Incidence Of Intracellular Ice Formation Research Articles

Cryobiological parameters of multipotent stromal cells obtained from different sources

Stem cells are important for regenerative medicine mainly due to their multilineage differentiation capacity. However, the cells rapidly loose this capability during culturing. Cryopreservation preserves the differentiation potential of the cells, until they are needed. In this study, specific cell properties of multipotent stromal cells (MSCs), from the common marmoset monkey Callithrix jacchus MSCs derived from amnion (Am) and bone marrow (Bm) were studied in order to predict optimal cooling rates for cryopreservation. Cell volume behaviour in anisotonic media, hydraulic membrane permeability at supra as well as subzero temperatures, and time point of intracellular ice formation (IIF) were investigated by Coulter Counter and cryomicroscopy. Cryopreservation outcome was studied using the predicted and experimentally determined cooling rate followed by 24 h re-cultivation. Little differences in osmotically inactive volume were found between amnion (0.27 × Vo) and bone marrow (0.28 × Vo) derived MSCs. The activation energy for water transport at suprazero temperature was found to be similar for both cell types; 4.4 ± 0.2 and 5.0 ± 0.15 kcal mol−1 for amnion and bone marrow derived MSCs, respectively. At subzero temperatures in the absence of dimethyl sulfoxide (Me2SO), the activation energy for water transport increased to 24.8 ± 3 kcal mol−1 and 27.4 ± 0.9 kcal mol−1 for Am and BmMSCs respectively. In the presence of Me2SO, activation energies were found to be 11.6 ± 0.3 kcal mol−1 and 19.5 ± 0.5 kcal mol−1 respectively. Furthermore, Me2SO was found to decrease the incidence of intracellular ice formation. The predicted optimal cooling rates of 11.6 ± 0.9 °C/min (AmMSCs) and 16.3 ± 0.5 °C/min (BmMSCs) resulted in similar post-thaw viability values compared to the experimentally determined optimal cooling profiles of 7.5 °C/min to −30 °C, followed by 3 °C/min to −80 °C.

Effect of supercooling and cell volume on intracellular ice formation

Intracellular ice formation (IIF) has been linked to death of cells cryopreserved in suspension. It has been assumed that cells can be supercooled by 2 to 10°C before IIF occurs, but measurements of the degree of supercooling that cells can tolerate are often confounded by changing extracellular temperature and solutions of different osmolality (which affect the cell volume). The purpose of this study was to examine how the incidence of IIF in the absence of cryoprotectants is affected by the degree of supercooling and cell volume. Human umbilical vein endothelial cells were suspended in isotonic (300mOsm) and hypertonic (∼600 to 700mOsm) solutions and exposed to supercooling ranging from 2 to 10°C before extracellular ice was nucleated. The number of cells undergoing IIF was examined in a cryostage (based on the darkening of cells upon intracellular freezing (“flashing”)) as a function of the degree of supercooling, and cell survival post-thaw was assessed using a membrane integrity assay. We found that while the incidence of IIF increased with supercooling in both isotonic and hypertonic solutions, it was higher in the isotonic solution at any given degree of supercooling. Since cells in hypertonic solution were shrunken due to water efflux, we hypothesized that the difference in IIF behavior could be attributed to the decreased volume of cells in the hypertonic solution. Our results confirm that cells with a smaller diameter before extracellular ice nucleation have a decreased probability of IIF and suggest that cell volume could play a more significant role in the incidence of IIF than the extracellular ice nucleation temperature.

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

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

Freezing-induced cellular and membrane dehydration in the presence of cryoprotective agents

FTIR and cryomicroscopy have been used to study mouse embryonic fibroblast cells (3T3) during freezing in the absence and presence of DMSO and glycerol. The results show that cell volume changes as observed by cryomicroscopy typically end at temperatures above -15°C, whereas membrane phase changes may continue until temperatures as low as -30°C. This implies that cellular dehydration precedes dehydration of the bound water surrounding the phospholipid head groups. Both DMSO and glycerol increase the membrane hydraulic permeability at subzero temperature and reduce the activation energy for water transport. Cryoprotective agents facilitate dehydration to continue at low subzero temperatures thereby decreasing the incidence of intracellular ice formation. The increased subzero membrane hydraulic permeability likely plays an important role in the cryoprotective action of DMSO and glycerol. In the presence of DMSO water permeability was found to be greater compared to that in the presence of glycerol. Two temperature regimes were identified in an Arrhenius plot of the membrane hydraulic permeability. The activation energy for water transport at temperature ranging from 0 to -10°C was found to be greater than that below -10°C. The non-linear Arrhenius behavior of Lp has been implemented in the water transport model to simulate cell volume changes during freezing. At a cooling rate of 1°C min(-1), ∼5% of the initial osmotically active water volume is trapped inside the cells at -30°C.

