The dominance of warming rate over cooling rate in the survival of mouse oocytes subjected to a vitrification procedure

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 °C at rates ranging from 37 to 1827 °C/min between 20 and −120 °C, and for each cooling rate, warmed at rates ranging from 139 to 2950 °C/min between −70 and −35 °C. The results are unambiguous. If the samples were warmed at the highest rate, survivals were >80% over cooling rates of 187–1827 °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.     

Extra- and intra-cellular ice formation of red seabream (Pagrus major) embryos at different cooling rates

The ice crystal formation is assumed as the most lethal factor for the failure of fish embryo cryopreservation and intracellular ice formation (IIF) plays a central role in cell injury during cooling. The objectives were to observe the morphological changes of red seabream (Pagrus major) embryo during the cooling–thawing process, and to examine the effect of cryoprotectant and cooling rate on the temperatures of oil globule ice formation (T_OIF), extra-cellular ice formation (T_EIF) and intracellular ice formation (T_IIF) using cryomicroscope. After thawing, the morphological changes of embryos were observed and recorded by the video attachment and monitor under the microscope. During the cooling process, three representative phenomena were observed: oil globule gradually turned bright firstly, then the whole field of view flashed and the embryo blackened. Cooling rate affect the temperature of both extra- and intra-cellular ice formations. T_EIF and T_IIF at high cooling rate were much lower than that at low cooling rate. And the value of T_EIF − T_IIF increased from 0.45 to 11.11 °C with the increase of cooling rate from 3 to130 °C/min. Taken together, our results suggested that high cooling rate with proper cryoprotectant would be a good option for red seabream embryo cryopreservation.     

Thermal properties of ethylene glycol aqueous solutions

Preventing ice crystallization by transforming liquids into an amorphous state, vitrification can be considered as the most suitable technique allowing complex tissues, and organs cryopreservation. This process requires the use of rapid cooling rates in the presence of cryoprotective solutions highly concentrated in antifreeze compounds, such as polyalcohols. Many of them have already been intensively studied. Their glass forming tendency and the stability of their amorphous state would make vitrification a reality if their biological toxicity did not reduce their usable concentrations often below the concentrations necessary to vitrify organs under achievable thermal conditions. Fortunately, it has been shown that mixtures of cryoprotectants tend to reduce the global toxicity of cryoprotective solutions and various efficient combinations have been proposed containing ethanediol. This work reports on the thermal properties of aqueous solutions with 40, 43, 45, 48, and 50% (w/w) of this compound measured by differential scanning calorimetry. The glass forming tendency and the stability of the amorphous state are evaluated as a function of concentration. They are given by the critical cooling rates v(ccr)above which ice crystallization is avoided, and the critical warming rates v(cwr) necessary to prevent ice crystallization in the supercooled liquid state during rewarming. Those critical rates are calculated using the same semi-empirical model as previously. This work shows a strong decrease of averaged critical cooling and warming rates when ethanediol concentration increases, V(ccr) and V(cwr) = 1.08 x 10 (10) K/min for 40% (w/w) whereas V(ccr) = 11 and V(cwr) = 853 K/min for 50% (w/w). Those results are compared with the corresponding properties of other dialcohols obtained by the same method. Ethylene glycol efficiency is between those of 1,2-propanediol and 1,3-propanediol.     

Effect of different cryoprotectants and vitrificant solutions on the hatching rate of turbot embryos (Scophthalmus maximus)

