Intracellular Ice Formation Temperature Research Articles

Visualization of intracellular ice formation and growth in mouse oocytes

BACKGROUND: Characterization of intracellular ice formation (IIF) in oocytes during the freezing and thawing processes will contribute to optimizing their cryopreservation. However, the observation of the ice formation process in oocytes is limited by the spatiotemporal resolution of the cryomicroscope systems. OBJECTIVE: To observe the intracellular icing of oocytes during cooling and rewarming, and to study the mechanism of formation and growth of intracellular ice in oocytes. MATERIALS AND METHODS: Mouse oocytes were frozen at different cooling rates to induce intracellular ice formation using a cryomicroscopy system consisting of a microscope equipped with a cryogenic cold stage, an automatic cooling system, a temperature control system, and a high-speed camera. The growth patterns of intracellular ice in oocytes were analyzed from the images recorded. Finally, the growth rate of intracellular ice formation in oocytes was calculated using an automatic intracellular ice tracking method.RESULTS: The IIF temperature decreased gradually with the increase in cooling rate. Initiation sites of IIF could be classified into three categories: marginal type, internal type and coexisting type. There was a strong predominance for ice crystal initiation site in the oocytes, with up to 80% of the initiation sites located in the marginal region. The intracellular ice growth modes of darkening and twitching cells were characterized by “spreading” and “clustering”, respectively. In addition, twitching cells started to recrystallize during rewarming, while darkening cells did not. The instantaneous maximal growth rate of ice crystals in twitching cells was about 10 times higher than that in darkening cells. CONCLUSION: By visualising the growth of ice crystals in mouse oocytes during cooling and rewarming, we obtained valuable information on the kinetics of ice formation and melting in these cells. This information can help us understand how ice formation and melting affect the viability and quality of oocytes after cryopreservation.

017 Cryomicroscopic analysis of damage mechanisms during freezing and thawing of a human diploid fibroblast cell line

Human diploid cell strains are useful for commercial production of viral vaccines. For example, the MRC-5 cell line (lung fibroblast) is used to produce vaccines against chickenpox and other childhood diseases. To support the safe mass production of such vaccines, it is necessary to cryopreserve standardized batches of concentrated cell suspension, thus ensuring a consistent supply of cells for manufacturing. However, the cryopreservation process can result in deleterious intracellular ice formation (IIF), or trigger other mechanisms that also lead to cell damage. Therefore, towards improving the efficiency of MRC-5 cryopreservation techniques, the IIF phenomenon was investigated in cell suspensions using a high-speed video cryomicroscopy system. Previous high-speed imaging studies have visualized the IIF process in adherent cells, but not in suspended cells. In the present study, the effect of cooling rate on IIF in MRC-5 cells in suspension was determined. High-speed videos were captured at recording rates of 540–1200 frames/s during freezing to −60 °C at various rates of cooling (1–120 °C/min). In the suspended cells, intracellular ice formation manifested as an advancing solidification front, with minimal change in cell transparency; the median duration of such IIF events was 1 ms during rapid cooling (120 °C/min), demonstrating the importance of high-speed imaging techniques to detect intracellular freezing. The cumulative incidence of IIF increased with increasing cooling rate, from 0% at 1 °C/min to 96% at 120 °C/min. The mean temperature of IIF was −21.7 °C ( n = 75) and −33.0 °C ( n = 853) during cooling at 15 °C/min and 120 °C/min, respectively. For MRC-5 cells cooled at 120 °C/min in the presence of 5% Me 2 SO, the mean IIF temperature was depressed to −46.1 °C. Whereas the majority of cells did not darken significantly during cooling, prominent cell darkening was often observed upon warming of the frozen cells. Video recordings were acquired during warming from −60 °C to 4 °C at a warming rate of 120 °C/min. In cells with intracellular ice, the probability of darkening during warming was greater than 50%, and darkening during warming appeared to be more likely for cells that had been frozen at a higher cooling rate. In slowly cooled samples, another phenomenon was observed during rapid warming: prior to melting of extracellular ice, a transient swelling was seen in many cells. Because the magnitude of these volume excursions was often large (e.g., doubling the original cell volume), it is likely that such events are damaging, and may represent a new form of slow-cooling injury. Source of funding: Funded in part by a gift from Merck & Co., Inc. to Villanova University. Conflict of interest: None declared. jens.karlsson@villanova.edu