Effects of freezing on membranes and proteins in LNCaP prostate tumor cells

Fourier transform infrared spectroscopy (FTIR) and cryomicroscopy were used to define the process of cellular injury during freezing in LNCaP prostate tumor cells, at the molecular level. Cell pellets were monitored during cooling at 2 °C/min while the ice nucleation temperature was varied between − 3 and − 10 °C. We show that the cells tend to dehydrate precipitously after nucleation unless intracellular ice formation occurs. The predicted incidence of intracellular ice formation rapidly increases at ice nucleation temperatures below − 4 °C and cell survival exhibits an optimum at a nucleation temperature of − 6 °C. The ice nucleation temperature was found to have a great effect on the membrane phase behavior of the cells. The onset of the liquid crystalline to gel phase transition coincided with the ice nucleation temperature. In addition, nucleation at − 3 °C resulted in a much more co-operative phase transition and a concomitantly lower residual conformational disorder of the membranes in the frozen state compared to samples that nucleated at − 10 °C. These observations were explained by the effect of the nucleation temperature on the extent of cellular dehydration and intracellular ice formation. Amide-III band analysis revealed that proteins are relatively stable during freezing and that heat-induced protein denaturation coincides with an abrupt decrease in α-helical structures and a concomitant increase in β-sheet structures starting at an onset temperature of approximately 48 °C.

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 °C). Samples were then cooled at 1 °C/min to the experimental temperature (−5 to −40 °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 °C, supporting the concept that intracellular ice confers cryoprotection by preventing cell dehydration during subsequent slow cooling.

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°C. At temperatures above −3°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°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.

Osmometric Behavior, Hydraulic Conductivity, and Incidence of Intracellular Ice Formation in Bovine Oocytes at Different Developmental Stages

Bovine oocytes that were immature (IMM), matured in vitro (IVM) or in vivo (MAT), or matured and fertilized in vitro (IVF) were studied using a microscope diffusion chamber to estimate osmotic parameters and a cryomicroscope to characterize intracellular ice formation (11F). Linear Boyle van't Hoff relationships were observed with all four types of oocytes between 0.265 and 0.799 osm NaCl. At 20°C, estimates of hydraulic conductivity ( L p ) were significantly higher for IVM oocytes than IMM and MAT oocytes (0.84 μm/(min · atm) vs 0.45 and 0.47, respectively). IVM oocytes also tended to have higher L p values than IVF oocytes (0.55 μm/(min · atm)). At 5°C, the L p of IVM oocytes decreased to 0.36 μm/min · atm) corresponding to an Arrhenius activation energy of 7.84 kcal/mol. The incidence of IIF in MAT oocytes suspended in salt solution and subjected to linear cooling to - 60°C was 45% at 4°C/min, 75% at 8°C/min, and 93% at 16°C/min; with IVF oocytes, the incidence of IIF was 40% at 4°C/min. 92% at 8°C/min, and 100% at 16°C/min. Comparisons involving median IIF temperatures (THF 50s) and the distributions of the observed IIF temperatures for IMM (Myers et al., Cryo-Lett. 8, 260), IVM (Chandrasekaran et al., Cryobiology 27, 676), MAT and IVF oocytes indicated that the IIF incidence in IMM oocytes cooled at 4°C/min was greater than that of oocytes at the other developmental stages cooled at the same rate. The THF 50s of IVM and IVF oocytes were lowered by equilibration in 1.5 M ethylene glycol (EG), glycerol, or propylene glycol (PG) prior to cooling, with EG tending to lower the THF 50s more than glycerol or PG. For all three cryoprotectants, the THF 50s and IFF temperature distributions were cooling-rate dependent. The Weibull probability distribution was fitted to the distributions of the IIF temperatures of oocytes suspended in salt solutions with and without cryoprotectants yielding R 2 values ranging from 0.70 to 0.98.

