Intracellular Ice Formation Kinetics Research Articles

C-10: High-speed video imaging of ice crystal formation in cells, tissues and droplets

Attempts to elucidate the causes of cryoinjury have benefited from nearly two centuries of microscopic observation of cell and tissue response during exposure to cold temperatures. However, the mechanisms of intracellular ice formation, a phenomenon that is strongly associated with irreversible cell damage, remains poorly understood: even in suspended, isolated cells, it has not been possible to obtain experimental evidence that conclusively supports or contradicts any of the competing hypotheses of intracellular ice formation (viz., the surface-catalyzed nucleation model, pore theory, or membrane rupture hypothesis). In part, this lack of progress has been due to the limited temporal resolution of conventional cryomicroscopy technology. Until recently, image acquisition rates in video cryomicroscopy studies have typically not exceeded 30 frames per second (fps), yielding a temporal resolution of 33 ms. Because the transformation of cell water to ice is completed in approximately 1 ms or less during rapid-cooling experiments, intracellular ice formation events cannot usually be detected using conventional video-micrography. As a result, cryomicroscopists have relied on the observation of cytoplasmic opacity changes as a proxy for intracellular freezing, an approach that can cause significant errors in estimates of intracellular ice formation kinetics. To detect the initial appearance and growth of ice crystals inside supercooled cells, and to quantify the phase transformation kinetics, it is necessary to use high-speed video cryomicroscopy to acquire micrographs at sub-millisecond resolution. In particular, to accurately measure intracellular ice formation kinetics in rapidly cooled somatic cells, image acquisition rates in the range 10 3 –10 4 fps are required, whereas shutter speeds should be faster than ∼0.1 ms to prevent motion blur. We have implemented such a high-speed imaging cryomicroscopy system, and have successfully used it to visualize non-equilibrium ice crystal formation and growth inside cells, tissue constructs, and microscale water droplets.

Measurement of intracellular ice formation kinetics by high-speed video cryomicroscopy.

Quantitative information about the kinetics and cumulative probability of intracellular ice formation is necessary to develop minimally damaging freezing procedures for the cryopreservation of cells and tissue. Conventional cryomicroscopic assays, which rely on indirect evidence of intracellular freezing (e.g., opacity changes in the cell cytoplasm), can yield significant errors in the estimated kinetics. In contrast, the formation and growth of intracellular ice crystals can be accurately detected using temporally resolved imaging methods (i.e., video recording at sub-millisecond resolution). Here, detailed methods for the setup and operation of a high-speed video cryomicroscope system are described, including protocols for imaging of intracellular ice crystallization events, and stochastic analysis of the ice formation kinetics in a cell population. Recommendations are provided for temperature profile design, sample preparation, and configuration of the video acquisition parameters. Throughout this chapter, the protocols incorporate best practices that have been drawn from over a decade of experience with high-speed video cryomicroscopy in our laboratory.

Effect of intercellular junction protein expression on water transport during freezing of MIN6 cells

A mouse insulinoma (MIN6) strain in which connexin expression has been inhibited by antisense technology holds promise as an experimental model system for investigating the role of gap junctions in intercellular ice propagation. However, to properly interpret measurements of intracellular ice formation kinetics, the effects of cell dehydration on cytoplasmic supercooling must be determined. Thus, the cell membrane water permeability in monolayer cultures of the antisense-transfected MIN6 strain was measured using a fluorescence quenching method. By repeating the experiments at 4°C, 12°C, 21°C, and 37°C, the activation energy for water transport was determined to be Ea=51±3kJ/mol. Although differences between membrane permeability measurements in theantisense and wild-type strains were not statistically significant, simulation of water transport during rapid freezing (130°C/min) predicted that intracellular supercooling in the genetically modified MIN6 strain may become significantly larger than the supercooling in wild-type cells at temperatures below −15°C.