Effects of Intercellular Junction Protein Expression on Intracellular Ice Formation in Mouse Insulinoma Cells

The development of cryopreservation procedures for tissues has proven to be difficult in part because cells within tissue are more susceptible to intracellular ice formation (IIF) than are isolated cells. In particular, previous studies suggest that cell-cell interactions increase the likelihood of IIF by enabling propagation of ice between neighboring cells, a process thought to be mediated by gap junction channels. In this study, we investigated the effects of cell-cell interactions on IIF using three genetically modified strains of the mouse insulinoma cell line MIN6, each of which expressed key intercellular junction proteins (connexin-36, E-cadherin, and occludin) at different levels. High-speed video cryomicroscopy was used to visualize the freezing process in pairs of adherent cells, revealing that the initial IIF event in a given cell pair was correlated with a hitherto unrecognized precursor phenomenon: penetration of extracellular ice into paracellular spaces at the cell-cell interface. Such paracellular ice penetration occurred in the majority of cell pairs observed, and typically preceded and colocalized with the IIF initiation events. Paracellular ice penetration was generally not observed at temperatures >−5.65°C, which is consistent with a penetration mechanism via defects in tight-junction barriers at the cell-cell interface. Although the maximum temperature of paracellular penetration was similar for all four cell strains, genetically modified cells exhibited a significantly higher frequency of ice penetration and a higher mean IIF temperature than did wild-type cells. A four-state Markov chain model was used to quantify the rate constants of the paracellular ice penetration process, the penetration-associated IIF initiation process, and the intercellular ice propagation process. In the initial stages of freezing (>−15°C), junction protein expression appeared to only have a modest effect on the kinetics of propagative IIF, and even cell strains lacking the gap junction protein connexin-36 exhibited nonnegligible ice propagation rates.

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 4.0M DMSO in sea water. As 2.0M 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 2.0M 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 2.0M 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.

Effect of the expression of aquaporins 1 and 3 in mouse oocytes and compacted eight-cell embryos on the nucleation temperature for intracellular ice formation

The occurrence of intracellular ice formation (IIF) is the most important factor determining whether cells survive a cryopreservation procedure. What is not clear is the mechanism or route by which an external ice crystal can traverse the plasma membrane and cause the heterogeneous nucleation of the supercooled solution within the cell. We have hypothesized that one route is through preexisting pores in aquaporin (AQP) proteins that span the plasma membranes of many cell types. Since the plasma membrane of mature mouse oocytes expresses little AQP, we compared the ice nucleation temperature of native oocytes with that of oocytes induced to express AQP1 and AQP3. The oocytes were suspended in 1.0 M ethylene glycol in PBS for 15 min, cooled in a Linkam cryostage to −7.0 °C, induced to freeze externally, and finally cooled at 20 °C/min to −70 °C. IIF that occurred during the 20 °C/min cooling is manifested by abrupt black flashing. The mean IIF temperatures for native oocytes, for oocytes sham injected with water, for oocytes expressing AQP1, and for those expressing AQP3 were −34, −40, −35, and −25 °C respectively. The fact that the ice nucleation temperature of oocytes expressing AQP3 was 10–15 °C higher than the others is consistent with our hypothesis. AQP3 pores can supposedly be closed by low pH or by treatment with double-strandedAqp3RNA. However, when morulae were subjected to such treatments, the IIF temperature still remained high. A possible explanation is suggested.

Rationalizing the equilibration and cooling stages of cryopreservation: The effect of cell size distribution