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

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

Characterization of intracellular ice formation in Drosophila melanogaster embryos

Cryomicroscopy and differential scanning calorimetry (DSC) were used to characterize the incidence of intracellular ice formation (IIF) in 12- to 13-hr-old embryos of Drosophila melanogaster (Oregon-R strain P2) as influenced by the state of the eggcase (untreated, dechorionated, or permeabilized), the composition of the suspending medium (with and without cryoprotectants), and the cooling rate. Untreated eggs underwent IIF over a very narrow temperature range when cooled at 4 or 16 °C/min with a median temperature of intracellular ice formation (TIIF 50) of −28 °C. The freezable water volume of untreated eggs was approximately 5.4 nl as determined by DSC. IIF in dechorionated eggs occurred over a much broader temperature range (−13 to −31 °C), but the incidence of IIF increased sharply below −24 °C, and the cumulative incidence of IIF at −24 °C decreased with cooling rate. In permeabilized eggs without cryoprotectants (CPAs), IIF occurred at much warmer temperatures and over a much wider temperature range than in untreated eggs, and the TIIF 50 was cooling rate dependent. At low cooling rates (1 to 2 °C/min), TIIF 50 increased with cooling rate; at intermediate cooling rates (2 to 16 °C/min), TIIF 50 decreased with cooling rate. The total incidence of IIP in permeabilized eggs was 54% at 1 °C/min, and volumetric contraction almost always occurred during cooling. Decreasing the cooling rate to 0.5 °C/min reduced the incidence of IIF to 43%. At a cooling rate of 4 °C/min, ethylene glycol reduced the TIIF 50 by about 12 °C for each unit increase in molarity of CPA (up to 2.0 M) in the suspending medium. The TIIF 50 was cooling rate dependent when embryos were preequilibrated with 1.0 M propylene glycol or ethylene glycol, but was not so in x1.0 M DMSO. For embryos equilibrated in 1.5 M ethylene glycol and then held at −5 °C for 1 min before further cooling at 1 °C/min, the incidence of IIF was decreased to 31%. Increasing the duration of the isothermal hold to 10 min reduced the incidence of IIF to 22% and reduced the volume of freezable water in embryos when intracellular ice formation occurred. If the isothermal hold temperature was −7.5 or −10 °C, a 10- to 30-min holding time was required to achieve a comparable reduction in the incidence of IIF.

Effect of Cold Acclimation on Intracellular Ice Formation in Isolated Protoplasts

When cooled at rapid rates to temperatures between -10 and -30 degrees C, the incidence of intracellular ice formation was less in protoplasts enzymically isolated from cold acclimated leaves of rye (Secale cereale L. cv Puma) than that observed in protoplasts isolated from nonacclimated leaves. The extent of supercooling of the intracellular solution at any given temperature increased in both nonacclimated and acclimated protoplasts as the rate of cooling increased. There was no unique relationship between the extent of supercooling and the incidence of intracellular ice formation in either nonacclimated or acclimated protoplasts. In both nonacclimated and acclimated protoplasts, the extent of intracellular supercooling was similar under conditions that resulted in the greatest difference in the incidence of intracellular ice formation-cooling to -15 or -20 degrees C at rates of 10 or 16 degrees C/minute. Further, the hydraulic conductivity determined during freeze-induced dehydration at -5 degrees C was similar for both nonacclimated and acclimated protoplasts. A major distinction between nonacclimated and acclimated protoplasts was the temperature at which nucleation occurred. In nonacclimated protoplasts, nucleation occurred over a relatively narrow temperature range with a median nucleation temperature of -15 degrees C, whereas in acclimated protoplasts, nucleation occurred over a broader temperature range with a median nucleation temperature of -42 degrees C. We conclude that the decreased incidence of intracellular ice formation in acclimated protoplasts is attributable to an increase in the stability of the plasma membrane which precludes nucleation of the supercooled intracellular solution and is not attributable to an increase in hydraulic conductivity of the plasma membrane which purportedly precludes supercooling of the intracellular solution.

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.

An experimental study of freezing in erythrocytes.

A unique experimental apparatus (termed cryomicroscope) has been developed for the study of freezing and thawing processes in living cells and has been used specifically to determine the conditions requisite for intracellular freezing in human erythrocytes. Careful measurements of the incidence of intracellular ice formation have been correlated directly with the magnitude of the cooling rate during the freezing process and degree of extracellular supercooling prior to nucleation of ice. The probability for intracellular freezing increases with the magnitudes of both the cooling rate and the extracellular supercooling. A 3-dimensional contour has been defined experimentally by which the frozen state of human erythrocytes can be predicted as a function of these two parameters.