86. An improved method for quantifying intracellular ice formation kinetics when multiple competing mechanisms are active

86. An improved method for quantifying intracellular ice formation kinetics when multiple competing mechanisms are active

Biotransport Phenomena in Freezing Mammalian Oocytes

Water transport across the cell plasma membrane and intracellular ice formation (IIF)-the two biophysical events that may cause cell injury during cryopreservation-were studied by cryomicroscopy and modeling using mammalian (Peromyscus) oocytes. Unusually high activation energy for water transport across the cell plasma membrane was identified indicating that the water transport process is unusually sensitive to temperature (and cooling rate). Although literally all studies on IIF were conducted using protocols with ice-seeding (seeding extracellular ice usually at≥-7°C), it is not used for cell cryopreservation by vitrification that is becoming increasingly popular today. In this article, we show that ice-seeding has a significant impact on IIF. With ice-seeding and cooling at 60°C/min, IIF was observed to occur over a wide range from approximately -8 to -48°C with a clear change of the ice nucleation mechanism (from surface- to volume-catalyzed nucleation) at approximately -43°C. On the contrary, without ice-seeding, IIF occurred over a much narrower range from approximately -19 to -27°C without a noticeable change of the nucleation mechanism. Moreover, the kinetics of IIF without ice-seeding was found to be strongly temperature (and cooling rate) dependent. These findings indicate the importance of quantifying the IIF kinetics in the absence of ice-seeding during cooling for development of optimal vitrification protocols of cell cryopreservation.

Thermal Injury Prediction During Cryoplasty Through In Vitro Characterization of Smooth Muscle Cell Biophysics and Viability

Restenosis in peripheral arteries is a major health care problem in the United States. Typically, 30-40% of angioplasties result in restenosis and hence alternative treatment techniques are being actively investigated. Cryoplasty, a novel technique involving simultaneous stretching and freezing of the peripheral arteries (e.g., femoral, iliac, popliteal) using a cryogen-filled balloon catheter, has shown the potential to combat restenosis. However, evaluation of the thermal and biophysical mechanisms that affect cellular survival during cryoplasty is lacking. To achieve this, the thermal history in arteries was predicted for different balloon temperatures using a thermal model. Cellular biophysical responses (water transport (WT) and intracellular ice formation (IIF)) were then characterized, using in vitro model systems, based on the thermal model predictions. The thermal and biophysical effects on cell survival were eventually determined. For this study, smooth muscle cells (SMC) isolated from porcine femoral arteries were used in suspensions and attached in vitro systems (monolayer and fibrin gel). Results showed that for different balloon temperatures, the thermal model predicted cooling rates from 2200 to 5 degrees C/min in the artery. Biophysical parameters (WT & IIF) were higher for SMCs in attached systems as compared to suspensions. The "combined" fit WT parameters for SMCs in suspension (at 5, 10, and 25 degrees C/min) are L (pg) = 0.12 microm/(min atm) and E (Lp) = 24.1 kcal/mol. Individual WT parameters for SMCs in attached cell systems at higher cooling rates are approximately an order of magnitude higher compared to suspensions (e.g., at 130 degrees C/min, WT parameters in monolayer and fibrin TE systems are L (pg) = 18.6, 19.4 microm/(min atm) and E (Lp) = 112, 127 kcal/mol, respectively). Similarly, IIF parameters assessed at 130 degrees C/min are higher for SMCs in attached systems than suspensions (Omega 0 = 1.1, 354, 378 (x 10(8) (1/m(2) s)) and kappa(o) = 1.6, 1.8, 2.1 (x 10(9) K(5)) for suspensions, monolayer, and fibrin TE, respectively). One possible reason for the differences in IIF kinetics was verified to be the presence of gap junctions, which facilitate cell-cell connections through which ice can propagate. This is reflected by the change in the predicted IIF parameters when a gap junction inhibitor was added and tested in monolayer (Omega 0 (1/m(2) s)); kappa(o) = 2.1 x 10(9) K(5)). SMC viability was affected by the model system (lower viability in attached systems), the thermal conditions and the biophysics. For e.g., IIF is lethal to cells and SMC viability was verified to be the least in fibrin TE (most % IIF) and the most in suspensions (least % IIF) at all cooling rates. Using the results from the fibrin TE (suggested as the best in vitro system to mimic a restenosis environment), conservative estimates of injury regimes in the artery during cryoplasty is predicted. The results can be used to suggest future optimizations and modifications during cryoplasty and also to design future in vivo studies.

29. Cell perimeter modulates the kinetics of intracellular ice formation and frequency of paracellular ice formation in micropatterned endothelial cells

29. Cell perimeter modulates the kinetics of intracellular ice formation and frequency of paracellular ice formation in micropatterned endothelial cells

Kinetics of Intracellular Ice Formation in One-Dimensional Arrays of Interacting Biological Cells

