Intracellular Freezing Research Articles

Measurement of intracellular ice formation kinetics by high-speed video cryomicroscopy.

Quantitative information about the kinetics and cumulative probability of intracellular ice formation is necessary to develop minimally damaging freezing procedures for the cryopreservation of cells and tissue. Conventional cryomicroscopic assays, which rely on indirect evidence of intracellular freezing (e.g., opacity changes in the cell cytoplasm), can yield significant errors in the estimated kinetics. In contrast, the formation and growth of intracellular ice crystals can be accurately detected using temporally resolved imaging methods (i.e., video recording at sub-millisecond resolution). Here, detailed methods for the setup and operation of a high-speed video cryomicroscope system are described, including protocols for imaging of intracellular ice crystallization events, and stochastic analysis of the ice formation kinetics in a cell population. Recommendations are provided for temperature profile design, sample preparation, and configuration of the video acquisition parameters. Throughout this chapter, the protocols incorporate best practices that have been drawn from over a decade of experience with high-speed video cryomicroscopy in our laboratory.

Molecular analysis of the cold tolerant Antarctic nematode, Panagrolaimus davidi.

Isolated and established in culture from the Antarctic in 1988, the nematode Panagrolaimus davidi has proven to be an ideal model for the study of adaptation to the cold. Not only is it the best-documented example of an organism surviving intracellular freezing but it is also able to undergo cryoprotective dehydration. As part of an ongoing effort to develop a molecular understanding of this remarkable organism, we have assembled both a transcriptome and a set of genomic scaffolds. We provide an overview of the transcriptome and a survey of genes involved in temperature stress. We also explore, in silico, the possibility that P. davidi will be susceptible to an environmental RNAi response, important for further functional studies.

Intracellular freezing in the infective juveniles of Steinernema feltiae: an entomopathogenic nematode.

Taking advantage of their optical transparency, we clearly observed the third stage infective juveniles (IJs) of Steinernema feltiae freezing under a cryo-stage microscope. The IJs froze when the water surrounding them froze at −2°C and below. However, they avoid inoculative freezing at −1°C, suggesting cryoprotective dehydration. Freezing was evident as a sudden darkening and cessation of IJs' movement. Freeze substitution and transmission electron microscopy confirmed that the IJs of S. feltiae freeze intracellularly. Ice crystals were found in every compartment of the body. IJs frozen at high sub-zero temperatures (−1 and −3°C) survived and had small ice crystals. Those frozen at −10°C had large ice crystals and did not survive. However, the pattern of ice formation was not well-controlled and individual nematodes frozen at −3°C had both small and large ice crystals. IJs frozen by plunging directly into liquid nitrogen had small ice crystals, but did not survive. This study thus presents the evidence that S. feltiae is only the second freeze tolerant animal, after the Antarctic nematode Panagrolaimus davidi, shown to withstand extensive intracellular freezing.

Consideration of the reasons why dormant buds of trees have evolved extraorgan freezing as an adaptation for winter survival

Dormant buds of larch (Larix kaempferi) exhibit extraorgan freezing, in which extracellular ice crystals are formed in localized areas far from the primordia, as an adaptive response to freezing temperatures. In order to better understand why dormant buds have evolved such a complicated mechanism, the freezing behavior of isolated primordia from dormant buds of larch were examined using cryo-scanning electron microscopy (cryo-SEM) and by freeze-fracture replica electron microscopy (FR-EM). When isolated primordia were slowly frozen (5°C/day), extracellular ice crystals developed within primordia. Thus, isolated primordia serves as a useful model to examine the freezing response of primordia in which extracellular freezing occurs. Slow freezing (5°C/day) of isolated primordia resulted in a much lower survival rate (25% at −30°C) than in intact buds (100% at −30°C). In whole buds, exhibiting extraorgan freezing, cryo-SEM observations indicated that slow freezing to −30°C resulted in deep supercooling with a slight shrinkage of all primordial cells, whereas in isolated primordia, slow freezing to −30°C resulted in intracellular freezing of about half of the cells with other cells exhibiting extracellular freezing with significant shrinkage and deformation. FR-EM of whole buds subjected to slow freezing revealed the formation of intramembrane particle (IMP)-free areas with long rectangular shapes in the plasma membrane of many primordial cells at temperatures lower than −15°C and in a small number of cells in isolated primordia. In isolated primordia, the number of cells exhibiting intracellular freezing, which in most cases did not reveal membrane fracture faces, corresponded to the numbers of cells exhibiting IMP-free areas in whole buds. We speculate that the formation of IMP-free areas in plasma membranes may induce intracellular freezing when extracellular ice crystals have developed within primordial tissues. FR-EM also revealed the formation of fracture-jump lesions only in the plasma membranes of primordial cells of slowly-frozen, isolated primordia. We propose that such fracture-jump lesions are produced not only by osmotic dehydration but also by the deformation of cells due to the presence of extracellular ice crystals within primordial tissues in isolated primordia. The results indicate that the plasma membrane of primordial cells in ecodormant buds of larch are subject to a phase transition that may give rise to intracellular freezing, as well as increased sensitivity to dehydration, when extracellular ice crystals form within primordial tissues. We propose that extraorgan freezing represents a mechanism that prevents the formation of extracellular ice crystals within primordial tissues, thus lowering the risk of intracellular freezing and dehydrative stress.