Vitrification could provide a promising tool for the cryopreservation of fish embryos. However, in order to achieve a vitrifiable medium, a high concentration of permeable cryoprotectants must be employed, and the incorporation of high molecular weight compounds should also be considered. The toxicity of these permeable and non-permeable agents has to be assessed, particularly when high concentrations are required. In the present study, permeable and non-permeable cryoprotectant toxicity was determined in turbot embryos at two development stages (F stage-tail bud and G stage-tail bud free). Embryos treated with pronase (2mg/ml, 10 min at 22 degrees C) were incubated in dimethyl sulfoxide (Me2SO), methanol (Meth.) or ethylene glycol (EG) in concentrations ranging from 0.5 to 6M for periods of 10 or 30 min, and in 5, 10, and 15% polyvinylpyrrolidone (PVP), 10, 15, and 20% sucrose or 0.1, 1, and 2% X-1000 for 2 min. The embryos were then washed well and incubated in seawater until hatching. The toxicity of permeable cryoprotectants increased with concentration and exposure time. There were no significant differences between permeable cryoprotectants. However, embryos tolerated higher concentrations of Me2SO than other cryoprotectants. Exposure to permeable cryoprotectants did not affect the hatching rate except at G stage with X-1000 treatment and 20% sucrose. Taking into account the cryoprotectant toxicity and the vitrification ability of cryoprotectant mixtures, three vitrification solutions (V1, V2, and V3), and one protocol for stepwise incorporation were designed. The tested solutions contained 5M Me2SO+2M Meth+1M EG plus 5% PVP, 10% sucrose or 2% X-1000. The hatching rate of embryos that had been exposed to the the vitrification solutions was analyzed and no significant differences were noticed compared with the controls. Our results demonstrate that turbot embryos can be subject to this cryoprotectant protocol without deleterious effect on the hatching rate.     

The effect of warming velocity on motility and acrosomal integrity of boar sperm as influenced by the rate of freezing and glycerol level

The effect of thawing velocities ranging from 10°C/min to 1.800°C/min on the motility and acrosomal integrity of boar spermatozoa frozen at 1°C/min (suboptimal), 5°C/min, and 30°C/min (optimal) rate was studied with the sperm suspended for freezing in diluent containing 2, 4, or 6% of glycerol (v/v). The influence of thawing on sperm survival depends on the rate at which the sperm had been frozen. In semen frozen at a suboptimal rate of 1°C/min, the percentage of motile sperm (FMP) initially fell to 3.5–4.0% when the thawing rose to 200°C/ min, but, with further increases in thawing rate, increased and reached peak values (10.3–11.0% FMP) after thawing at 1,800°C/min. The percentage of sperm with normal apical ridge (NAR) also increased moderately with thawing rate, but the degree of improvement decreased as the glycerol level was increased. In semen frozen at 1°C/min, acrosomal integrity (NAR) was best maintained in 2% glycerol, reaching 22.9% NAR after thawing at 1,800°C/min. In semen frozen at the optimal rate of 30°C/min, the increases in thawing rates above 200°C/min substantially improved motility. Motility was generally higher in semen protected by 4 or 6% glycerol, with the peak values of 44 or 46% FMP, respectively, after thawing at 1,200°C/min. The proportion of sperm with NAR also increased with thawing rate, but as in the case of suboptimally frozen sperm it was influenced negatively by the glycerol concentration. The peak value 53% NAR was recorded in semen protected by 2% glycerol, frozen at 30°C/min, and thawed at 1,200°C/min. In view of the inverse relationship between FMP and NAR, selection of optimal conditions from among the interacting variables, freezing rate, glycerol concentration, and thawing rate requires compromising between maximal FMP and maximal NAR. Accordingly, we have adopted as optimal a protocol with a thawing rate of 1,200°C/min, a freezing rate of 30°C/min and concentrations of 3% glycerol. © 1993 Wiley-Liss, Inc.     

The cryopreservation of Chlorella 1. Interactions of rate of cooling,protective additive and warming rate

The cryoprotective additives glycerol and dimethylsulphoxide were found to be toxic to Chlorella cells at concentrations greater then 2.5% w/v. Polyvinylpyrrolidone, was not damaging up to a concentration of 15% w/v. Chlorella 211/7a had a recovery rate greater than 95% at all rates of cooling studied. With Chlorella 211/8h the survival was lower than 0.1% at all rates examined. The addition of dimethylsulphoxide (5% w/v) to Chlorella 211/8h increased the recovery, particularly at the faster rates of cooling; with polyvinylpyrrolidone (10% w/v) there was an optimum range of cooling rate.Cells of Chlorella 211/7a from the exponential phase of growth were found to be damaged both by a temperature reduction from 25°C to 0°C (thermal shock) and by freezing and thawing. In contrast cells from the stationary phase of growth were resistant to these stresses.Abbreviations DMSO dimethylsulphoxide - HEPES N-2-hydroxyethylpiperazine-N-2-ethansulphonic acid - PVP polyvinylpyrrolidone     

Differences among dogs in response of their spermatozoa to cryopreservation using various cooling and warming rates