Although cell-cell interactions are known to significantly affect the kinetics of intracellular ice formation (IIF) during tissue freezing, this effect is not well understood. Progress in elucidating the mechanism and role of intercellular ice propagation in tissue freezing has been hampered in part by limitations in experimental design and data analysis. Thus, using rapid-cooling cryomicroscopy, IIF was measured in adherent cells cultured in micropatterned linear constructs (to control cell-cell interactions and minimize confounding factors). By fitting a Markov chain model to IIF data from micropatterned HepG2 cell pairs, the nondimensional rate of intercellular ice propagation was found to be α = 10.4 ± 0.1. Using this measurement, a new generator matrix was derived to predict the kinetics of IIF in linear four-cell constructs; cryomicroscopic measurements of IIF state probabilities in micropatterned four-cell arrays conformed with theoretical predictions ( p < 0.05), validating the modeling assumptions. Thus, the theoretical model was extended to allow prediction of IIF in larger tissues, using Monte Carlo techniques. Simulations were performed to investigate the effects of tissue size and ice propagation rate, for one-dimensional tissue constructs containing up to 100 cells and nondimensional propagation rates in the range 0.1 ≤ α ≤ 1000.

Intracellular Ice Formation Is Affected by Cell Interactions

Cell-to-cell and cell-to-surface interactions are important to the structure and function of tissues. These interactions are also important determinants of low-temperature responses in tissues. Fourin vitromodels using hamster fibroblast cells in tissue culture were used to investigate the influence of cell–cell and cell–surface interactions on intracellular ice formation in these systems. The four models were: (a) single cells in suspension; (b) cells individually attached to glass with only cell-to-surface adhesion; (c) colonies of cells attached to glass with both cell–cell and cell–surface interactions; and (d) multicellular spheroids with extensive cell–cell contacts. Cryomicroscopy was used to monitor the prevalence and kinetics of intracellular ice formation after ice nucleation in the extracellular solution. The temperature for intracellular freezing in 50% of the cells was significantly affected by both cell–cell and cell–surface interactions. There was also evidence of intercellular nucleation through cell–cell interactions. The results indicate that cell–cell and cell–surface interactions play a significant role in the low-temperature response of tissue systems.

Fertilization and development of mouse oocytes cryopreserved using a theoretically optimized protocol.

Rational design of a cryopreservation protocol was demonstrated by using theoretical models of the cryopreservation process to develop an optimal freezing protocol for mouse oocytes. A coupled mechanistic model of the processes of freeze-induced cell dehydration and intracellular ice formation was developed, and cryomicroscopical measurements of intracellular ice formation kinetics were used to determine biophysical parameters required by the model, and to test model predictions of the freezing behaviour of mouse oocytes. A simple phenomenological model for oocyte damage resulting from exposure to concentrated electrolyte and cryoprotectant solutions during cryopreservation was obtained by defining a cost function equal to the duration of the freezing protocol. A two-step freezing protocol was theoretically optimized by using a sequential simplex algorithm to minimize the cost function, subject to the constraint that the predicted probability of intracellular ice formation remain below 5%, yielding a putative optimum at the cooling rate B = 0.59 degrees C/min, and plunge temperature Tp = -67 degrees C. By systematically varying B and Tp about these values in experiments with mouse oocytes cryopreserved in 1.5 M dimethyl sulphoxide, the maximal recovery of intact oocytes with a normal morphology (82%) was obtained for B = 0.5 degrees C/min and Tp = -80 degrees C. Further evaluation of the fertilizability and developmental capacity of oocytes cryopreserved using the optimized protocol yielded cleavage to the 2-cell stage in 65% of oocytes inseminated, and blastocyst formation in 50% of these 2-cell embryos.

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.

Erratum: ‘‘Thermodynamics and kinetics of intracellular ice formation during freezing of biological cells’’ [J. Appl. Phys. 6 7, 1582 (1990)

First Page

Cryopreservation of isolated hepatocytes: Intracellular ice formation under various chemical and physical conditions

Kinetics of intracellular ice formation (IIF) for isolated rat hepatocytes was studied using a cryomicroscopy system. The effect of the cooling rate on IIF was investigated between 20 and 400 °C/min in isotonic solution. At 50 °C/min and below, none of the hepatocytes underwent IIF; whereas at 150 °C/min and above, IIF was observed throughout the entire hepatocyte population. The temperature at which 50% of hepatocytes showed IIF ( 50 T IIF) was almost constant with an average value of −7.7 °C. Different behavior was seen in isothermal subzero holding temperatures in the presence of extracellular ice. 50 T IIF from isothermal temperature experiments was ~ −5 °C as opposed to −7.7 °C for constant cooling rate experiments. These experiments clearly demonstrated both the time and temperature dependence of IIF. On the other hand, in cooling experiments in the absence of extracellular ice, IIF was not observed until ~ −20 °C (at which temperature the whole suspension was frozen spontaneously) suggesting the involvement of the external ice in the initiation of IIF. The effect of dimethyl sulfoxide (Me 2SO) on IIF was also quantified. 50 T IIF decreased from −7.7 °C in the absence of Me 2SO to −16.8 °C in 2.0 M Me 2SO for a cooling rate of 400 °C/min. However, the cooling rate (between 75 and 400 °C/min) did not significantly affect 50 T IIF (−8.7 °C) in 0.5 M Me 2SO. These results suggest that multistep protocols will be required for the cryopreservation of hepatocytes.

