synthesized ternary phase diagrams for solutes of cryobiological interest, Cryobiology 54 (2007) 212-222]. Unfrozen fractions at the temperatures of IIF computed from these synthesized phase diagrams are about half of those calculated from the Woods et al. data, and are in close agreement with the computations for glycerol; i.e., IIF occurs when about 92-94% of the external water is frozen. A parallel paper was published by Guenther et al. [J.F. Guenther, S. Seki, F.W. Kleinhans, K. Edashige, D.M. Roberts, P. Mazur, Extra-and intra-cellular ice formation in Stage I and II Xenopus laevis oocytes, Cryobiology 52 (2006) 401-416] on IIF in oocytes of the frog Xenopus. It too examined whether the temperatures of IIF were related to the unfrozen fractions at those temperatures. It also used the Woods et al. ternary phase data to calculate the unfrozen fractions for EG solutions. As reported here, once again the values of these unfrozen fractions are substantially different from those calculated using synthesized phase diagrams. With the latter, the unfrozen fractions at IIF become very similar for EG and glycerol.

Phase diagrams are of great utility in cryobiology, especially, those consisting of a cryoprotective agent (CPA) dissolved in a physiological salt solution. These ternary phase diagrams consist of plots of the freezing points of increasing concentrations of solutions of cryoprotective agents (CPA) plus NaCl. Because they are time-consuming to generate, ternary diagrams are only available for a small number of CPAs. We wanted to determine whether accurate ternary phase diagrams could be synthesized by adding together the freezing point depressions of binary solutions of CPA/water and NaCl/water which match the corresponding solute molality concentrations in the ternary solution. We begin with a low concentration of a solution of CPA + salt of given R (CPA/salt) weight ratio. Ice formation in that solution is mimicked by withdrawing water from it which increases the concentrations of both the CPA and the NaCl. We compute the individual solute concentrations, determine their freezing points from published binary phase diagrams, and sum the freezing points. These yield the synthesized ternary phase diagram for a solution of given R. They were compared with published experimental ternary phase diagrams for glycerol, dimethyl sulfoxide (DMSO), sucrose, and ethylene glycol (EG) plus NaCl in water. For the first three, the synthesized and experimental phase diagrams agreed closely, with some divergence occurring as wt% concentrations exceeded 30% for DMSO and 55% for glycerol, and sucrose. However, in the case of EG there were substantial differences over nearly the entire range of concentrations which we attribute to systematic errors in the experimental EG data. New experimental EG work will be required to resolve this issue.

Improved methods for preservation of honey bee, Apis mellifera L., germplasm would be very welcome to beekeeping industry queen breeders. The introduction of two parasites and the emergence of an antibiotic resistant disease have increased demands for resistant stock. Techniques for artificial insemination of queens are available, and semen has been cryopreserved with limited success. However, cryopreservation of embryos for rearing queens would mesh well with current practices and also provide drones (haploid males). Eggs at five ages between twenty-four hours and sixty-two hours were exposed to 0, −6.6, and/or −15 °C for various times, and successful hatch measured. Honey bee embryos show chill sensitivity as do other insect embryos, and the rate of chill injury increases dramatically with decrease in holding temperature. The 48 h embryos in both groups showed the greatest tolerance to chilling, although 44 h embryos were only slightly less so.

The occurrence of intracellular ice formation (IIF) during freezing, or the lack there of, is the single most important factor determining whether or not cells survive cryopreservation. One important determinant of IIF is the temperature at which a supercooled cell nucleates. To avoid intracellular ice formation, the cell must be cooled slowly enough so that osmotic dehydration eliminates nearly all cell supercooling before reaching that temperature. This report is concerned with factors that determine the nucleation temperature in mouse oocytes. Chief among these is the concentration of cryoprotective additive (here, glycerol or ethylene glycol). The temperature for IIF decreases from -14 degrees C in buffered isotonic saline (PBS) to -41 degrees C in 1M glycerol/PBS and 1.5M ethylene glycol/PBS. The latter rapidly permeates the oocyte; the former does not. The initial extracellular freezing at -3.9 to -7.8 degrees C, depending on the CPA concentration, deforms the cell. In PBS that deformation often leads to IIF; in CPA it does not. The oocytes are surrounded by a zona pellucida. That structure appears to impede the growth of external ice through it, but not to block it. In most cases, IIF is characterized by an abrupt blackening or flashing during cooling. But in some cases, especially with dezonated oocytes, a pale brown veil abruptly forms during cooling followed by slower blackening during warming. Above -30 degrees C, flashing occurs in a fraction of a second. Below -30 degrees C, it commonly occurs much more slowly. We have observed instances where flashing is accompanied by the abrupt ejection of cytoplasm. During freezing, cells lie in unfrozen channels between the growing external ice. From phase diagram data, we have computed the fraction of water and solution that remains unfrozen at the observed flash temperatures and the concentrations of salt and CPA in those channels. The results are somewhat ambiguous as to which of these characteristics best correlates with IIF.

