Intracellular ice formation in insects: Unresolved after 50 years?

Many insects survive internal ice formation. The general model of freeze tolerance is of extracellular ice formation (EIF) whereby ice formation in the haemocoel leads to osmotic dehydration of the cells, whose contents remain unfrozen. However, survivable intracellular ice formation (IIF) has been reported in fat body and certain other cells of some insects. Although the cellular location of ice has been determined only in vitro, several lines of evidence suggest that IIF occurs in vivo. Both cell-to-cell propagation of intracellular ice and inoculation from the haemocoel may be important, although the route of ice into the cell is unclear. It is unclear why some cells survive IIF and others do not, but it is suggested that the shape, size, and low water content of fat body cells may predispose them towards surviving ice formation. We speculate that IIF may reduce water loss in some freeze tolerant species, but there are too few data to build a strong conceptual model of the advantages of IIF. We suggest that new developments in microscopy and other forms of imaging may allow investigation of the cellular location of ice in freeze tolerant insects in vivo.     

Intercellular ice propagation: experimental evidence for ice growth through membrane pores

Propagation of intracellular ice between cells significantly increases the prevalence of intracellular ice in confluent monolayers and tissues. It has been proposed that gap junctions facilitate ice propagation between cells. This study develops an equation for capillary freezing-point depression to determine the effect of temperature on the equilibrium radius of an ice crystal sufficiently small to grow through gap junctions. Convection cryomicroscopy and video image analysis were used to examine the incidence and pattern of intracellular ice formation (IIF) in the confluent monolayers of cell lines that do (MDCK) and do not (V-79W) form gap junctions. The effect of gap junctions on intracellular ice propagation was strongly temperature-dependent. For cells with gap junctions, IIF occurred in a directed wave-like pattern in 100% of the cells below -3 degrees C. At temperatures above -3 degrees C, there was a marked drop in the incidence of IIF, with isolated individual cells initially freezing randomly throughout the sample. This random pattern of IIF was also observed in the V-79W monolayers and in MDCK monolayers treated to prevent gap junction formation. The significant change in the low temperature behavior of confluent MDCK monolayers at -3 degrees C is likely the result of the inhibition of gap junction-facilitated ice propagation, and supports the theory that gap junctions facilitate ice nucleation between cells.     

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.     

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.     

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 alpha = 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 < or = alpha < or = 1000.     

Visualization of intracellular ice formation using high-speed video cryomicroscopy

A high-speed video cryomicroscopy system was developed, and used to observe the process of intracellular ice formation (IIF) during rapid freezing (130 °C/min) of bovine pulmonary artery endothelial cells adherent to glass substrates, or in suspension. Adherent cells were micropatterned, constraining cell attachment to reproducible circular or rectangular domains. Employing frame rates of 8000 frames/s and 16,000 frames/s to record IIF in micropatterned and suspended cells, respectively, intracellular crystal growth manifested as a single advancing front that initiated from a point source within the cell, and traveled at velocities of 0.0006-0.023 m/s. Whereas this primary crystallization process resulted in minimal change in cell opacity, the well-known flashing phenomenon (i.e., cell darkening) was shown to be a secondary event that does not occur until after the ice front has traversed the cell. In cells that were attached and spread on a substrate, IIF initiation sites were preferentially localized to the peripheral zone of the adherent cells. This non-uniformity in the spatial distribution of crystal centers contradicts predictions based on common theories of IIF, and provides evidence for a novel mechanism of IIF in adherent cells. A second IIF mechanism was evident in ∼20% of attached cells. In these cases, IIF was preceded by paracellular ice penetration; the initiation site of the subsequent IIF event was correlated with the location of the paracellular ice dendrite, indicating an association (and possibly a causal relationship) between the two. Together, the peripheral-zone and dendrite-associated initiation mechanisms accounted for 97% of IIF events in micropatterned cells.     

Intracellular ice formation and growth in MCF-7 cancer cells

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

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

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

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

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

An Improved Model for Nucleation-Limited Ice Formation in Living Cells during Freezing

Ice formation in living cells is a lethal event during freezing and its characterization is important to the development of optimal protocols for not only cryopreservation but also cryotherapy applications. Although the model for probability of ice formation (PIF) in cells developed by Toner et al. has been widely used to predict nucleation-limited intracellular ice formation (IIF), our data of freezing Hela cells suggest that this model could give misleading prediction of PIF when the maximum PIF in cells during freezing is less than 1 (PIF ranges from 0 to 1). We introduce a new model to overcome this problem by incorporating a critical cell volume to modify the Toner''s original model. We further reveal that this critical cell volume is dependent on the mechanisms of ice nucleation in cells during freezing, i.e., surface-catalyzed nucleation (SCN) and volume-catalyzed nucleation (VCN). Taken together, the improved PIF model may be valuable for better understanding of the mechanisms of ice nucleation in cells during freezing and more accurate prediction of PIF for cryopreservation and cryotherapy applications.     