AbstractA novel model able to quantitatively describe intracellular ice formation (IIF) as a function of temperature in a cell population characterized by a size distribution is proposed for the case when a permeant cryoprotectant agent (CPA) is present. As such, the model represents the ideal extension of the one recently proposed by the authors for interpreting the behavior of a size‐distributed population of cells during cryopreservation without CPA. To resemble the experimental procedure actually adopted, the former model is coherently modified for the simulation of the initial equilibration stage (i.e., a one‐step CPA permeation kinetics into cells initially under isotonic conditions to be suspended in a water/salt/CPA solution at ambient temperature) and of the subsequent cooling stage at constant rate down to −60°C. Specifically, the size distribution dynamics of a cell population in response to water osmosis, CPA permeation, and IIF is simulated by means of a suitable population balance approach. Water and CPA intracellular transport equations based on the classical Kedem and Katchalsky formalism are coupled to the quantitative description of nucleation and diffusion‐limited growth of ice crystals in the framework of a one‐dimensional population balance equation. The driving forces of all the physico‐chemical phenomena involved are evaluated by taking into account the relevant ternary phase diagram, whereas the viscosity of the cytoplasm solution is properly accounted as a function of the salt and CPA concentrations. After a reasoned choice of the values of model parameters, the behavior of isolated rat hepatocytes suspended in a water/sodium‐chloride/glycerol solution is simulated and analyzed at different, practicable (i.e., low) constant cooling rates and CPA equilibration concentrations. It is confirmed that differently sized cells in a single population exhibit different IIF temperatures under the same operative conditions even in the presence of CPA. Correspondingly, the probability of IIF results to be a function of the initial size distribution of the cell population. Depending on the specific operative conditions adopted, the size distribution and the osmotic properties of the cell lineage at hand, IIF at −60°C may be lethal for a fraction of the cell population (i.e., larger size classes), or it may not reach a dangerous level for the intermediate size class cells, while it will not even take place for the smaller ones. Finally, even if no comparison with experimental data is provided, an original and physically sound explanation for several, well‐known experimental evidences, which appeared in a number of articles available in the literature of cell cryopreservation with CPA, is comprehensively given. © 2010 American Institute of Chemical Engineers AIChE J, 2011

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

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

The Temperature and Type of Intracellular Ice Formation in Preimplantation Mouse Embryos as a Function of the Developmental Stage1

Our studies the past 5 yr have concentrated on intracellular ice formation (IIF) in mature mouse oocytes at the metaphase stage of meiosis II. Here we report an analogous investigation of the temperature of intracellular ice nucleation in preimplantation embryo stages from one-cell to early morula suspended in 1 M ethylene glycol/PBS and cooled at 20 degrees C/min to -70 degrees C. Physical modeling indicates that oocytes and preimplantation embryos undergo very little osmotic shrinkage at that cooling rate. As a consequence, their interior becomes increasingly supercooled until the supercooling is abruptly terminated by IIF. Four categories of IIF were observed. The first two were 1) those undergoing IIF at temperatures well below the temperature of external ice formation (EIF; -7.2 degrees C) vs. 2) those undergoing IIF within 1 degrees C of the EIF temperature. The other two categories were those multicellular stages in which 3) all the blastomeres underwent IIF simultaneously vs. 4) those in which blastomeres underwent IIF sequentially. Embryos in categories 1 and 3 constituted the majority (80-90%), and for them, the mean IIF temperatures of one-cell, two-cell, four- to six-cell, and early eight-cell ranged from -37 degrees C to -43 degrees C, temperatures that indicate that IIF is a consequence of homogeneous nucleation. However, the IIF nucleation temperature of early morulae in categories 1 and 3 was markedly higher; namely, -23.1 +/- 1.5 degrees C. This marked rise in nucleation temperature coincides with the appearance of aquaporin 3 and gap junctions in early morulae (compacted eight-cell), and is presumably causally related.

The effect of cell size distribution during the cooling stage of cryopreservation without CPA

AbstractA novel model capable of quantitatively describing and predicting intracellular ice formation (IIF) as a function of temperature in a cell population characterized by a size distribution is proposed. The model overcomes the classical approach which takes into account a population of identically sized cells. The size distribution dynamics of a cell population in response to water osmosis and IIF occurrence during the cooling stage of a standard cryopreservation protocol without using cryoprotective agent (CPA) is simulated by means of a suitable population balance approach. Specifically, the model couples the classical water transport equation developed by Mazur1 to the quantitative description of nucleation and diffusion‐limited growth of ice crystals in the framework of a 1‐D population balance equation (PBE). It is found that IIF temperature depends on the cell size, i.e., it is higher for larger cells. Correspondingly, the probability of IIF (PIIF) results to be dependent on the initial size distribution of the cell population. Model's parameters related to the osmotic behavior of the cell population and to IIF kinetics are obtained by comparison between theoretical results and suitable experimental data of isolated rat hepatocytes available in the literature. Model reliability is successfully verified by predicting the experimental data of PIIF at different, constant cooling rates with better accuracy as compared to the theoretical approaches available in the literature. © 2009 American Institute of Chemical Engineers AIChE J, 2010