Our laboratory has determined that the survival of mouse oocytes subjected to vitrification procedures depends far more on the warming rate than on the cooling rate, not only in a full-strength vitrification solution but also in dilutions of that solution. But what is not known is the state of the water in the cells and in the outside medium as a function of cooling rate and as a function of the concentration of solutes in the vitrification solution; i.e. whether vitrified or frozen. The main purpose of this study was to investigate the latter; namely, whether the lack of correlation between survival and cooling rate was mirrored by a lack of correlation between cooling rate and vitrification of the medium (EAFS), and between survival and the vitrification of the medium. The relative concentration of the EAFS vitrification solution (1×, 0.75×, 0.6×, 0.5×, 0.4×, 0.33×, and 0.25×) and the cooling rate (522, 1827, or 69,250 °C/min) had a major effect on whether the media vitrified or froze, but when those results were compared with previous morphological survival and membrane integrity data from Seki and Mazur [PLoS One (2012)], the survival showed little or no correlation with the vitrification or freezing of the medium. The procedure consisted of placing a 0.1–0.2 μ l droplet of the desired concentration of EAFS medium on a Cryotop and abruptly immersing the naked Cryotop in liquid nitrogen (LN2). This produces a cooling rate of 69,250 °C/min. For slower cooling, the Cryotops were insulated in various ways. Two questions arose. One was how much dehydration of the droplets of EAFS occurs between the time a droplet is placed on the Cryotop and the time cooling is initiated? The second question was what is the effect of droplet size on whether it freezes or vitrifies? The answer to the second question was that the percentage of droplets vitrifying did not differ significantly in drops ranging in volume from 0.1 to 1.0 μ l. The answer to the first question is that the droplets dehydrate rather rapidly in room-temperature air; namely, they lose 20–30% of their mass in the first 2 minutes. This dehydration increases the solute concentration of the EAFS which artifactually increases the likelihood of vitrification. Consequently, if it is not minimized, it can cause confounding. Source of funding: E. Paredes was sponsored by a fellowship from the Spanish government (FPU Research Stays). The research was supported by NIH grant 8R01 OD 011201, P. Mazur, P.I. Conflict of interest: None declared. pmazur@utk.edu

We have recently reported that the survival of mouse spermatozoa is decreased when they are warmed at a suboptimal rate after being frozen at an optimal rate. We proposed that this drop in survival is caused by physical damage derived from the recrystallization of extracellular ice during slow warming. The first purpose of the present study was to determine the temperatures over which the decline in survival occurs during slow warming and the kinetics of the decline at fixed subzero temperatures. The second purpose was to examine the effects of antifreeze proteins (AFP) on the survival of slowly warmed mouse spermatozoa, the rationale being that AFP have the property of inhibiting ice recrystallization. With respect to the first point, a substantial loss in motility occurred when slow warming was continued to higher than -50 degrees C and the survival of the sperm decreased with an increase in the temperature at which slow warming was terminated. In contrast, the motility of sperm that were warmed rapidly to these temperatures remained high initially but dropped with increased holding time. At -30 degrees C, most of the drop occurred in 5 min. These results are consistent with the hypothesis that damage develops as a consequence of the recrystallization of the external ice. AFP ought to inhibit such recrystallization, but we found that the addition of AFP-I, AFP-III, and an antifreeze glycoprotein at concentrations of 1-100 microg/ml did not protect the frozen-thawed cells; rather it led to a decrease in survival that was proportional to the concentration. There was no decrease in survival from exposure to the AFP in the absence of freezing. AFP are known to produce changes in the structure and habit of ice crystals, and some have reported deleterious consequences associated with those structural changes. We suggest that such changes may be the basis of the adverse effects of AFP on the survival of the sperm, especially since mouse sperm are exquisitely sensitive to a variety of mechanical stresses.