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-microm) 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 (T(EIF)) and IIF (TI(IF)) was less than 3 degrees C and the average T(IIF) was approximately between -4 and -6 degrees C. Neither partial cellular dehydration nor addition of the cryopreservative dimethyl sulfoxide significantly depressed the T(IIF). Under some conditions, we observed ice nucleation at multiple locations within the cytoplasm, suggesting that several factors contribute to the unusually high T(IIF) during controlled-rate freezing and thus vitrification may be a more suitable method for cryopreserving these cells.     

Protective effect of intracellular ice during freezing?

Injury results during freezing when cells are exposed to increasing concentrations of solutes or by the formation of intracellular ice. Methods to protect cells from the damaging effects of freezing have focused on the addition of cryoprotective chemicals and the determination of optimal cooling rates. Based on other studies of innocuous intracellular ice formation, this study investigates the potential for this ice to protect cells from injury during subsequent slow cooling. V-79W Chinese hamster fibroblasts and Madin-Darby Canine Kidney (MDCK) cells were cultured as single attached cells or confluent monolayers. The incidence of intracellular ice formation (IIF) in the cultures at the start of cooling was pre-determined using one of two different extracellular ice nucleation temperatures (-5 or -10 degrees C). Samples were then cooled at 1 degrees C/min to the experimental temperature (-5 to -40 degrees C) where samples were warmed rapidly and cell survival assessed using membrane integrity and metabolic activity. For single attached cells, the lower ice nucleation temperature, corresponding to increased incidence of IIF, resulted in decreased post-thaw cell recovery. In contrast, confluent monolayers in which IIF has been shown to be innocuous, show higher survival after cooling to temperatures as low as -40 degrees C, supporting the concept that intracellular ice confers cryoprotection by preventing cell dehydration during subsequent slow cooling.     

Intracellular ice formation during the freezing of hepatocytes cultured in a double collagen gel.

During freezing, intracellular ice formation (IIF) has been correlated with loss in viability for a wide variety of biological systems. Hence, determination of IIF characteristics is essential in the development of an efficient methodology for cryopreservation. In this study, IIF characteristics of hepatocytes cultured in a collagen matrix were determined using cryomicroscopy. Four factors influenced the IIF behavior of the hepatocytes in the matrix: cooling rate, final cooling temperature, concentration of Me2SO, and time in culture prior to freezing. The maximum cumulative fraction of cells with IIF increased with increasing cooling rate. For cultured cells frozen in Dulbecco's modified Eagle's medium (DMEM), the cooling rate for which 50% of the cells formed ice (B50) was 70 degrees C/min for cells frozen after 1 day in culture and decreased to 15 degrees C/min for cells frozen after 7 days in culture. When cells were frozen in a 0.5 M Me2SO + DMEM solution, the value of B50 decreased from 70 to 50 degrees C/min for cells in culture for 1 day and from 15 to 10 degrees C/min for cells in culture for 7 days. The value of the average temperature for IIF (TIIF) for cultured cells was only slightly depressed by the addition of Me2SO when compared to the IIF behavior of other cell types. The results of this study indicate that the presence of the collagen matrix alters significantly the IIF characteristics of hepatocytes. Thus freezing studies using hepatocytes in suspension are not useful in predicting the freezing behavior of hepatocytes cultured in a collagen matrix. Furthermore, the weak effect of Me2SO on IIF characteristics implies that lower concentrations of Me2SO (0.5 M) may be just as effective in preserving viability. Finally, the value of B50 measured in this study indicates that cooling rates nearly an order of magnitude faster than those previously investigated could be used for cryopreservation of the hepatocytes in a collagen gel.     

Cell-cell contact affects membrane integrity after intracellular freezing

The response of cells to freezing depends critically on the presence of an intact cell membrane. During rapid cooling, the cell plasma membrane may no longer be an effective barrier to ice propagation and can be breached by extracellular ice resulting in the nucleation of the supercooled cytoplasm. In tissues, the formation of intracellular ice is compounded by the presence of cell-cell and cell-surface interactions. Three different hamster fibroblast model systems were used to simulate structures found in organized tissues. Samples were supercooled to an experimental temperature on a cryostage and ice nucleated at the constant temperature. A dual fluorescent staining technique was used for the quantitative assessment of the integrity of the cell plasma membrane. A novel technique using the fluorescent stain SYTO was used for the detection of intracellular ice formation (IIF) in cell monolayers. The cumulative incidence of cells with a loss of membrane integrity and the cumulative incidence of IIF were determined as a function of temperature. Cells in suspension and individual attached cells showed no significant difference in the number of cells that formed intracellular ice and those that lost membrane integrity. For cells in a monolayer, with cell-cell contact, intracellular ice formation did not result in the immediate disruption of the plasma membrane in the majority of cells. This introduces the potential for minimizing damage due to IIF and for developing strategies for the cryoprotection of tissues during rapid cooling.     