Spermatozoa collected from the caudae epididymides of 16 dogs of various breeds were suspended in an isotonic salt solution (DIMI medium) containing 0.6 M glycerol, frozen in liquid nitrogen, and their "survival" was measured after thawing. In the first experimental series, duplicate samples of spermatozoa from each of 11 dogs were cooled at rates of 0.5, 3, 11, 58, or 209 degrees C/min, stored in liquid nitrogen, and the frozen samples warmed at approximately 830 or at 33 degrees C/min. Sperm "survival" was judged by microscopic assessments of motility and of membrane integrity, the latter as assayed with Fertilight, a double fluorescent stain. Motility of frozen spermatozoa that were thawed rapidly, averaged for 11 dogs, was low at low rates, increased to a maximum at 11 degrees C/min, and then decreased significantly at higher rates (P<0.01). This inverted V-shaped curve was also observed with slow thawing, although the apparent optimum cooling rate ranged from 3 to 11 degrees C/min. The integrity of sperm plasma membranes showed a similar dependence on cooling rate, although the percentages of spermatozoa with intact plasma membranes were higher than the percentages of motile spermatozoa. Motility of spermatozoa, as a function of cooling rate, varied considerably from male to male (P<0.01), whereas membrane integrity was much more consistent among the 11 dogs. In the second experimental series with spermatozoa from 5 dogs, motility of spermatozoa frozen at 0.5 degrees C/min and warmed at 3.6, 33, 140, or 830 degrees C/min also exhibited an inverted V-shaped survival curve, in this case as a function of warming rate. In summary, high survival of frozen-thawed canine epididymal spermatozoa depended on both cooling and warming rates, but spermatozoa from each dog exhibited their own sensitivity to cooling and warming rates.     

Schistosoma mansoni: Cryopreservation of schistosomula by two-step addition of ethanediol and rapid cooling

A simple, inexpensive, and highly effective technique for the Cryopreservation of schistosomula of Schistosoma mansoni is outlined by experiments designed to clarify the role of each of the steps involved. The technique consists of incubating schistosomula in 10% ($\text{v}\text{v}$) ethanediol for 10 min at 37 C followed by 5 min at 0 C and for a further 10 min in 35% ($\text{v}\text{v}$) ethanediol at 0 C. The schistosomular suspension is then aliquotted in 20-μl drops onto 40 × 5.5-mm glass slivers prepared from standard microscope coverslips, each drop being spread out to cover an area of approximately 15 × 4 mm. These glass slivers are then dropped directly into liquid nitrogen giving a cooling rate of approximately 5000 C min^?1. Survival is further improved if the schistosomula are at least 90 min old before Cryopreservation and if the frozen organisms are thawed in culture medium prewarmed to +42 C. Levels of survival obtained with this technique are consistently high: 44 to 60% as assessed by motility. From 400 ± 11 cryopreserved schistosomula injected intramuscularly into eight mice, a mean of 54.5 ± 16.3 adult worms were recovered representing an infection level of 13.7%, and which in turn represents 47.4% of the unfrozen control level.     

Simple, inexpensive attainment and measurement of very high cooling and warming rates

We have developed a simple, inexpensive system (<$300 US) for measuring cooling and warming rates of small (∼ 0.1 μl) aqueous samples at rates as high as 10⁵ °C/min. The measurement system itself, can track rates approaching one million °C/min. For temperature sensing, a Type T thermocouple with 50 μm wire was used. The thermocouple output voltage was read with an inexpensive USB based digital oscilloscope interfaced to a laptop computer, and the raw data were processed with MS Excel.     

Effects of different concentration and combinations of cryoprotectants on sperm quality,functional integrity in three Indian horse breeds