Vitrification is the most sought after route to the cryopreservation of animal oocytes and embryos and other cells of medical, genetic, and agricultural importance. Standard vitrification has been based on two firmly held premises. One is that the vitrification solution in which the cells are suspended must have a very high concentration of a mixture of non-electrolytic solutes, some of which can permeate the cell and some of which cannot. We have used EAFS10/10, a solution developed by Pedro et al., where E, A, F, and S refer to ethylene glycol (EG), acetamide, Ficoll, and sucrose. The total molality is 7.38 molal, of which 6.5 molal is permeating (EG and acetamide), and the remainder are non-permeating. The second premise is that avoiding ice formation in cells and obtaining high survivals demands the highest of cooling rates. Consequently, a series of devices have been developed over the past decades that achieve cooling rates of ⩾10,000 °C/min by using very small volumes of cell suspensions. One example is the Cryotop. So, current thinking is that successful vitrification requires that cells be suspended in high concentrations of protective solutes and that they be cooled at very high rates to below -100°C. We report here that neither of these beliefs hold for mouse oocytes. Rather, we find that if mouse oocytes are suspended in media that produce considerable osmotic dehydration before vitrification and are subsequently warmed at ultrarapid rates (10,000,000 °C/min) achieved by a laser pulse, nearly 100% will survive even when cooled rather slowly and when the concentration of solutes in the medium is only one third of the standard. Full paper published on-line 3/22/14 at http://dx.doi.org/10.1016/j.cryobiol.2014.02.005 . Research supported by NIH grant R01OD011201 .

Jin, Kleinhans, and Mazur have reported in this meeting that up to 100% of oocytes and 2-cell embryos survive vitrification in a 3-fold dilution of EAFS medium if warmed at 10,000,000 °C/min by an IR laser pulse. The usual cooling rate was 69,000 °C/min, but 95% also survived when cooled at 10,000 °C/min. Thus, survival is far more dependent on the rate of warming than on the rate of cooling, the reason being that very high warming rates prevent or impede the recrystallization of intracellular ice. In all our experiments, the oocytes or embryos were held for 2.0 min in 1/3 × EAFS before vitrification That time was chosen because Pedro (1997) found it to produce the best survival after vitrification. EAFS media are strongly hyperosmotic so that oocytes/embryos placed in them, undergo an osmotic shrink-swell pattern with time. There is an initial rapid shrinkage to a minimum volume due to water loss, followed by a much slower re-swelling due to the slow influx of the permeating solutes. Based on Pedro’s data, that minimum occurs near 2 min, and osmotic theory argues that at that point, the cells have lost 85% of their original water and very little permeating solute has entered them. The strong inference, then, is that the high subsequent survivals are a consequence of the 85% osmotic dehydration and are not due to the intracellular presence of permeating solutes. Supporting that conclusion is Jin and Mazur’s report that 77% of oocytes and 95% of 2-cell and 8-cell embryos survive vitrification when suspended in media containing only the non-permeating solutes sucrose provided they are warmed at 1 × 107 °C/min by a pulse from our IR laser. None survived when warmed 100× more slowly in the absence of the laser. These findings are at odds with orthodox beliefs about the causes of injury from vitrification. They could possibly open the way to the successful vitrification of important cell types that to date have been partially or fully refractory to this approach.

In a companion presentation at this meeting and in a full paper published 3/22/14 on line in Cryobiology , we have reported that when Metaphase II mouse oocytes or 2-cell mouse embryos are suspended in EAFS vitrification media that contain only one-third the concentration of solutes as standard, over 90% survive when assessed by osmotic/morphological criteria and up to 85% survive when assessed by the functional ability to undergo IVF and/or develop in vitro to expanded blastocysts, provided that they are warmed at 10,000,000 °C/min by a powerful pulse from an IR laser. The base 0.3× EAFS medium that we used contains ethylene glycol (EG) [0.70 molal], acetamide [0.71 molal], 70,000 MW Ficoll [0.0013 molal], sucrose [0.16 molal], and PBS salts [0.15 molal]. Variants included 0, 0.16, 0.25, 0.5, and 1.0 molal sucrose, and 0.0062 molal Ficoll. Sucrose, Ficoll, and the salts cannot penetrate the cells; the EG and acetamide can readily do so, but the 2-min exposure time is too short for this to occur. As a consequence, the cells are strongly dehydrated osmotically when vitrification is initiated. This raises the question of whether permeating solutes are needed to achieve high survivals after vitrification. In the companion presentation, we argued that the most important factor aside from ultra-rapid warming was the total molality (and hence, osmolality) of the medium. In the present presentation we address the matter of survival of not only oocytes, but of 2-cell and 8-cell embryos, and morluae and blastocysts vitrified in the total absence of permeating solutes. The morphological and functional survivals of vitrified 2-cell and 8-cell embryos were 92%; the survivals of vitrified oocytes and blastocysts were above 70%. All four of these survivals were obtained with laser-induced warming at 10,000,000 °C/min. If warming was slowed 100-fold to the previous standard maximum of 100,000 °C/min, morphological and functional survivals were zero. Since no one has heretofore warmed oocytes and embryos at the ultra-high rates used here, we believe that this is the first time it has been shown that these cells can be cryopreserved in a solution that is free of any permeating compounds.