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.     

Experimental study of intracellular ice growth in human umbilical vein endothelial cells

Study of the intracellular ice formation (IIF) and growth is essential to the mechanistic understanding of cellular damage through freezing. In the aid of high speed and high resolution cryo-imaging technology, the transient intracellular ice formation and growth processes of the attached human umbilical vein endothelial cells (HUVEC) were successfully captured during freezing. It was found that the intracellular ice nucleation site was on the cell membrane closer to the nucleus. The ice growth was directional and toward the nucleus, which covered the whole nucleus before growing into the cytoplasm. The crystal growth rate in the nucleus was much larger than that in the cytoplasm, and its morphology was influenced by the cooling rate. During the thawing process, small crystals fused into larger ones inside the nucleus. Moreover, the cumulative fraction of the HUVEC with IIF was mainly dependent on the cooling rate not the confluence of the cells attached.     

Extra- and intracellular ice formation in mouse oocytes

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

Numerical Study of Cell Cryo-Preservation: A Network Model of Intracellular Ice Formation

In this study, a new intracellular ice formation network model, coupled with an improved cell dehydration model has been developed. The non-uniform dehydration of the cell during freezing is simulated with moving boundary condition. Internal cell structures like cell nucleus are taken into consideration. The IIF network model is developed from classic diffusion limited IIF model in order to simulate spatial ice growth pattern inside cells. Simulation results suggest that cell nuclear plays a significant role in cryo-dehydration and would affect water/CPA concentration gradient inside the cell. At the same time, the ice growth pattern of exogenous IIF hypothesis is examined in the model. It is consistent with our previous experiments, in which we witnessed the intracellular ice first grown into the nucleus before spreading to the whole intercellular space. According to this model, the water concentration difference between nucleus and cytoplasm during cryo-dehydration could partly explain why ice crystal in the nucleus grows faster. However, it is not the dominate factor. Higher diffusion coefficient in cell nucleus might play a more important role in the phenomenon.     

Survival of Pacific oyster, Crassostrea gigas, oocytes in relation to intracellular ice formation

The effect of IIF in Pacific oyster oocytes was studied using cryo and transmission electron microscopy (TEM). The viability of oocytes at each step of a published cryopreservation protocol was assessed in an initial experiment. Two major viability losses were identified; one when oocytes were cooled to −35 °C and the other when oocytes were plunged in liquid nitrogen. Although the cryomicroscope showed no evidence of IIF in oocytes cooled with this protocol, TEM revealed that these oocytes contained ice crystals and were at two developmental stages when frozen, prophase and metaphase I. To reduce IIF, the effect of seven cooling programmes involving cooling to −35 or −60 °C at 0.1 or 0.3 °C min⁻¹ and holding for 0 or 30 min at −35 or −60 °C was evaluated on post-thaw fertilization rate of oocytes. Regardless of the cooling rate or holding time, the fertilization rate of oocytes cooled to −60 °C was significantly lower than that of oocytes cooled to −35 °C. The overall results indicated that observations of IIF obtained from cryomicroscopy are limited to detection of larger amounts of ice within the cells. Although the amount of cellular ice may have been reduced by one of the programmes, fertilization was reduced significantly; suggesting that there is no correlation between the presence of intracellular ice and post-thaw fertilization rate. Therefore, oyster oocytes may be more susceptible to the effect of high solute concentrations and cell shrinkage than intracellular ice under the studied conditions.     

Kinetics and mechanism of intercellular ice propagation in a micropatterned tissue construct

Understanding the effects of cell-cell interaction on intracellular ice formation (IIF) is required to design optimized protocols for cryopreservation of tissue. To determine the effects of cell-cell interactions during tissue freezing, without confounding effects from uncontrolled factors (such as time in culture, cell geometry, and cell-substrate interactions), HepG2 cells were cultured in pairs on glass coverslips micropatterned with polyethylene glycol disilane, such that each cell interacted with exactly one adjacent cell. Assuming the cell pair to be a finite state system, being either in an unfrozen state (no ice in either cell), a singlet state (IIF in one cell only), or a doublet state (IIF in both cells), the kinetics of state transitions were theoretically modeled and cryomicroscopically measured. The rate of intercellular ice propagation, estimated from the measured singlet state probability, increased in the first 24 h of culture and remained steady thereafter. In cell pairs cultured for 24 h and treated with the gap junction blocker 18beta-glycyrrhetinic acid before freezing, the intercellular ice propagation rate was lower than in untreated controls (p < 0.001), but significantly greater than zero (p < 0.0001). These results suggest that gap junctions mediate some, but not all, mechanisms of ice propagation in tissue.