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.     

Nucleation and growth of ice crystals inside cultured hepatocytes during freezing in the presence of dimethyl sulfoxide.

A three-part, coupled model of cell dehydration, nucleation, and crystal growth was used to study intracellular ice formation (IIF) in cultured hepatocytes frozen in the presence of dimethyl sulfoxide (DMSO). Heterogeneous nucleation temperatures were predicted as a function of DMSO concentration and were in good agreement with experimental data. Simulated freezing protocols correctly predicted and explained experimentally observed effects of cooling rate, warming rate, and storage temperature on hepatocyte function. For cells cooled to -40 degrees C, no IIF occurred for cooling rates less than 10 degrees C/min. IIF did occur at faster cooling rates, and the predicted volume of intracellular ice increased with increasing cooling rate. Cells cooled at 5 degrees C/min to -80 degrees C were shown to undergo nucleation at -46.8 degrees C, with the consequence that storage temperatures above this value resulted in high viability independent of warming rate, whereas colder storage temperatures resulted in cell injury for slow warming rates. Cell damage correlated positively with predicted intracellular ice volume, and an upper limit for the critical ice content was estimated to be 3.7% of the isotonic water content. The power of the model was limited by difficulties in estimating the cytosol viscosity and membrane permeability as functions of DMSO concentration at low temperatures.     

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.     

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 degrees C/min in isotonic solution. At 50 degrees C/min and below, none of the hepatocytes underwent IIF; whereas at 150 degrees C/min and above, IIF was observed throughout the entire hepatocyte population. The temperature at which 50% of hepatocytes showed IIF (50TIIF) was almost constant with an average value of -7.7 degrees C. Different behavior was seen in isothermal subzero holding temperatures in the presence of extracellular ice. 50TIIF from isothermal temperature experiments was approximately -5 degrees C as opposed to -7.7 degrees 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 approximately -20 degrees 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 (Me2SO) on IIF was also quantified. 50TIIF decreased from -7.7 degrees C in the absence of Me2SO to -16.8 degrees C in 2.0 M Me2SO for a cooling rate of 400 degrees C/min. However, the cooling rate (between 75 and 400 degrees C/min) did not significantly affect 50TIIF (-8.7 degrees C) in 0.5 M Me2SO. These results suggest that multistep protocols will be required for the cryopreservation of hepatocytes.     

Cryobiophysical characteristics of genetically modified hematopoietic progenitor cells

The freezing responses of hematopoietic progenitor cells isolated from normal donors and from donors with mucopolysaccharidosis type I (MPS I) were determined using cryomicroscopy and analyzed using theoretical models for water transport and intracellular ice formation. The cells from donors with MPS I used in this investigation were cultured and transduced with a retroviral vector for the alpha-l-iduronidase (IDUA) enzyme in preclinical studies for human gene therapy. The water transport and intracellular ice formation (IIF) characteristics were determined at different time points in the culture and transduction process for hematopoietic progenitor cells expressing CD34 antigen from donors with MPS I and from normal donors. There were statistically significant changes in water transport, osmotically inactive cell volume fraction, and permeability between cells from different sources (normal donors vs donors with MPSI) and different culture conditions (freshly isolated vs cultured and transduced). Specifically, Lpg and Ea increased after ex vivo culture of the cells and the changes in permeability parameters were observed after as little as 3 days in culture. Similarly, the IIF characteristics of hematopoietic progenitor cells can also be influenced by the culture and transduction process. The IIF characteristics of freshly isolated cells from donors with MPS I were statistically distinct from those of cultured and transduced cells from the same donor. The ability to cryopreserve cells which are cultured ex vivo for therapeutic purposes will require an understanding of the biophysical changes resulting from the culture conditions and the manner in which these changes influence viability.     

Gene transfection of multicellular spheroid of hepatocytes on an artificial substrate

The handling of hepatocytes, a major cell population in the liver, is an important technique in both liver tissue engineering and hepatology. However, these cells are so fragile that it has been impossible to harvest hepatocytes with high viability from tissue culture dishes after a period of culture in vitro. In this study, we employed an artificial substrate for transfection of multilayer hepatocytes and harvested these cells with high viability after transfection. Hepatocytes cultured on an amphiphilic artificial substrate form multilayer aggregates (spheroids) in the presence of growth factors during gene transfection with cation liposomes. Compared to cells cultured on a collagen-coated plate, these spheroids are easily harvested with high viability by pipetting in EDTA solution. In addition, these spheroids rapidly spread on collagen after transfer from the artificial substrate, demonstrating that hepatocytes in the center of the spheroids were viable. Epidermal growth factor (EGF) increased the transfection efficiency into hepatocytes while hepatocyte growth factor (HGF) alone did not increase the efficiency. However, HGF synergestically increased the effect of EGF on transfection. Interestingly, this transfection required the process of spheroid formation because the gene was not transfected once the spheroid formation completed or under conditions where hepatocytes did not form spheroids. This method using spheroidal hepatocytes for in vitro transfection is promising for the development of ex vivo gene therapy.     