This presentation will attempt to relate some fundamental aspects of cryobiology to the problems of cryosurgery. Much understanding of the fundamentals has come from studies on cryopreservation, but cryopreservation and cryosurgery differ in important ways aside from their ultimate aims. The following are the topics to be discussed: A. Extrinsic factors or variables: • Extracellular medium • Cooling rate • Can be constant in cryopreservation • * Can not be constant in cryosurgical freezing • Temperature • Minimum temperature • Usually uniform in cryopreservation • Can not be uniform in cryosurgical freezing • Time at subzero temperature • Warming rate • Number of repeat freezing thawing cycles B. Physical events: • Extra-cellular and inter-cellular ice formation • Osmotic dehydration • Permeation of permeating solutes • Cell supercoiling • Is cell position perturbed by ice front? • Intracellular ice formation • Vitrification • Recrystallization of ice • Post-thaw osmotic excursions C. Intrinsic cellular factors • Cell size • Permeability to water • Permeability to solutes. • Possession of aquaporins or not • Chill sensitivity • Sensitivity to osmotic excursions D. Major differences between: Cryopreservation freezing Cryosurgical freezing Cooling is from outside-In Cooling is from inside-out All parts of the system reach equilibrium with temperature of −196 °C throughout System reaches steady state with temps.varying from −160 °C near probe to −0.5 °C at ice-ball surface Cryoprotective agents are always added Cryoprotective agents are never added. But.. Survivals of ⩾50% are usually acceptable Survivals of

Cryopreserved mouse sperm are beginning to be used to meet the demand of a reliable cost-effective method for maintaining the rapidly expanding numbers of lines of mutant mice. However, successful and reproducible cryopreservation has proven to be a difficult problem. Furthermore, the underlying factors responsible for success or failure are mostly obscure. Several contributors to these difficulties have been identified. Our laboratory has found that mouse sperm are extremely susceptible to the mechanical stresses associated with pipetting, mixing, and centrifugation, and others have found that they are severely limited in their tolerance to osmotic volume changes. We have hypothesized two other contributors to the difficulties. One is that the concentrations of glycerol used in published protocols are substantially lower than those found to be optimal for most mammalian cells. The other hypothesis relates to the fact that mouse sperm membranes are especially susceptible to damage from oxygen-derived free radicals. That damage may reduce their ability to survive freezing. If so, survival ought to increase if the concentration of oxygen is kept low throughout the procedure. To achieve low levels, we have incorporated an Escherichia coli membrane fraction, Oxyrase, into all media. A previous report showed a protective effect. That is confirmed here under a broader range of conditions. The conditions studied have been the individual and interactive effects of the concentrations of glycerol, raffinose, and phosphate-buffered saline (PBS) on motility after freezing at 21 degrees C/min to -70 degrees C. Cryoprotection increased with increasing raffinose concentration, provided that the concentration of PBS was appropriately reduced to hold the total osmolality of nonpermeating solutes to within tolerated limits. Surprisingly, the best results were achieved in the total absence of glycerol. The highest motilities to date (68 +/- 8%) after freezing to -70 degrees C have been achieved using media containing Oxyrase, 0 M glycerol, and 18% raffinose in 14x strength modified PBS. We also determined the motility loss after freezing to intermediate temperatures, i.e., -10 and -30 degrees C. The major motility loss occurred by -10 degrees C, especially in the absence of Oxyrase. These results suggest that a major problem in the freezing of mouse sperm is the physical stress resulting from extracellular ice crystal formation. Oxyrase appears to lessen that damage substantially.