Intracellular ice and cell survival in cryo-exposed embryonic axes of recalcitrant seeds of Acer saccharinum: an ultrastructural study of factors affecting cell and ice structures.

Cryopreservation is the only long-term conservation strategy available for germplasm of recalcitrant-seeded species. Efforts to cryopreserve this form of germplasm are hampered by potentially lethal intracellular freezing events; thus, it is important to understand the relationships among cryo-exposure techniques, water content, structure and survival. Undried embryonic axes of Acer saccharinum and those rapidly dried to two different water contents were cooled at three rates and re-warmed at two rates. Ultrastructural observations were carried out on radicle and shoot tips prepared by freeze-fracture and freeze-substitution to assess immediate (i.e. pre-thaw) responses to cooling treatments. Survival of axes was assessed in vitro. Intracellular ice formation was not necessarily lethal. Embryo cells survived when crystal diameter was between 0·2 and 0·4 µm and fewer than 20 crystals were distributed per μm(2) in the cytoplasm. Ice was not uniformly distributed within the cells. In fully hydrated axes cooled at an intermediate rate, the interiors of many organelles were apparently ice-free; this may have prevented the disruption of vital intracellular machinery. Intracytoplasmic ice formation did not apparently impact the integrity of the plasmalemma. The maximum number of ice crystals was far greater in shoot apices, which were more sensitive than radicles to cryo-exposure. The findings challenge the accepted paradigm that intracellular ice formation is always lethal, as the results show that cells can survive intracellular ice if crystals are small and localized in the cytoplasm. Further understanding of the interactions among water content, cooling rate, cell structure and ice structure is required to optimize cryopreservation treatments without undue reliance on empirical approaches.

131 Proposal of reaction kinetic model for cell death due to extracellular freezing based on two-factor hypothesis

Post-thaw cell viability (CV) as a function of cooling rate generally shows the bell-shaped distribution with an optimal cooling rate. The characteristic distribution is understood on the basis of a two-factor hypothesis, i.e. the cell damage is caused by two factors: the intracellular freezing (IIF) and the extracellular freezing (EIF) without IIF [Mazur, P. et al., Experimental Cell Research 71 (1972) 345–355]. Mathematical description and prediction of the cell survival curve (CSC) is one of important problems in the area of cryobiology. Contribution to the cell death by IIF was already modelled successfully based on nucleation in the cells [Toner, M. et al., J. Appl. Phys., 67(3) (1990) 1582–1593]. In the present study, a reaction kinetic model was proposed to describe the cell death caused due to EIF without IIF. According to the two-factor hypothesis, the post-thaw cells are classified by freezing pattern into two categories: those with IIF experience and those with only EIF and no IIF experience. The former is regarded as dead, and the latter consists of live and dead cells. Regarding the cells with only EIF as a population, the model to describe mathematically CV of the population as a function of cooling rate was developed on the basis of phenomenological investigation of CV without considering the mechanisms of cell damage and death. Four assumptions for the modelling were made as follows: (1) Cell damage and death are described as one overall reaction kinetic process, (2) Cells have three states: normal, damaged (but still alive) (intermediate state) and dead, (3) Cells change from normal state through damaged into dead, (4) Change rate of the number of cells is expressed by the first-order reaction. Rate constants for the changes in the number of cells were assumed to depend on the time elapsed during the freezing and thawing. Also, the simplest formulation was investigated for comparison: cells changed from normal directly into dead. Conservation equations of the number of cells in each state, were expressed by a set of simultaneous first-order linear ordinary differential equations. Under the condition that all the cells were initially normal, analytical solutions of CV of the population were found for the rate constants depending on time. On condition of constant cooling rate, the time integral of rate constant in the solutions was changed into a temperature integral of it, and a value of the latter was defined as a model constant. Therefore, a basic function of cooling rate for CSC was found. Further, three functions with cooling rate modified by another model constant for CSC were also proposed. A parameter study of model constants was performed to evaluate CSC as a function cooling rate. The model constants were also determined by inverse problem analysis based on the experiment with human erythrocytes [MAZUR, P., Am. J. Physiol. 247, C125–C142 (1984)] as the experimental data on the viability of the cells with only EIF were unavailable. The experiment and prediction by the model were compared on CSC. The result shows that EIF-induced cell death is successfully described by the reaction kinetic mode. Source of funding: Grant-in-Aid for Scientific Research #24360083 and #21360098 to H.I. from Japan Society for the Promotion of Science. Conflict of interest: None declared. ishiguro@life.kyutech.ac.jp