94. The temperature of intracellular ice formation in mouse embryos as a function of the developmental stage

94. The temperature of intracellular ice formation in mouse embryos as a function of the developmental stage

14. The temperature of intracellular ice formation in mouse oocytes vs. the unfrozen fraction at that temperature

14. The temperature of intracellular ice formation in mouse oocytes vs. the unfrozen fraction at that temperature

Relationship between intracellular ice formation in oocytes of the mouse and Xenopus and the physical state of the external medium—A revisit

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.

The temperature of intracellular ice formation in mouse oocytes vs. the unfrozen fraction at that temperature

We have previously reported [Cryobiology 51 (2005) 29–53] that intracellular ice formation (IIF) in mouse oocytes suspended in various concentrations of glycerol and ethylene glycol (EG) occurs at temperatures where the percentage of unfrozen water is about 6% and 12%, respectively, even though the IIF temperatures varied from −14 to −41 °C. However, because of the way the solutions were prepared, the concentrations of salt and glycerol or EG in that unfrozen fraction at IIF were also rather tightly grouped. The experiments reported in the present paper were designed to separate the effects of the unfrozen fraction at IIF from that of the solute concentration in the unfrozen fraction. This separation makes use of two facts. One is that the concentration of solutes in the residual liquid at a given subzero temperature is fixed regardless of their concentration in the initial unfrozen solution. However, second, the fraction unfrozen at a given temperature is dependent on the initial solute concentration. Experimentally, oocytes were suspended in solutions of glycerol/buffered saline and EG/buffered saline of varying total solute concentration with the restriction that the mass ratios of glycerol and EG to salts are held constant. The oocytes were then cooled rapidly enough (20 °C/min) to avoid significant osmotic shrinkage, and the temperature at which IIF occurred was noted. When this is done, we find, as previously that the fraction of water remaining unfrozen at the temperature of IIF remains nearly constant at 5–8% for both glycerol and EG even though the IIF temperatures vary from −14 to −50 °C. But unlike the previous results, the salt and CPA concentrations in the unfrozen fraction vary by a factor of three. The present procedure for preparing the solutions produces a potentially complicating factor; namely, the cell volumes vary substantially prior to freezing: substantially greater than isotonic in some solutions; substantially smaller in others. However, the data in toto demonstrate that cell volume is not a determining factor in the IIF temperature.

Analysis of intracellular ice nucleation in Xenopus oocytes by differential scanning calorimetry

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 2 M 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 °C/min) cooled. EIF in the 1, 1.5, and 2 M EG occurred at −6.4, −7.8, and −8.9 °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 1.5 M EG, T IIF was −21.1, −25, and −26.6 °C in stages I–III, but was −17 and −8.5 °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.

Effects of hold time after extracellular ice formation on intracellular freezing of mouse oocytes

MII mouse oocytes in 1 and 1.5 M ethylene glycol(EG)/phosphate buffered saline have been subjected to rapid freezing at 50 °C/min to −70 °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 1 M EG, the IIF temperatures are −23.7 and −39.2 °C with 0 and 2 min hold times; in 1.5 M EG, the corresponding IIF temperatures are −29.1 and −40.8 °C.

Starfish Oocytes Form Intracellular Ice at Unusually High Temperatures

Starfish oocytes, eggs, and embryos are popular models for studying meiotic maturation, fertilization, and embryonic development. Their large (170- to 200-μm) oocytes are obtainable in copious amounts and are amenable to manipulations that mammalian oocytes are not. The most formidable obstacle to working with marine oocytes is their seasonal availability, yet a successful means of preserving them for use during the nonreproductive season has not been reported. The aim of this study was to investigate the response of starfish oocytes to freezing with rapid and slow cooling rates under a variety of conditions to develop a cryopreservation protocol for these cells. Cryomicroscopic observation revealed that starfish oocytes in isotonic medium undergo intracellular ice formation (IIF) at very high subzero temperatures, such that the mean difference between the temperature of extracellular ice formation (TEIF) and IIF (TIIF) was less than 3°C and the average TIIF was approximately between −4 and −6°C. Neither partial cellular dehydration nor addition of the cryopreservative dimethyl sulfoxide significantly depressed the TIIF. Under some conditions, we observed ice nucleation at multiple locations within the cytoplasm, suggesting that several factors contribute to the unusually high TIIF during controlled-rate freezing and thus vitrification may be a more suitable method for cryopreserving these cells.