The cryobiological preservation of mouse spermatozoa has presented difficulties in the form of poor motilities or irreproducibility. We have hypothesized several underlying problems. One is that published studies have used concentrations of the cryoprotectant glycerol that are substantially lower (0.3 M) than the approximately 1 M concentrations that are optimal for most mammalian cells. Another may arise from the known high susceptibility of mouse sperm to free radical damage. We have been able to obtain high motilities in 0.8 M glycerol provided that the exposure time is held to approximately 5 min to minimize toxicity and provided that the glycerol is added and removed stepwise to minimize osmotic shock. Since free radical damage in mouse sperm is proportional to the oxygen concentrations, we have determined the consequences of reducing the oxygen to3% of atmospheric by maintaining the sperm in contact with an Escherichia coli membrane preparation, Oxyrase, from the moment of collection throughout the assessment of motility. Prior studies have shown that the procedure significantly reduces damage from centrifugation and osmotic shock. In the experiments reported here we obtained approximately 50% motility relative to untreated controls when suspensions containing 3.8% Oxyrase were exposed approximately 5 min to a solution of 0.8 M glycerol and 0.17 M (10%) raffinose in a supplemented PBS and then frozen at approximately 25 degrees C/min to -75 degrees C. In the absence of Oxyrase, the normalized motility dropped to 31%. The protection by Oxyrase was in part a consequence of minimizing centrifugation damage, but in part it reflected a reduction in freeze-thaw damage. Preliminary experiments indicate that the number of motile sperm after cryopreservation in Oxyrase is higher when the sperm are collected without swim-up than when they are collected by swim-up. This is in part due to the fact that more cells are collected in the absence of swim-up and in part due to a greater protective effect of Oxyrase on those cells. The minimum temperature in these initial experiments was limited to -75 degrees C to avoid the potential contribution of other injurious factors between -75 and -196 degrees C.

The cryobiological preservation of mouse spermatozoa has presented difficulties in the form of poor motilities or irreproducibility. We have identified several likely underlying problems. One is that published studies have used concentrations of the cryoprotectant glycerol that are substantially lower (0.3 M) than the approximately 1 M concentrations that are optimal for most cells. Another may arise from the known high susceptibility of mouse sperm to free radical damage. We have identified two contributors to damage from higher concentrations of glycerol, namely, chemical toxicity proportional to concentration and exposure time and osmotic damage arising from too rapid an addition and removal of the glycerol. When toxicity is minimized by restricting the exposure time to 1 or 5 min and osmotic shock is minimized by adding and removing the glycerol stepwise, relatively high percentages of the sperm survive contact with 0.8 M glycerol. Free-radical damage in mouse sperm is known to be proportional to the oxygen concentration. We have determined the consequences of reducing the oxygen to

Our laboratory has been investigating the effect of warming rate on survival of mouse oocytes after very rapid cooling. Cooling and warming rates are affected by sample thermal mass, surface area, thermal impedance, and delta T. To maximize these rates we use 0.1 ul samples placed on Cryotops and plunged directly into LN2 for cooling and into room temperature buffer for warming. The maximum warming rate obtained is ∼ 100,000 C/min and it yields the highest survivals. However, extrapolation of the experimental data suggests faster warming would yield higher survivals. Here we report on the use of an IR laser pulse to achieve warming rates 3.5 to 100 times faster. Our objective was to choose a laser wavelength which would warm the cryo buffer while leaving the cells ‘untouched’, thus leading to indirect warming of the cells. Optical tweezer studies show that a wide variety of cell types can tolerate very high intensities of 1064 nm IR energy emitted by Nd:YAG lasers and so this was the type chosen. Calculations indicate the laser intensity for cryo melting to be significantly less than that used for optical tweezers. Most cryo buffers, including the EAFS we use are relatively transparent in the IR. The buffer IR absorption is easily increased by adding a little India ink. India ink consists primarily of carbon particles from 0.1 to 1 um in diameter, and at the concentration we use, 0.25% (V/V), is harmless to the oocytes. The concentration is carefully chosen to allow most of the IR energy to pass completely through the sample. This yields relatively even sample heating from front to back. We estimate ink-free samples absorb ∼ 9% of the incident energy, and the added ink absorbs ∼ 25% of the remaining energy, yielding a total absorption of ∼ 30%. We have developed a small cryo ‘jig’ which lifts the sample out of an LN2 bath, covers the bath, and fires a laser pulse, all in less than one sec. This assures that the sample is rapidly warmed by the laser pulse rather than by slower air warming. Thus, the samples are warmed from ∼ −180°C to ∼ 0°C by the laser pulse. We have computed the required laser energy for melting, but there are so many variables that we have found it preferable to empirically determine the required laser energy to just melt the samples for a given experiment. This is done by direct visual observation with a 15 x stereo microscope built into the laser system. We use a 40 joule welding laser (for long pulse durations!) with a beam diameter of 2 mm and a pulse length of 0.5 to 30 msec. This yields warming rates from 3.5 x 10 5 to 2 x 10 7 C/min. However, modeling indicates that rates higher than 1 x 10 7 C/min yield unacceptably large thermal gradients across the 70 um diameter oocytes and are not used. Preliminary experiments at a warming rate of 3.5 x 10 5 C/min yield survivals comparable to our traditional methods. Source of Funding: NIH Grant 8R01 OD 011201, Peter Mazur, PI. E. Paredes FPU Research Stays Fellowship from the Spanish Government, 1 November 2012-31 January 2013. Conflict of Interest: None declared. fkleinha@iupui.edu