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.     

Water transport and IIF parameters for a connective tissue equivalent

Understanding the biophysical processes that govern freezing injury of a tissue equivalent (TE) is an important step in characterizing and improving the cryopreservation of these systems. TEs were formed by entrapping human dermal fibroblasts (HDFs) in collagen or in fibrin gels. Freezing studies were conducted using a Linkam cryostage fitted to an optical microscope allowing observation of the TEs cooled under controlled rates between 5 and 130 degrees C/min. Typically, freezing of cellular systems results in two biophysical processes that are both dependent on the cooling rate: dehydration and/or intracellular ice formation (IIF). Both these processes can potentially be destructive to cells. In this study, the biophysics of freezing cells in collagen and fibrin TEs have been quantified and compared to freezing cells in suspension. Experimental data were fitted in numerical models to extract parameters that governed water permeability, E(Lp) and L(pg), and intracellular ice nucleation, omega(o) and kappa(o). Results indicate that major differences exist between freezing HDFs in suspension and in a tissue equivalent. During freezing, 55% of the HDFs in suspension formed IIF as compared to 100% of HDFs forming IIF in collagen and fibrin TE at a cooling rate of 130 degrees C/min. Also, both the water permeability and the IIF parameters were determined to be higher for HDFs in TEs as compared to cell suspensions. Between the TEs, HDFs in fibrin TE exhibited higher values for the biophysical parameters as compared to HDFs in collagen TE. The observed biophysics seems to indicate that cell-cell and cell-matrix interactions play a major role in ice propagation in TEs.     

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.     

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.     

Effects of medium composition on the morphology and function of rat hepatocytes cultured as spheroids and monolayers

Primary hepatocytes cultured as monolayers or as spheroids were studied to compare the effects of four different culture media (Williams' E, Chee's, Sigma Hepatocyte, and HepatoZYME medium). Rat hepatocytes were cultured as conventional monolayers for 3 d or as spheroids for 2 wk. For spheroid formation a method was emplOyed that combined the use of a nonadherent substratum with rotation of cultures. Hepatocyte integrity and morphology were assessed by light and electron microscopy and by reduced glutathione content. Hepatocyte function was measured by albumin secretion and 7-ethoxycoumarin metabolism. Chee's medium was found to be optimal for maintenance of hepatocyte viability and function in monolayers, but it failed to support spheroid formation. For spheroid formation and for the maintenance of spheroid morphology and function, Sigma HM was found to be optimal. These results demonstrate that the medium requirements of hepatocytes differ markedly depending on the culture model employed. Spheroid culture allowed better preservation of morphology and function of hepatocytes compared with conventional monolayer culture. Hepatocytes in spheroids formed bile canaliculi. and expressed an actin distribution resembling that found in hepatocytes in vivo. Albumin secretion was maintained at the same level as that found during the first d in primary culture, and 7-ethoxycoumarin metabolism was maintained over 2 wk in culture at approximately 30% of the levels found in freshly isolated hepatocytes. The improved morphology and function of hepatocyte cultures as spheroids may provide a more appropriate in vitro model for certain applications where the maintenance of liver-specific functions in long-term culture is crucial.     

Subzero water permeability parameters of mouse spermatozoa in the presence of extracellular ice and cryoprotective agents.

