Mechanisms of intracellular ice formation.

The phenomenon of intracellular freezing in cells was investigated by designing experiments with cultured mouse fibroblasts on a cryomicroscope to critically assess the current hypotheses describing the genesis of intracellular ice: (a) intracellular freezing is a result of critical undercooling; (b) the cytoplasm is nucleated through aqueous pores in the plasma membrane; and (c) intracellular freezing is a result of membrane damage caused by electrical transients at the ice interface. The experimental data did not support any of these theories, but was consistent with the hypothesis that the plasma membrane is damaged at a critical gradient in osmotic pressure across the membrane, and intracellular freezing occurs as a result of this damage. An implication of this hypothesis is that mathematical models can be used to design protocols to avoid damaging gradients in osmotic pressure, allowing new approaches to the preservation of cells, tissues, and organs by rapid cooling.     

Mechanism of initiation of intracellular ice formation

Intracellular ice crystallization was studied by the method of cryomicroscopy in the systems modeling a biological suspension, such as erythrocyte concentrates. Initiation of crystallization by extracellular ice through hydrophilic channels has been shown to be the most probable mechanism of intracellular ice formation.     

The formation of ice in plant tissues

Summary The formation of ice in the petioles ofSolanum acaule andSolanum tuberosum has been studied by light microscopy and by continuous recordings of latent heat production. Frost hardy material ofS. acaule, when slightly turgor deficient, freezes in two distinct stages. The first short freezing is due to the crystallisation of liquid in the vascular tissues and adjacent intercellular spaces. The second major phase shows the gradual formation of layers of ice in the sub-hypodermal regions of the material after the withdrawal of fluid from the cells of the interior tissues. InS. tuberosum, a frost susceptible plant, the two stages of freezing are less well defined, and are confluent; during the second stage of freezing the ice is laid down at scattered loci through the tissues of the petiole. The behaviour of the petiole ofBrassica oleracea was similar to that ofS. acaule, whilstBegonia rex, an extremely frost sensitive plant, froze similarly toS. tuberosum, but without any indication of a normal double cooling curve.It is proposed that during the freezing process of hardy plants an extensive thaw, initiated by a release of solutes from the protoplasts, occurs. The thaw, which may take place in two stages, is to be seen during the second stage of freezing when petioles ofS. acaule are frozen. The release of solutes and a lowering of turgor pressure within the cells may account for the subsequent formation of the extensive layers of ice formed in the outer regions of hardy plants. The release would be favoured by the high permeabilities of resistant cells. In frost susceptible plants the extent of thawing appears to be too small to permit a wholesale movement of fluid to the outer regions of the tissues and, consequently, ice forms at random points within the material and probably causes a greater degree of mechanical injury than is found in hardy tissues.

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Zusammenfassung Die Eisbildung im Blattstiel vonSolanum acaule undS. tuberosum-Pflanzen ist bei laufender lichtmikroskopischer Beobachtung und dauerndem Messen der Kristallisationswärme untersucht worden. Frostresistente Gewebe, die schwach ausgetrocknet sind, frieren in zwei deutlichen Phasen. Die erste kurze Gefrierphase beschreibt die Erstarrung der Flüssigkeit in den Gefäßen und Intercellularräumen. In der zweiten Phase tritt als Folge einer Flüssigkeitsbewegung von den inneren Zellen her eine allmähliche Eisbildung unter dem Hypoderm des Blattstiels ein. Im frostempfindlichenS. tuberosum fließen die zwei Phasen des Gefrierens zusammen: Eisbildung findet in vereinzelten Stellen der Blattstielgewebe statt. Das Verhalten des Blattstiels vonBrassica oleracea war ähnlich dem vonS. acaule. Begonia rex, eine sehr empfindliche Pflanze, verhält sich gleichartig wieS. tuberosum, aber ohne jeglichen Hinweis auf eine normale doppelt gekniete Gefrierkurve.Wahrscheinlich findet in frostresistenten Pflanzen in der zweiten Gefrierphase ein zeitweiliges Tauen statt als Folge einer plötzlichen Abgabe gelöster Stoffe durch das Protoplasma. Dieses Tauen, das vermutlich in zwei Schritten vor sich geht, kann während der zweiten Gefrierphase in vielen Abkühlungskurven unter gleichzeitiger mikroskopischer Untersuchung besonders beiS. acaule beobachtet werden. Die Abgabe von gelösten Stoffen und die dadurch bewirkte Senkung des Turgors in den Zellen trägt zur Erklärung der Bildung von ausgedehnten Eisschichten bei, die in frostharten Pflanzen gefunden werden. Die hohe Durchlässigkeit der frostresistenten Zellen würde solch eine Entbindung begünstigen. In frostempfindlichen Pflanzen, wo Eisbildung an vereinzelten Stellen der Gewebe stattfindet, scheint der Grad des Auftauens zu gering, um eine völlige Bewegung der Flüssigkeit zum äußeren Teil der Gewebe zu erlauben; daher ist die mechanische Schädigung größer als in frostresistenten Pflanzen.