Seki and Mazur have recently shown [PLoS One. 2012;7(4)] that about 90% of MII mouse oocytes survive vitrification in solutions of EAFS 10/10 that contain only half the usual concentration of solutes, provided that the warming rate is extremely high (117,000 °C/min). They reported that fewer than 10% survived if the concentration of EAFS were further reduced to 33% of normal. We are currently investigating whether high survivals could be obtained in 0.33× and 0.25× EAFS if the warming rates were still higher. For this purpose we have cooled MII oocytes in these media on Cryotops to −196 °C and warmed them 5 times more rapidly at ∼600,000 °C/min by subjecting them to a 13 joule pulse of 15 msec duration from a Nd:YAG laser. Kleinhans et al in a companion paper at this meeting discuss the physics attributes of the laser and the interactions of the pulse with the sample, as well as the jig he developed to permit the manipulation of the Cryotop before and during its exposure to the laser pulse. The osmotic/morphological survival of 31 samples suspended in 0.25× EAFS was 15.2 ± 22% (standard deviation). The survival of two samples in 0.33× EAFS was 30%. The total molality of these solutions is 1.27 and 1.72, respectively. These are in the range used in standard slow-freeze cryopreservation. For comparison, note that the total molality of full strength 1x EAFS is 7.37 molal, 5.8 and 4.3-fold higher. Although these preliminary results are encouraging, we have two caveats. One is that the variability of the former is high as evidenced by the large standard errors. The other is that the survival of controls subjected to the same cooling procedure but warmed at 117,000 °C/min (no laser) was close to the same (13.3% vs. 15.2%). Source of funding: NIH Grant 8R01 OD 011201, Peter Mazur, PI. Conflict of interest: None declared. pmazur@utk.edu

Intraembryonic freezing (IEF) in Anopheles mosquito embryos has been evaluated by differential scanning calorimetry with respect to embryo age, temperature, rate and duration of cooling, and absence or presence of extraembryonic ice. The initial temperatures for intraembryonic ice nucleation were -30.1 +/- 0.3, -28.4 +/- 0.4, and -29.1 +/- 0.2 degree C for embryos incubated for 15 h at 17 degrees C, 15 h at 26 degrees C, and 24 h at 26 degrees C, respectively, after oviposition. The first value is slightly but significantly lower than the latter two. These values were obtained on embryos in which the surface water was removed by brief drying; however, the values were nearly identical when external water and ice were present. Not only were the embryos of all three ages able to supercool at least transiently to -26 degrees C, but they could remain supercooled for up to 4 h at -20 degrees C after being cooled to -20 degrees C at 10 degrees C/min or (in the case of embryos incubated for 15 h at 26 degrees C) at 100 degrees C/min. The amount of freezable water in single embryos has been calculated from the differential scanning calorimetry measurements to be 3.45 +/- 0.08, and 3.53 +/- 0.06 microgram for embryos incubated for 15 h at 17 degrees C, 15 h at 26 degrees C, and 24 h at 26 degrees C, respectively. The differences are not significant. The corresponding values for the total water contents for embryos of the three ages were 4.04 +/- 0.20, 3.72 +/- 0.16, and 3.98 +/- 0.10 microgram, values that also did not differ significantly. Water thus makes up approximately 74% of the total weight of the embryo (approximately 5.3 micrograms) and about 91% of that water is freezable. Total water contents were determined gravimetrically after extensive air and vacuum drying. The kinetics of dehydration were determined during the air drying. They differed substantially among the three ages. The embryos incubated for 15 h at 17 degrees C lost water at about four times the rate of those incubated for 15 h at 26 degrees C and 10 times the rate of the embryos incubated for 24 h at 26 degrees C.