Traditionally Glycerol (Gly) is being used as major cryoprotectant and its toxicity could be a reason for the variation on stallion sperm freezability and fertility. In an effort to minimize Gly toxicity alternative cryoprotective agents like Di-methyl Formamide (DMF) have been investigated. The effect of the cryoprotectant and dose of cryoprotective agent varies from breed to breed and also from stallion to stallion within the same breed. Considering these factors a study was designed to study the effects of Gly and DMF at different concentrations and combinations on the plasma membrane, acrosome and DNA integrity as well as other post thaw seminal characteristics of semen of three Indigenous stallion breeds. In the current study, semen was collected from apparently healthy 4–6 years old 3 Marwari, 3 Manipuri and 3 Zanskari breed stallions. After semen collection and evaluation of fresh semen, each semen sample was extended with semen extender containing different concentrations and combinations of Gly and DMF cryoprotectants (i.e. 5% Gly, 5% DMF, 2% Gly, 2% DMF, 2.5% Gly +2.5% DMF and 1% Gly +1% DMF) and frozen. Post thaw semen evaluation was done on the basis of post thaw motility, live sperm count, hypo osmotic swelling test, acrosomal integrity and DNA integrity. Frozen thawed semen showed that the values of plasma membrane integrity, acrosome integrity and DNA integrity parameters were significantly higher (P < 0.05) with 5% DMF than the other cryoprotectants levels and combinations of Gly and DMF. From the present study, it was inferred that the combination of cryoprotectants at different concentrations (Gly and DMF @ 2.5 and 1%) also could not show better enhancement compared to the single cryoprotectant i.e DMF @5% in various post thaw seminal characteristics of Indigenous stallion semen. DMF at 5% concentration gave better protection to the plasma membrane and retained the acrosome and DNA integrity of the spermatozoa. Hence it can be concluded that DMF at 5% can be used for the cryopreservation of the Indigenous stallion with better preservation of the seminal quality.     

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.     

Extreme rapid warming yields high functional survivals of vitrified 8-cell mouse embryos even when suspended in a half-strength vitrification solution and cooled at moderate rates to −196 °C

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., ?1000 °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 83%, 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.     

Combined effect of glycerol concentration and cooling velocity on motility and acrosomal integrity of boar spermatozoa frozen in 0.5 ml straws

The interaction of glycerol concentrations of 0-10% and cooling rates from 1 to 1,500 degrees C/min with boar spermatozoa motility and acrosomal integrity (proportion of spermatozoa with normal apical ridge) was studied after thawing 0.5 ml straws at a constant rate. While increasing the glycerol concentration from 0 to 4% progressively improved motility, the percentage of spermatozoa with a normal apical ridge gradually decreased. The magnitudes of the respective changes depended on cooling rate. A peak value of 48.1% and rating 3.8 were obtained in semen protected with 4% glycerol, frozen at 30 degrees C/min. Increasing the glycerol levels above 6% resulted in a gradual decrease in motility. The proportion of spermatozoa with normal apical ridge was highest in semen protected with 0-1% glycerol after cooling at 30 degrees C/min (64.4% and 66.1%, respectively), but at these glycerol concentrations the percentage of motile spermatozoa was low. At the 30 degrees C/min cooling rate, the decline in the proportion of cells with normal apical ridge due to increasing the glycerol levels to 3 and 4% was relatively slow (57.3% and 49.4%, respectively). Cooling at 1 degrees C/min was detrimental to acrosomal integrity, which decreased with increasing glycerol concentration, in contrast to increasing motility, which even at its maximum, remained low. The direct plunging of straws into liquid nitrogen (1,500 degrees C/min) resulted in damaged acrosomes in all spermatozoa with the total loss of motility. Balancing motility and acrosomal integrity, freezing boar semen protected with 3% glycerol by cooling at 30 degrees C/min resulted in optimal survival for boar semen frozen in 0.5 ml French straws.     

Intracellular ice formation in confluent monolayers of human dental stem cells and membrane damage

Dental pulp stem cells (DPSCs) are of interest to researchers and clinicians due to their ability to differentiate into various tissue types and potential uses in cell-mediated therapies and tissue engineering. Currently DPSCs are cryopreserved in suspension using Me₂SO. However, preservation as two and three dimensional constructs, along with the elimination of toxic Me₂SO, may be required. It was shown that intracellular ice formation (IIF), lethal to cells in suspensions, may be innocuous in cell monolayers due to ice propagation between cells through gap junctions that results in improved post-thaw recovery. We hypothesized that innocuous IIF protects confluent DPSC monolayers against injury during cryopreservation. The objective was to examine the effects of IIF on post-thaw viability of both confluent monolayers and suspensions of DPSCs. Confluent DPSC monolayers were assessed for the expression of gap junction protein Connexin-43. IIF was induced on the cryostage and in the methanol bath at different subzero temperatures. Membrane integrity and colony-forming ability were assessed post-thaw. Confluent DPSC monolayers expressed Connexin-43. In cell suspensions, 85.9 ± 1.7% of cells were damaged after 100% IIF. In cell monolayers, after 100% IIF, only 25.5 ± 5.5% and 14.8 ± 3.3% of cells were damaged on the cryostage and in the methanol bath respectively. However, DPSC monolayers exposed to 100% IIF showed no colony-forming ability. We conclude that confluent monolayers of DPSCs express the gap junction-forming protein Connexin-43 and upon IIF retain membrane integrity, however lose the ability to proliferate.     

