Contribution of extracellular ice formation and the solution effects to the freezing injury of PC-3 cells suspended in NaCl solutions

The mechanism of cell injury during slow freezing was examined using PC-3 human prostate adenocarcinoma cells suspended in NaCl solutions. The objective was to evaluate contribution of extracellular ice and the 'solution effects' to freezing injury separately. The solution effects that designate the influence of elevated concentration were evaluated from a pseudo-freezing experiment, where cells were subjected to the milieu that simulated a freeze-thaw process by changing the NaCl concentration and the temperature at the same time. The effect of extracellular ice formation on cell injury was then estimated from the difference in cell survival between the pseudo-freezing experiment and a corresponding freezing experiment. When cells were frozen to a relatively higher freezing temperature at -10 degrees C, about 30% of cells were damaged mostly due to extracellular ice formation, because the concentration increase without ice formation to 2.5-M NaCl, i.e., the equilibrium concentration at -10 degrees C, had no effect on cell survival. In contrast, in the case of the lower freezing temperature at -20 degrees C, about 90% of cells were injured by both effects, particularly 60-80% by the solution effects among them. The present results suggested that the solution effects become more crucial to cell damage during slow freezing at lower temperatures, while the effect of ice is limited to some extent.     

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 2M ethylene glycol (EG) in frog Ringers plus SnoMax. A multistep freezing protocol was used in which the samples were cooled until extracellular ice formation (EIF) occurred, partially remelted, slowly recooled through the EIF temperature, and then rapidly (10 degrees C/min) cooled. EIF in the 1, 1.5, and 2M EG occurred at -6.4, -7.8, and -8.9 degrees C, respectively. Freezing exotherms of individual stage I-VI oocytes were readily visible. A general trend was observed in which the IIF temperature of the early stage oocytes (I-III) was well below T(EIF) while the later stages (IV-VI) froze at temperatures much closer to T(EIF). Thus, in 1.5M EG, T(IIF) was -21.1, -25, and -26.6 degrees C in stages I-III, but was -17 and -8.5 degrees C for stage IV and V-VI. Concurrently, the percentage of oocytes in which IIF was observed fell dramatically from a high of 40 to 72% in early stages (I-III) to a low of only 7% in stage V-VI because, particularly in the later stages, IIF was hidden in the EIF exotherm. We conclude that early stage oocytes are a good model system in which to investigate modulators of IIF, but that late stage oocytes are damaged during EIF and infrequently supercool.     

High ice nucleation temperature of zebrafish embryos: slow-freezing is not an option

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

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.     

The application of convolution neural network based cell segmentation during cryopreservation

For most of the cells, water permeability and plasma membrane properties play a vital role in the optimal protocol for successful cryopreservation. Measuring the water permeability of cells during subzero temperature is essential. So far, there is no perfect segmentation technique to be used for the image processing task on subzero temperature accurately. The ice formation and variable background during freezing posed a significant challenge for most of the conventional segmentation algorithms. Thus, a robust and accurate segmentation approach that can accurately extract cells from extracellular ice that surrounding the cell boundary is needed. Therefore, we propose a convolutional neural network (CNN) architecture similar to U-Net but differs from those conventionally used in computer vision to extract all the cell boundaries as they shrank in the engulfing ice. The images used was obtained from the cryo-stage microscope, and the data was validated using the Hausdorff distance, means ± standard deviation for different methods of segmentation result using the CNN model. The experimental results prove that the typical CNN model extracts cell borders contour from the background in its subzero state more coherent and effective as compared to other traditional segmentation approaches.     

A microscale model for prediction of breast cancer cell damage during cryosurgery

The morphology of cancerous breast tissue is characterized by tightly packed groups of small malignant cells, as found in most duct cell carcinoma. This special structure affects the osmotic responses of the cells to freezing and hence their probability of damage from cellular dehydration or intracellular ice formation. A mathematical model has been developed to study the microscale damage to these breast cancer cells during cryosurgery by accounting for their special structure. The model is based on a spherical unit comprised of an extracellular region that surrounds several layers of cancer cells, as experimentally observed of breast duct cell carcinoma by other researchers. Temperature transients in the breast cancer undergoing cryosurgery are calculated numerically using the Pennes equation. When subjected to various thermal histories, both cellular dehydration and intracellular ice formation in the unit structure are examined by considering the cell-to-cell contact and water transport at the microscale level. It is found that the cells in the inner layers hardly dehydrated while those in the outermost layer do greatly. The results help interpret the previously observed experimental phenomena that breast cancer tissues exhibit intracellular ice formation even at a slow cooling rate of -3 degrees C/min. In the attempt to better define an optimal procedure for breast cancer cryosurgery, various freezing protocols are simulated. The constant heat flux protocol induces greater cellular dehydration and higher intracellular ice formation probability simultaneously compared to the other protocols studied.     