As an initial step in the development of cryopreservation methods for Anopheles gambiae embryos, we determined the permeability of intact and dechorionated A. gambiae embryos to water and ethylene glycol and compared those permeabilities with those of Drosophila melanogaster embryos. Most studies were conducted on embryos allowed to develop for 8 h at 26 degrees C or 15 h at 17 degrees C. Intact A. gambiae embryos are some 50 times more permeable to water vapor than are D. melanogaster embryos; e.g., when air dried, half of 15 h/17 degrees C A. gambiae embryos become severely dehydrated in 3. 5-4.0 min, whereas comparably aged D. melanogaster embryos require approximately 4 h or more. Comparable differences between the two species exist with respect to the loss of liquid water under an osmotic driving force. After 90 min in 0.75 M sucrose in 0.26 osmolal D-20 Drosophila medium, 25-40% of intact A. gambiae embryos underwent extensive shrinkage, whereas D. melanogaster embryos showed no shrinkage. The chorion of both species can be removed by a 2.5-min exposure to 50% household bleach (Clorox). Dechorionation increases the rate of water loss two- to sixfold during air drying in both species and during exposure to hyperosmotic sucrose in A. gambiae. Dechorionated D. melanogaster embryos, however, show no shrinkage in hyperosmotic sucrose even after 6 h. The water permeability of dechorionated A. gambiae embryos thus remains many-fold higher than that of dechorionated D. melanogaster embryos. Although moderately permeable to water, neither intact nor dechorionated A. gambiae embryos are permeable to ethylene glycol, as evidenced by the failure of dehydrated embryos to reexpand in 120-180 min in that solution.

Authors of the above manuscript regret to inform about a mistake in the originally published paper. The corrected information is: Kitazato Cryotops are 0.7 mm wide: Cryotop sticks were used in this work and their size has previously been reported as 0.1 mm thick 0.4 mmwide 20 mm long [1]; the size we used in our analyses. However, we recently discovered that the Cryotops we have are, in fact, 0.7 mm wide. This increases the size, mass, and corresponding thermal mass of the Cryotops which affects the calculations in Table 1, the scale bar in Fig. 2, and a few sentences throughout. Specifically

Although sperm were successfully cryopreserved in 1950, another 22 years elapsed before the successful preservation of a mammalian embryo. In 1963, I had carried out physical–chemical analyses indicating that if cells were cooled too rapidly, they would undergo lethal intracellular ice formation (IIF), and defined what is meant by “too rapidly” for a particular cell. To avoid IIF, the cell had to dehydrate during cooling, and the required rate of cooling depended chiefly on the permeability of a particular cell to water and on the temperature coefficient of that permeability. Between 1968 and 1972, Stanley Leibo and I turned our attention to the survival of yeast, mouse marrow stem cells, and Chinese Hamster tissue culture cells as functions of cooling rate and temperature. We found that plots of survival vs. cooling rate formed an inverted “U”, with high survivals at a medium rate, and lower survivals after very slow and very rapid cooling. We proposed a “Two-Factor” hypothesis which stated that the right hand arm of the inverted U was a result of IIF at high cooling rates. Interestingly, the optimum cooling rate for maximum survival varied over a wide range for the different cells. This was chiefly because the cells differed widely in water permeability and the temperature coefficient of that permeability. In fact if the known values of these parameters were used to generate the theoretical curves, the predicted relation between IIF and cooling rate agreed quite closely with the observed drop in survival vs. cooling rate. In 1971 David Whittingham reported the successful cryopreservation of mouse 8-cell embryos cooled at 50 °C/min in PVP. Leibo and I were excited but perplexed about these results – perplexed because our computations indicated that mouse embryos would undergo lethal IIF when cooled at that rate. We calculated that the cooling rate would have to be about 1 °C/min.. That turned out to be the case. With that change and the substitution of Me 2 SO for PVP, David, Stanley and I obtained very good in vitro and in vivo survival of 8-cell embryos cooled to −196 °C (or −269 °C). The ability to cryopreserve mouse embryos quickly had important applied consequences and important fundamental consequences. Perhaps, the most important direct application was that it permitted the long-term maintenance of thousands of mutant lines of mice in the form of preimplantation embryos in a few liquid nitrogen tanks. The indirect applications stemmed from the fact that essentially the same procedures worked for early embryos of over 20 other mammals, including humans. In the last decade, clinical interest has been emphasizing the cryopreservation of human oocytes. They have proved more difficult to freeze and so interest has shifted somewhat to cryopreserving them by vitrification. As discussed, one way to avoid lethal IIF is to cool cells slowly enough so that the freezable water in the cell flows out of the cell and freezes externally. An alternative approach is to avoid intracellular ice by vitrifying cellular water. Vitrification involves converting water into a non-crystalline or amorphous glass. To achieve this conversion, it has been the strong belief that cells have to be in solutions containing a very high concentration of glass-inducing solutes and have to be cooled very rapidly. In recent work, however, we have found that neither of these is true for mouse oocytes. The important factor is not the cooling rate – it is the warming rate, and the warming rate has to be very high. Furthermore, if the warming rate is very high, one can cut in half the concentration of solutes in the vitrification solution and still obtain high survivals.