Water Permeability and Its Activation Energy for Individual Hamster Pancreatic Islet Cells

Coupled with the rapid development of clinical pancreatic islet transplantation, there is an increasing requirement for cryopreservation of viable islets. Fundamental cryobiology requires determination of several cryobiophysical parameters to predict optimal cryopreservation procedures. These include water permeability or hydraulic conductivity (Lp) and its activation energy (Ea), the permeability of the cell plasma membrane to a cryoprotectant(s) (Ps) and its Ea, the osmotically inactive fraction of cell volume (Vb), and the intracellular ice formation temperature. For islet cells, these parameters have not previously been reported. In the present studies, the Lp. its Ea , and Vb were determined for isolated individual golden hamster pancreatic islet cells. The Lp and Vb parameters were also measured for corresponding exocrine cells. Both islet and the exocrine cells appeared to be ideal osmometers over the experimental range when examined by the Boyle Van't-Hoff relationship (linear regression, r = 0.99 for both types of cells). Extrapolation of these plots generaled Vb values of 0.40 for the islet cells and 0.45 for the pancreatic exocrine cells. To determine the Lp , kinetic changes of cell volume over time (dv/dt) in response to anisoosmotic conditions (ranging from 145 mOsm/kg to 1.35 Osm/kg) were measured using an electronic particle counter. The experimental data were fitted to generate the Lp values by least squares curve fitting to a differential equation describing osmotic water movement across the plasma membrane. For pancreatic islet cells, the Lp was determined to be 0.25 ± 0.03 μm/min/atm (X̄ ± SD, n = 14) at 22°C, 0.54 ± 0.07 (n = 10), 0.06 ± 0.008 (n = 9), and 0.01 ± 0.001 (n = 9) at 37, 8, and 0°C, respectively. The Ea for Lp was calculated from the slope of the Arrhenius plot based upon the mean Lp values at the four different temperatures. The Ea was 16.21 Kcal/mol between 0 and 37°C. Based upon these values, an optimal cooling rate for cryopreserving pancreatic islet cells is predicted to be approximately 0.5°C min. The Lp for the individual exocrine cells was determined to be 3.73 ± 1.75 μm/min/atm (n = 13) at 22°C, which was approximately 10 times the Lp value of the corresponding islet cells.

Effect of Electroporation on Intracellular Ice Formation in Rye Leaf Protoplasts

Cryomicroscopy was used to study the statistical behavior of intracellular ice formation (IIF) in protoplasts isolated from rye leaves (Secale cereale L cv. Aroostook) and subjected to prior electrical treatment ("electroporation"). Protoplasts were suspended in isotonic (530 mOsm) or hypotonic (265 mOsm) saline and exposed to exponentially decaying electric fields of 50 to 500 V/cm and capacitances of 1 × 850, 2 × 850, or 3 × 850 μF. Treatment conditions were selected by determining apparent cell lysis, viability, and increased permeability to fluorescent dyes. For each condition, protoplasts were subjected to linear cooling at 5, 10, 20, or 40°C/min, or to cooling at 40°C/min followed by an isothermal hold at -6, -9, -12, or -15°C. In linear cooling, field strengths above 150 V/cm and capacitances above 1 × 850 μF resulted in a 1 to 9°C shift to higher median IIF temperatures. Maximum IIF incidence was unaffected. Under the weakest condition (100 V/cm, 1 × 850 μF), median IIF temperatures during linear cooling were shifted 3 to 7°C below those of the control. In isothermal periods, mean IIF times of electroporated protoplasts were 50 to 300% smaller than the control, implying faster IIF kinetics in electroporated protoplasts. In hypotonic saline the effects of electroporation were suppressed. Based on a comparison of stochastic model parameters, electropotation affected IIF during linear cooling and isothermal periods similarly.