Effect of freezing and thawing rates on denaturation of proteins in aqueous solutions

The freeze denaturation of model proteins, LDH, ADH, and catalase, was investigated in absence of cryoprotectants using a microcryostage under well-controlled freezing and thawing rates. Most of the experimental data were obtained from a study using a dilute solution with an enzyme concentration of 0.025 g/l. The dependence of activity recovery of proteins on the freezing and thawing rates showed a reciprocal and independent effect, that is, slow freezing (at a freezing rate about 1 degrees C/min) and fast thawing (at a thawing rate >10 degrees C/min) produced higher activity recovery, whereas fast freezing with slow thawing resulted in more severe damage to proteins. With minimizing the freezing concentration and pH change of buffer solution by using a potassium phosphate buffer, this phenomenon could be ascribed to surface-induced denaturation during freezing and thawing process. Upon the fast freezing (e.g., when the freezing rate >20 degrees C/min), small ice crystals and a relatively large surface area of ice-liquid interface are formed, which increases the exposure of protein molecules to the ice-liquid interface and hence increases the damage to the proteins. During thawing, additional damage to proteins is caused by recrystallization process. Recrystallization exerts additional interfacial tension or shear on the entrapped proteins and hence causes additional damage to the latter. When buffer solutes participated during freezing, the activity recovery of proteins after freezing and thawing decreased due to the change of buffer solution pH during freezing. However, the patterns of the dependence on freezing and thawing rates of activity recovery did not change except for that at extreme low freezing rates (<0.5 degrees C/min). The results exhibited that the freezing damage of protein in aqueous solutions could be reduced by changing the buffer type and composition and by optimizing the freezing-thawing protocol.     

Effect of cooling rates on the cold hardiness and cryoprotectant profiles of locust eggs

To examine the relationship between cooling rate and cold hardiness in eggs of the migratory locust, Locusta migratoria, the survival rates and cryoprotectant levels of three embryonic developmental stages were measured at different cooling rates (from 0.05 to 0.8 degrees C min(-1)) in acclimated and non-acclimated eggs. Egg survival rate increased with decreasing cooling rate. The concentration of cryoprotectants (myo-inositol, trehalose, mannitol, glycerol, and sorbitol) increased in non-acclimated eggs, but varied significantly in response to different cooling rates in acclimated eggs. The acclimation process (5 degrees C for 3 days) did not increase eggs resistance to quick cooling ("plunge" cooling and 0.8 degrees C min(-1)). Earlier stage embryos were much more sensitive than later stage embryos to the same cooling rates. Time spent at subzero temperatures also had a strong influence on egg survival.     

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.     

Measurement of cooling and warming rates in vitrification‐based plant cryopreservation protocols

Cryopreservation protocols include the use of additives and pretreatments aimed to reduce the probability of ice nucleation at all temperatures, mainly through micro‐viscosity increase. Still, there is a risk of ice formation in the temperature region comprised between the equilibrium freezing (T_f) and the glass transition (T_G) temperatures. Consequently, fast cooling and warming, especially in this region, is a must to avoid ice‐derived damage. Vitrification and droplet‐vitrification techniques, frequently used cryopreservation protocols based in fast cooling, were studied, alongside with the corresponding warming procedures. A very fast data acquisition system, able to read very low temperatures, down to that of liquid nitrogen, was employed. Cooling rates, measured between ?20°C and ?120°C, ranged from ca. 5°C s^?1 to 400°C s^?1, while warming rates spanned from ca. 2°C s^?1 to 280°C s^?1, for the different protocols and conditions studied. A wider measuring window (0°C to ?150°C) produced lower rates for all cases. The cooling and warming rates were also related to the survival observed after the different procedures. Those protocols with the faster rates yielded the highest survival percentages. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1177–1184, 2014