Optimization of techniques for cryopreservation of mammalian sperm is limited by a lack of knowledge regarding water permeability characteristics during freezing in the presence of extracellular ice and cryoprotective agents (CPAs). Cryomicroscopy cannot be used to measure dehydration during freezing in mammalian sperm because they are highly nonspherical and their small dimensions are at the limits of light microscopic resolution. Using a new shape-independent differential scanning calorimeter (DSC) technique, volumetric shrinkage during freezing of ICR mouse epididymal sperm cell suspensions was obtained at cooling rates of 5 and 20 degrees C/min in the presence of extracellular ice and CPAs. Using previously published data, the mouse sperm cell was modeled as a cylinder (122-microm long, radius 0.46 microm) with an osmotically inactive cell volume (V(b)) of 0.61V(o), where V(o) is the isotonic cell volume. By fitting a model of water transport to the experimentally obtained volumetric shrinkage data, the best-fit membrane permeability parameters (L(pg) and E(Lp)) were determined. The "combined best-fit" membrane permeability parameters at 5 and 20 degrees C/min for mouse sperm cells in solution are as follows: in D-PBS: L(pg) = 1.7 x 10(-15) m(3)/Ns (0.01 microm/min-atm) and E(Lp) = 94.1 kJ/mole (22.5 kcal/mole) (R(2) = 0.94); in "low" CPA media (consisting of 1% glycerol, 6% raffinose, and 15% egg yolk in D-PBS): L(pg)[cpa] = 1.7 x 10(-15) m(3)/Ns (0.01 microm/min-atm) and E(Lp)[cpa] = 122.2 kJ/mole (29.2 kcal/mole) (R(2) = 0.98); and in "high" CPA media (consisting of 4% glycerol, 16% raffinose, and 15% egg yolk in D-PBS): L(pg)[cpa] = 0.68 x 10(-15) m(3)/Ns (0.004 microm/min-atm) and E(Lp)[cpa] = 63.6 kJ/mole (15.2 kcal/mole) (R(2) = 0.99). These parameters are significantly different than previously published parameters for mammalian sperm obtained at suprazero temperatures and at subzero temperatures in the absence of extracellular ice. The parameters obtained in this study also suggest that damaging intracellular ice formation (IIF) could occur in mouse sperm cells at cooling rates as low as 25-45 degrees C/min, depending on the concentrations of the CPAs. This may help to explain the discrepancy between the empirically determined optimal cryopreservation cooling rates, 10-40 degrees C/min, and the numerically predicted optimal cooling rates, greater than 5000 degrees C/min, obtained using suprazero mouse sperm permeability parameters that do not account for the presence of extracellular ice. As an independent test of this prediction, the percentages of viable and motile sperm cells were obtained after freezing at two different cooling rates ("slow" or 5 degrees C/min; "fast," or 20 degrees C/min) in both the low and high CPA media. The greatest sperm motility and viability was found with the low CPA media under fast (20 degrees C/min) cooling conditions.     

Differences in the uptake of transferrin bound 239Pu and 59Fe into multicellular spheroids of hepatocytes from adult male rats

Hepatocytes were cultured as monolayers and multicellular spheroids, respectively. The uptake of both transferrin-bound metals, iron and plutonium, differed significantly between these two culture systems. The uptake into the multicellular spheroids for plutonium was about 30 times greater, and for iron about 4 times greater, than in monolayer-cultured hepatocytes, which is not a consequence of proliferation and/or de-differentiation of the hepatocytes in the multicellular spheroid culture system. A comparison of the iron and plutonium uptake showed that plutonium was delivered to the cells to an 8-fold greater extent than iron if the hepatocytes were cultured as spheroids. Additionally, the binding of plutonium was not inhibited by preincubation of the spheroids with the iron-transferrin complex. Therefore, we propose that there are two different binding sites for iron and plutonium on hepatocyte membranes.     

Encapsulation of rat hepatocyte spheroids for the development of artificial liver

Hepatocyte spheroids and hepatocyte were immobilized in chitosan/alginate capsules formed by the electrostatic interactions between chitosan and alginate. After encapsulation, there was a 10% decrease in the viability of spheroids due to the exposure of the cells to a pH 6 during the encapsulation process. However, the encapsulated hepatocyte spheroids maintained over 50% viability and liver specific functions for 2 weeks while the encapsulated hepatocytes, free hepatocytes and free hepatocyte spheroids showed low viability and liver specific functions. Therefore, encapsulated hepatocyte spheroid might be applied to the development of a bioartificial liver.     

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.     

Cryoprotectant-dependent control of intracellular ice recrystallization in hepatocytes using small molecule carbohydrate derivatives

To promote the recovery of cells that undergo intracellular ice formation (IIF), it is imperative that the recrystallization of intracellular ice is minimized. Hepatocytes are more prone to IIF than most mammalian cells, and thus we assessed the ability of novel small molecule carbohydrate-based ice recrystallization inhibitors (IRIs) to permeate and function within hepatocytes. HepG2 monolayers were treated with N-(4-chlorophenyl)-d-gluconamide (IRI 1), N-(2-fluorophenyl)-d-gluconamide (IRI 2), or para-methoxyphenyl-β-D-glycoside (IRI 3) and fluorescent cryomicroscopy was used for real time visualization of intracellular ice recrystallization. Both IRI 2 and IRI 3 reduced rates of intracellular recrystallization, whereas IRI 1 did not. IRI 2 and IRI 3, however, demonstrated a marked reduction in efficiency in the presence of the most frequently used permeating cryoprotectants (CPAs): glycerol, propylene glycol (PG), dimethyl sulfoxide (DMSO), and ethylene glycol (EG). Nevertheless, IRI 3 reduced rates of intracellular recrystallization relative to CPA-only controls in the presence of glycerol, PG, and DMSO. Interestingly, IRI preparation in trehalose, a commonly used non-permeating CPA, did not impact the activity of IRI 3. However, trehalose did increase the activity of IRI 1 while decreasing that of IRI 2. While this study suggests that each of these compounds could prove relevant in hepatocyte cryopreservation protocols where IIF would be prominent, CPA-mediated modulation of intracellular IRI activity is apparent and warrants further investigation.     

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.     

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.     

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.