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. Four in vitro models 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.     

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.     

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.     

Investigating cryoinjury using simulations and experiments. 1: TF-1 cells during two-step freezing (rapid cooling interrupted with a hold time)

There is significant interest in designing a cryopreservation protocol for hematopoietic stem cells (HSC) which does not rely on dimethyl sulfoxide (Me₂SO) as a cryoprotectant. Computer simulations that describe cellular osmotic responses during cooling and warming can be used to optimize the viability of cryopreserved HSC; however, a better understanding of cellular osmotic parameters is required for these simulations. As a model for HSC, the erythroleukemic human cell line TF-1 was used in this study. Simulations, based on the osmotic properties of TF-1 cells and on the solution properties of the intra- and extracellular compartments, were used to interpret cryoinjury associated with a two-step cryopreservation protocol. Calculated intracellular supercooling was used as an indicator of cryoinjury related to intracellular ice formation. Simulations were applied to the two-step cooling protocol (rapid cooling interrupted with a hold time) for TF-1 cells in the absence of Me₂SO or other cryoprotectants and optimized by minimizing the indicator of cryoinjury. A comparison of simulations and experimental measurements of membrane integrity supports the concept that, for two-step cooling, increasing intracellular supercooling is the primary contributor to potential freezing injury due to the increase in the likelihood of intracellular ice formation. By calculating intracellular supercooling for each step separately and comparing these calculations with cell recovery data, it was demonstrated that it is not optimal simply to limit overall supercooling during two-step freezing procedures. More aptly, appropriate limitations of supercooling differ from the first step to the second step. This study also demonstrates why high cell recovery after cryopreservation could be achieved in the absence of traditional cryoprotectants.     

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 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.     

Equilibrium,quasi-equilibrium,and nonequilibrium freezing of mammalian embryos

The first successful freezing of early embryos to −196°C in 1972 required that they be cooled slowly at ∼1°C/min to about −70°C. Subsequent observations and physical/chemical analyses indicate that embryos cooled at that rate dehydrate sufficiently to maintain the chemical potential of their intracellular water close to that of the water in the partly frozen extracellular solution. Consequently, such slow freezing is referred to as equilibrium freezing. In 1972 and since, a number of investigators have studied the responses of embryos to departures from equilibrium freezing. When disequilibrium is achieved by the use of higher constant cooling rates to −70°C, the result is usually intracellular ice formation and embryo death. That result is quantitatively in accord with the predictions of the physical/chemical analysis of the kinetics of water loss as a function of cooling rate. However, other procedures involving rapid nonequilibrium cooling do not result in high mortality. One common element in these other nonequilibrium procedures is that, before the temperature has dropped to a level that permits intracellular ice formation, the embryo water content is reduced to the point at which the subsequent rapid nonequilibrium cooling results in either the formation of small innocuous intracellular ice crystals or the conversion of the intracellular solution into a glass. In both cases, high survival requires that subsequent warming be rapid, to prevent recrystallization or devitrification. The physical/ chemical analysis developed for initially nondehydrated cells appears generally applicable to these other nonequilibrium procedures as well.     

Cell shape and localisation of ice in leaves of overwintering wheat during frost stress in the field

Wheat leaf pieces were excised and freeze-fixed in the field, preparatory to low-temperature scanning electron microscopy to study distribution of ice within leaf blades, and associated cell shapes, during natural frosts. Pieces of leaf blades from wheat plants (Triticum aestivum L. 7942H1-20-8) overwintering in Indiana, USA (January, 1991), were excised and immediately freeze-fixed by manually plunging in melting freon. Cells in controls were turgid and extracellular ice was absent. The leaves of the frost-stressed plants froze at about — 2.4° C, and at that temperature extracellular ice was mainly located sub-epidermally, including in the substomatal cavity, and occupied about 14% of the fracture faces. The frequency of ice particles per unit leaf area in two specimens was 14 and 210 · mm^–2 (about 140 and 2100 · g^–1 leaf fresh-weight basis). At -9.0° C, ice filled the extracellular spaces, occupying 61% of the fracture faces. Cells were somewhat collapsed at -2.4° C and were much more collapsed at -9.0° C. The epidermal cells were more collapsed than the mesophyll cells. Tissue structure (connections with adjacent cells), wall flexibility, and ice growth may all have influenced the shapes of the collapsing cells. The experiments demonstrate the feasibility of freeze-fixation in the field. The sub-epidermal location of most ice indicates that in the field either (i) ice is nucleated sub-epidermally (implying both the presence of nucleators and the presence of liquid water in the sub-epidermal spaces) or (ii) ice is nucleated on the leaf surface, then propagates into the leaf probably through stomata.Abbreviations LTSEM low-temperature scanning electron microscopy     

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.     