It has been brought to notice that the name of the authors for the above mentioned abstract and the funding statement for this abstract has been missed during the typesetting. Hence, please find below the corrected versions of the abstract with all the details. The publisher apologizes for any inconvenience caused by the error. The correct abstract: 85. Intracellular ice formation in mouse zygotes and early morulae vs. cooling rate and temperature–Experimental vs. theory. ⁄Bo Jin, Peter Mazur, Fundamental and Applied Cryobiology Group, Department of Biochemistry and Cellular and Molecular Biology, The University of Tennessee, Knoxville, TN 37996-0840, USA. In 1972, Whittingham, Leibo, and Mazur reported successful cryopreservation of 8-cell mouse embryos. They found that plots of their survival vs. cooling rate (CR) take the form of an inverted U. They also reported on the survival of 2-cell embryos and blastocysts as function of CR. These two stages also yielded an inverted U with a somewhat similar shape. They hypothesized that the drop in survival above CR of 1 C/min was due to intracellular ice formation (IIF). Subsequent papers showed that hypothesis to be correct for 8-cell embryos, but it has never been demonstrated for zygotes and morulae. That was the purpose of the work reported here. In this study, mature female mice of the ICR strain were induced to superovultate, mated, and collected at either zygote or early morula stages. Embryos suspended in 1 M EG in PBS containing 10 mg/ LSnomax for 15 min, then transferred in sample holder to Linkam

Because of their high susceptibility to chilling injury, permeabilized Drosophila embryos can not be cryobiologically preserved by slow freezing at rates low enough to prevent the formation of intraembryonic ice. Calculations indicated that to outrun the chilling injury they must be cooled and warmed rapidly at an estimated 20,000 degrees C/min or faster. Ordinarily, such cooling rates would inevitably produce lethal intracellular ice. To prevent this, embryos must contain and be surrounded by sufficiently high concentrations of glass-promoting solutes to induce vitrification on cooling and prevent devitrification on warming. Like Steponkus et al. (Nature 345, 170, 1990) we have used ethylene glycol as the solute and have exposed permeabilized 12-h embryos to it in two steps. (Permeabilization was effected by exposing dechorionated embryos to a mixture of 0.3% 1-butanol in n-heptane for 90 or 110 s.) The two steps were (i) a 30-min exposure to 2 M ethylene glycol at 23 degrees C and (ii) a 5-min exposure to 8.5 M ethylene glycol [+/- 10% polyvinylpyrrolidone (PVP)] at 5 degrees C. The volumetric response to the first step indicates that full permeation of the 2 M glycol has been approached by 30 min. The point of the second step is to raise the intraembryonic concentration of ethylene glycol to near 8.5 M ethylene glycol by osmotic dehydration. Survival based on hatching is some 45% at this point. When 12-h embryos in 8.5 M glycol containing 10% PVP are then cooled to -205 degrees C at approximately 100,000 degrees C/min and warmed at about that rate, an average of about 12% survive (hatch), although in about half the runs 15-29% survive. Survivals in the absence of PVP are usually poorer but have been as high as 40%. Currently, 5% of the surviving larvae develop to adult flies (Steponkus et al. reported 18% hatching and 3% development to adult). Embryos that develop but do not hatch show readily detectable abnormalities in mouth parts and dorsal closure. Very high warming rates are much more critical to survival than are very high cooling rates; for example, none survive when warming is 2000 degrees C/min. The deleterious effect of slow warming is exerted between -80 and -40 degrees C. The lack of reciprocity between the effects of time spent cooling and time spent warming argues against ascribing death to chilling injury. Rather, it and other data argue for ascribing death to the devitrification during warming of cytoplasm that vitrified during cooling.(ABSTRACT TRUNCATED AT 250 WORDS)