Performance of a kinetic model for intracellular ice formation based on the extent of supercooling

Cryomicroscopy was used to study the incidence of intracellular ice formation (IIF) in protoplasts isolated from rye ( Secale cereale) leaves during subfreezing isothermal periods and in in vitro mature bovine oocytes during cooling at constant rates. IIF in protoplasts occurred at random times during isothermal periods, and the kinetics of IIF were faster as isothermal temperature decreased. Mean IIF times decreased from approximately 1700 s at −4.0 °C to <1 s at −18.5 °C. Total incidence of IIF after 200 s increased from 4% at −4.0 °C to near 100% at −15.5 °C. IIF behavior in protoplasts was qualitatively similar to that for Drosophila melanogaster embryos over the same temperature ranges (Myers et al., Cryobiology 26, 472–484, 1989), but the kinetics of IIF were about five times faster in protoplasts. IIF observations in linear cooling of bovine oocytes indicated a median IIF temperature of −11 °C at 16 °C/min and total incidences of 97%, 50%, and 19% at 16, 8, and 4 °C/min, respectively. A stochastic model of IIF was developed which preserved certain features of an earlier model (Pitt et al. Cryobiology 28, 72–86, 1991), namely Weibull behavior in IIF temperatures during rapid linear cooling, but with a departure from the concept of a supercooling tolerance. Instead, the new model uses the osmotic state of the cell, represented by the extent of supercooling, as the independent variable governing the kinetics of IIF. Two kinetic parameters are needed for the model: a scale factor τ 0 dictating the sensitivity to supercooling, and an exponent ρ dictating the strength of time dependency. The model was fit to the data presented in this study as well as those from Myers et al. and Pitt et al. for D. melanogaster embryos with and without cryoprotectant, and from Toner et al. ( Cryobiology 28, 55–71, 1991) for mouse oocytes. In protoplasts, D. melanogaster embryos, and mouse oocytes, the parameters were estimated from IIF times in the early stages of isothermal periods, while the osmotic state of the cell was relatively constant. In bovine oocytes, the parameters were estimated from linear cooling data. Without further calibration, the model was used to predict total IIF incidence under different cooling regimes. For protoplasts, D. melanogaster embryos, and bovine oocytes, the model's predictions were quite accurate compared to the actual data. In mouse oocytes, adjustment of the hydraulic permeability coefficient ( L p) at 0 °C was required to yield realistic behavior. Thus, the two-parameter model for IIF was able to predict IIF incidence without the use of a supercooling tolerance, but precise knowledge of subfreezing volumetric behavior was required. An implication of the model is that the extent and duration of thermodynamic disequilibrium were the primary driving forces for ice propagation across cell membranes for all the systems studied, ρ varied from 2.4 to 2.8 for mouse oocytes and D. melanogaster embryos but was 3.8 for rye protoplasts, indicating that the strength of time dependency of IIF varied, for unknown reasons, among the different systems.

Subfreezing volumetric behavior and stochastic modeling of intracellular ice formation in Drosophila melanogaster embryos

Cryomicroscopic observations were made of the volumetric behavior and kinetics of intracellular ice formation (IIF) in Drosophila melanogaster embryos in a modified cell culture medium (BD.20) or BD.20 + 2 M ethylene glycol. After rapid cooling to a given temperature, transient volumetric contraction of the embryos during the isothermal period was quantified by computerized video image analysis. Fitting these data to the numerical solution of the volume flux equation yielded estimates of the hydraulic permeability coefficient ( L p) for individual embryos at various subfreezing temperatures. L p approximately followed an Arrhenius relation between −2 and −9 °C, with a value of 0.168 μm/(min-atm) extrapolated to 0 °C and an apparent activation energy Δ E of 38.9 kcal/mol. IIF during an isothermal period occurred at random times whose characteristic temperature range and kinetics were affected by the presence of ethylene glycol. A stochastic process model developed to fit these data indicated the influence of both time-dependent and instantaneous components of IIF, presumed to be the result of seeding and heterogeneous nucleation, respectively. The presence of 2 M ethylene glycol depressed the characteristic temperature of instantaneous IIF by about 12 °C and reduced the rate constant for time-dependent IIF. Comparison with observed incidences of IIF yielded an estimate of the supercooling tolerance of 3 to 5°C.