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.

Cryomicroscopic analysis of intracellular ice formation during freezing of mouse oocytes without cryoadditives

Kinetics of intracellular ice formation (IIF) under various freezing conditions was investigated for mouse oocytes at metaphase II obtained from B 6D 2F 1 mice. A new cryostage with improved optical performance and “isothermal” temperature field was used for nucleation experiments. The maximum thermal gradient across the window was less than 0.1 °C/10 mm at sample temperatures near 0 °C. The dependence of IIF on the initial concentration of the suspending medium was found to be pronounced. The mean IIF temperatures were found to be −9.56, −12.49, −17.63, −22.20 °C for freezing at 120 °C/min in 200, 285, 510, and 735 mosm phosphate-buffered saline, respectively. For concentrations higher than 735 mosm, the kinetics of IIF showed a break point at ~ −31 °C. Below −31 °C, all the remaining unfrozen oocytes underwent IIF almost immediately over a temperature range of less than 3 °C. This dramatic shift in the kinetics of IIF suggests that there were two distinct mechanisms responsible for IIF during freezing. The effect of the cooling rate on the kinetics of IIF was also investigated in isotonic PBS. At 1 °C/min none of the oocytes contained ice, whereas, at 5 °C/min all the oocytes contained ice. The mean IIF temperatures for cooling rates between 1 and 120 °C/min were almost constant with an average of −12.82 ± 0.6 °C (SEM). In addition, constant temperature experiments were conducted in isotonic PBS. The percentages of oocytes with IIF were 0, 50, 60, and 95% for −3.8, −6.4, −7.72, and −8.85 °C. In undercooling experiments, IIF was not observed until ~ −20 °C (at which temperature the whole suspension was frozen spontaneously), suggesting the involvement of the external ice in the initiation of IIF between $ ̃ −5 and −31 °C during freezing of oocytes.

Thermodynamics and kinetics of intracellular ice formation during freezing of biological cells

A physicochemical model based on the modified classical heterogeneous nucleation theory was proposed to analyze the ice formation inside biological cells during freezing. According to this model, intracellular ice formation (IIF) may be catalyzed either by the plasma membrane via the effects of the external ice on the plasma membrane, called surface-catalyzed nucleation (SCN), or by the intracellular particles, called volume-catalyzed nucleation (VCN), depending on the freezing conditions. The model for IIF was coupled with the model describing the kinetics of water transport to predict the thermodynamic state of the cytoplasm. A method to estimate the nucleation frequency from the observed probability of nucleation was suggested. This method was based on the assumption that each cell has the same heteronucleating particles with identical properties to alter the nucleation kinetics in an identical way. This allowed the correlation of the experimental probability of IIF with the nucleation rate. It was suggested that the uncertainties in the experimental conditions could be minimized if cells were cooled at rapid rates to prevent water transport during freezing. The model was applied to mouse oocytes at metaphase II. Correlation of the theory with the experimental kinetics of the cumulative fraction of IIF revealed preexponentional factors of 3.56×108 (m2 s)−1 and 1.84×1050 (m3 s)−1, and exponential factors of 4.6×109 and 1.08×1012 K5 for SCN and VCN, respectively. The contact angle between the internal surface of the plasma membrane and the ice cluster for SCN, θ, was determined to be 35° for freezing in isotonic solutions (285 mosm) increasing linearly to 47° in hypertonic solutions (1035 mosm). SCN was active between ∼−5 and −20 °C in the presence of external ice. It is suggested that the presence of external ice alters the plasma membrane through its chemical, electrical, mechanical, ionic, and thermal effects, to make it an energetically favorable site for heterogeneous nucleation. For VCN, lower and upper limits for θ and R were found to be 60°&amp;lt;θ&amp;lt;100° and 10−7&amp;lt;R&amp;lt;4×10−7 cm, respectively. VCN was active between ∼−30 and −35 °C.