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With 6 Figures in the Text     

Biofilm formation in an ice cream plant

The sites of biofilm formation in an ice cream plant were investigated by sampling both the production line and the environment. Experiments were carried out twice within a 20-day period. First, stainless steel coupons were fixed to surfaces adjacent to food contact surfaces, the floor drains and the doormat. They were taken for the analysis of biofilm at three different production stages. Then, biofilm forming bacteria were␣enumerated and also presence of Listeria monocytogenes was monitored. Biofilm forming isolates were selected on the basis of colony morphology and Gram’s reaction; Gram negative cocci and rod, Gram positive cocci and spore forming isolates were identified. Most of the biofilm formations were seen on the conveyor belt of a packaging machine 8 h after the beginning of the production, 6.5 × 10³ cfu cm⁻². Most of the Gram negative bacteria identified belong to Enterobacteriaceae family such as Proteus, Enterobacter, Citrobacter, Shigella, Escherichia, Edwardsiella. The other Gram negative microflora included Aeromonas, Plesiomonas, Moraxella, Pseudomonas or Alcaligenes spp. were also isolated. Gram positive microflora of the ice cream plant included Staphyloccus, Bacillus, Listeria and lactic acid bacteria such as Streptococcus, Leuconostoc or Pediococcus spp. The results from this study highlighted the problems of spread of pathogens like Listeria and Shigella and spoilage bacteria. In the development of cleaning and disinfection procedures in ice cream plants, an awareness of these biofilm-forming bacteria is essential for the ice cream plants.     

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.     

Diatom assemblages promote ice formation in large lakes

We present evidence for the directed formation of ice by planktonic communities dominated by filamentous diatoms sampled from the ice-covered Laurentian Great Lakes. We hypothesize that ice formation promotes attachment of these non-motile phytoplankton to overlying ice, thereby maintaining a favorable position for the diatoms in the photic zone. However, it is unclear whether the diatoms themselves are responsible for ice nucleation. Scanning electron microscopy revealed associations of bacterial epiphytes with the dominant diatoms of the phytoplankton assemblage, and bacteria isolated from the phytoplankton showed elevated temperatures of crystallization (T_c) as high as −3 °C. Ice nucleation-active bacteria were identified as belonging to the genus Pseudomonas, but we could not demonstrate that they were sufficiently abundant to incite the observed freezing. Regardless of the source of ice nucleation activity, the resulting production of frazil ice may provide a means for the diatoms to be recruited to the overlying lake ice, thereby increasing their fitness. Bacterial epiphytes are likewise expected to benefit from their association with the diatoms as recipients of organic carbon excreted by their hosts. This novel mechanism illuminates a previously undescribed stage of the life cycle of the meroplanktonic diatoms that bloom in Lake Erie and other Great Lakes during winter and offers a model relevant to aquatic ecosystems having seasonal ice cover around the world.     

Influence of lipids on ice formation in cryoprotective media

Cryomicroscopic analysis demonstrated that two lipid preparations from marine vertebrates (<0.1%) and liposomes prepared from rainbow trout sperm lipids (<0.5%) efficiently hindered the growth of ice crystals during freezing of multicomponent cryoprotective media used for trout sperm cryopreservation. At higher lipid concentrations, crystals either did not form at all or had altered shape and blurred boundaries. Addition of egg yolk (10%) together with these lipids increased the size of crystal structures and markedly changed their shape.     

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 degrees C/min with a median temperature of intracellular ice formation (TIIF50) of -28 degrees 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 degrees C), but the incidence of IIF increased sharply below -24 degrees C, and the cumulative incidence of IIF at -24 degrees 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 TIIF50 was cooling rate dependent. At low cooling rates (1 to 2 degrees C/min), TIIF50 increased with cooling rate; at intermediate cooling rates (2 to 16 degrees C/min), TIIF50 decreased with cooling rate. The total incidence of IIF in permeabilized eggs was 54% at 1 degree C/min, and volumetric contraction almost always occurred during cooling. Decreasing the cooling rate to 0.5 degree C/min reduced the incidence of IIF to 43%. At a cooling rate of 4 degrees C/min, ethylene glycol reduced the TIIF50 by about 12 degrees C for each unit increase in molarity of CPA (up to 2.0 M) in the suspending medium. The TIIF50 was cooling rate dependent when embryos were preequilibrated with 1.0 M propylene glycol or ethylene glycol, but was not so in 1.0 M DMSO. For embryos equilibrated in 1.5 M ethylene glycol and then held at -5 degrees C for 1 min before further cooling at 1 degree 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 degrees C, a 10- to 30-min holding time was required to achieve a comparable reduction in the incidence of IIF.     