Spatio-temporal tumour model for analysis and mechanism of action of intracellular drug accumulation

We have developed a one-dimensional tumour simulator to describe the biodistribution of chemotherapeutic drugs to a tumoral lesion and the tumour cell’s response to therapy. A three-compartment model is used for drug dynamics within the tumour. The first compartment represents the extracellular space in which cells move, the second corresponds to the intracellular fluid space (including cell membrane) which is in direct equilibrium with the extracellular space, and the third is a non-exchangeable compartment that represents sequestered drug which is trapped in the nucleus to damage the cellular DNA, directly triggering cell death. Analytical and numerical techniques (Finite Element Method) are used to describe the tumour’s response to therapy and the effect of parameter variation on the drug concentration profiles in the three compartments.     

Kinetics of rouleau formation. II. Reversible reactions.

Red blood cells aggregate face-to-face to form long, cylindrical, straight chains and sometimes branched structures called rouleaux. Here we extend a kinetic model developed by R. W. Samsel and A. S. Perelson (1982, Biophys. J. 37:493-514) to include both the formation and dissociation of rouleaux. We examine thermodynamic constraints on the rate constants of the model imposed by the principle of detailed balance. Incorporation of reverse reactions allows us to compute mean sizes of rouleaux and straight chain segments within rouleaux, as functions of time and at equilibrium. Using the Flory - Stockmayer method from polymer chemistry, we obtain a closed-form solution for the size distribution of straight chain segments within rouleaux at any point in the evolution of the reaction. The predictions of our theory compare favorably with data collected by D. Kernick , A.W.L. Jay , S. Rowlands , and L. Skibo (1973, Can. J. Physiol. Pharmacol. 51:690-699) on the kinetics of rouleau formation. When rouleaux grow large, they may contain rings or loops and take on the appearance of a network. We demonstrate the importance of including the kinetics of ring closure in the development of realistic models of rouleaux formation.     

The relevance of ice crystal formation for the cryopreservation of tissues and organs

This paper discusses the role of ice crystal formation in causing or contributing to the difficulties that have been encountered in attempts to develop effective methods for the cryopreservation of some tissues and all organs. It is shown that extracellular ice can be severely damaging but also that cells in situ in tissues can behave quite differently from similar cells in a suspension with respect to intracellular freezing. It is concluded that techniques that avoid the formation of ice altogether are most likely to yield effective methods for the cryopreservation of recalcitrant tissues and vascularised organs.     

Freezing injury: the special case of the sperm cell

The cellular damage that spermatozoa encounter at rapid rates of cooling has often been attributed to the formation of intracellular ice although no convincing evidence of intracellular ice formation has ever been obtained. We demonstrate that the high intracellular protein content together with the osmotic shrinkage associated with extracellular ice formation leads to intracellular vitrification of spermatozoa during cooling. At rapid rates of cooling the cell damage to spermatozoa is a result of an osmotic imbalance encountered during thawing, not intracellular ice formation. The osmotic imbalance occurs at rapid cooling rates due to a diffusion limited ice crystallisation in the extracellular fluid, i.e. the amount of ice forming during the cooling is less than expected from the equilibrium phase diagram. This explanation allows insights into other aspects of the cryobiology of spermatozoa and it is anticipated that this understanding will lead to specific improved methods of conventional cryopreservation for mammalian spermatozoa. It is also likely that this model will be relevant to the development of novel technologies for sperm preservation including vitrification and freeze drying.     

Intracellular freezing of glycerolized red cells.

The response of glycerolized human red blood cells to freezing has been evaluated in terms of the thermodynamic state of the frozen intracellular medium. The physiochemical conditions requisite for intracellular freezing, characterized by the cooling rate and the degree of extracellular supercooling, are altered appreciably by the prefreezing addition of glycerol to the cells.Fresh human erythrocytes were suspended in an isotonic glycerol solution yielding a final cryophylactic concentration of either 1.5 or 3.0 m. Subsequently the cell suspension was frozen on a special low temperature stage, mounted on a light microscope, at controlled constant cooling rates with varying degrees of extracellular supercooling (ΔT_sc). The formation of a pure intracellular ice phase was detected by direct observation of the cells.The addition of glycerol produced several significant variations in the freezing characteristics of the blood. As in unmodified cells, the incidence of intracellular freezing increased with the magnitudes of both the cooling rate and the extracellular supercooling. However, the glycerolized cells exhibited a much greater tendency to supercool prior to the initial nucleation of ice. Values of ΔT_sc > ?20 °C were readily obtained. Also, the transition from 0 to 100% occurrence of intracellular ice covered a cooling rate spectrum in excess of 300 to 600 °K/min, as compared with 10 °C/min for unmodified cells. Thus, the incidence of intracellular ice formation was significantly increased in glycerolized cells.