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.     

Quench cooling and ice crystal formation in biological tissues

The rates of cooling of tissue packages quenched in liquid nitrogen were investigated using microthermocouples. By assembling tissue packages from a standard 200-μm tissue slice a microthermocouple could be positioned at different depths within the package. Results showed that for a given mass of tissue the rates of cooling at different depths were the same. When the tissue mass was varied the rates of cooling at a fixed depth decreased with increasing tissue mass.The ice crystal formations produced when tissues are quenched in liquid nitrogen were investigated using freeze substitution. Assemblies of rabbit cornea of different thicknesses were quenched in liquid nitrogen and freeze substituted. The size of the ice crystal cavities produced during the quenching increased with increasing tissue mass, exhibiting a saturation size for the larger tissue masses. There was no obvious size distribution of the ice crystal cavities across the thickness of the corneas.The results suggest an “isotherm” model for the quenching conditions used in these experiments, there being small or negligible temperature gradients through the tissue which uniformly cools at a fixed rate.     

Applications of gray-level variation detection method to intracellular ice formation

Intracellular ice formation (IIF) is the major cause of death in cells subjected to freezing. The occurrence of intracellular ice prevents the penetration of light into the camera and makes the image dark. Therefore, the gray-level variation can reflect the IIF. However, cell deformation is accompanied with IIF, especially for larger cells. It is necessary to account this entire phenomenon together in a single method. In this paper, the normalized parameter C defined by the gray-level variation depending on the displacement was defined to reflect the gray-level change of each pixel point in the region of interest of the image. The process of IIF of onion epidermal cells and 293T cells was analyzed by this method.     

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.     

Effect of cold acclimation on intracellular ice formation in isolated protoplasts

When cooled at rapid rates to temperatures between −10 and −30°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°C at rates of 10 or 16°C/minute. Further, the hydraulic conductivity determined during freeze-induced dehydration at −5°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°C, whereas in acclimated protoplasts, nucleation occurred over a broader temperature range with a median nucleation temperature of −42°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.     

The influence of hydroxyethyl starch on ice formation in aqueous solutions

Differential scanning calorimetry, and, in some supplementary experiments, X-ray diffractometry and cryomicroscopy, were applied to study the influence of concentration (< 70 wt%) and cooling/warming rates (< 320 K/min) on ice formation in aqueous solutions of HES. The calorimetric measurements of the quantity of crystallizing water indicated that a mass fraction ? = 0.522 (i.e., grams water per gram HES) remained unfrozen. These results are in good agreement with our earlier extrapolations from ternary phase diagram data and tend to support the proposed cryoprotective mechanism. The value of ? determined during warming was essentially independent of composition up to the corresponding saturation concentration. It was observed that solutions containing 60 wt% HES or more remained wholly amorphous during cooling even at rates as low as 2.5 K/min (down to 120 K). Such glassy solutions are subject to devitrification at temperatures T_d which depend on the warming rate. The concentrations close to 55 wt% HES mark a transitional range exhibiting two crystallization peaks, probably due to different mechanisms of nucleation, the portion of ice formed during cooling being related to the imposed cooling rate. All samples showed a recrystallization transition at 257.5 K which was also observed cryomicroscopically. Glass transitions, however, could not be detected by the methods applied in this study. The X-ray diffraction patterns contained the structure of only one solid phase, namely hexagonal ice. A comparison of various modifications of HES, PEG, and PVP involving bound water and melting temperature did not reveal marked differences. Minimum initial HES concentrations preventing lethal salt enrichment were computed for both binary and ternary mass fractions of NaCl as biologically relevant parameters, yielding 24.1 and 10.8 wt% HES, respectively.     

Influence of lipids on ice formation during the freezing of cryoprotective medium

The influence of lipids on ice formation during the freezing of cryoprotective medium for the semen of rainbow trout has been studied by the cryomicroscopy technique. It was shown that the lipids extracted from marine vertebrates and liposomes from the lipids of trout sperm effectively inhibit the ice formation in cryoprotective solutions during freezing, fundamentally changing the form and size of ice crystals. At high concentrations of lipids, either the crystallization does not occur in the cryoprotective medium or, even if ice crystals are formed, they have a broken shape and blurred borders. The addition of egg yolk sligthly increases the size and essentially changes the shape of ice crystals during the freezing of solution.