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

089 Inhibitory effects on low temperature injury by flavonoids (glycosides)

Our previous studies showed that xylem parenchyma cells (XPCs) in trees, which adapt to winter cold by deep supercooling, contained several kinds of supercooling promoting substances [Kasuga et al., Cryobiology, 55 (2007) 305–314; Wang et al., Planta, 235 (2012) 747–759]. It is suggested that the presence of supercooling-promoting substances keeps liquid state of water in XPCs near −40 °C. In the presence of such supercooling-promoting substances in XPCs, although occurrence of lethal intracellular freezing is prevented, it is noted that cells are subjected to extremely low temperature under liquid state of water. It is, thus, speculated that deep supercooling XPCs may also contain any substances which protect cellular components from such extremely low temperatures. It is thought that such inhibitory substances may be applied for an inhibitor of low temperature injury in a wide variety of biological materials. We examined effects of several compounds from deep-supercooling XPCs to find protection effect on low temperature injury using cultured mammal cells as experimental materials. Among the compounds examined, several kinds of flavonoids (glycosides) including quercetin 3-glycoside (Q3G), which existed in deep supercooling XPCs as supercooling-promoting substances, exhibited inhibitory effect of low temperature injury at 4 °C in very low concentrations ranging from 0.1 to 100 μ/mL to human promyelocytic leukemia cells (HL60), primary mouse embryo fibroblast, human foreskin fibroblast (Hs68) and human umbilical vein endothelial cells (HUVEC). It is worth to note, however, that inhibitory effect of low temperature injury by these flavonoids (glycosides) is greatly influenced depending on constituents of incubation medium. Although we examined relation between inhibitory effect of low temperature injury and degree of antioxidant activities in flavonoids (glycosides), no relation between them was found. Therefore, it is thought that inhibitory effect of low temperature injury by flavonoids (glycosides) is developed by mechanisms other than antioxidant effect. It is suggested that these flavonoids (glycosides) are useful to prevent low temperature injury of biological materials and we are currently examining their inhibitory effects under subzero-temperatures. Source of funding: None declared. Conflict of interest: None declared. sfuji@for.agr.hokudai.ac.jp

Hydrurus‐related golden algae (Chrysophyceae) cause yellow snow in polar summer snowfields

SummaryIn polar regions, melting snow fields can be occupied by striking blooms of chrysophycean algae, which cause yellowish slush during summer. Samples were harvested at King George Island (South Shetland Islands, Maritime Antarctica) and at Spitsbergen (Svalbard archipelago, High Arctic). The populations live in an ecological niche, where water‐logged snow provides a cold and ephemeral ecosystem, possibly securing the survival of psychrophilic populations through the summer. A physiological adaptation to low temperatures was shown by photosynthesis measurements. The analysis of soluble carbohydrates showed the occurrence of glycerol and sugars, which may play a role in protection against intracellular freezing. Although both populations were made of unicells with Ochromonas‐alike morphology, investigation by molecular methods (18S rDNA sequencing) revealed unexpectedly a very close relationship to the mountain‐river dwelling Hydrurus foetidus (Villars) Trevisan. However, macroscopic thalli typical for the latter species were never found in snow, but are known from nearby localities, and harvested samples of snow algae exposed to dryness evolved a similar pervading, ‘fishy’ smell. Moreover, in both habitats tetrahedal zoospores with four elongate spikes were found, similar to what is known from Hydrurus. Our molecular results go along with earlier reports, where chrysophycean sequences of the same taxonomic affiliation were isolated from snow. This points to a distinct group of photoautotrophic, Hydrurus‐related chrysophytes, which are characteristic for long‐lasting, slowly melting snow packs in certain cold regions of the world.

A Low Temperature Limit for Life on Earth

There is no generally accepted value for the lower temperature limit for life on Earth. We present empirical evidence that free-living microbial cells cooling in the presence of external ice will undergo freeze-induced desiccation and a glass transition (vitrification) at a temperature between −10°C and −26°C. In contrast to intracellular freezing, vitrification does not result in death and cells may survive very low temperatures once vitrified. The high internal viscosity following vitrification means that diffusion of oxygen and metabolites is slowed to such an extent that cellular metabolism ceases. The temperature range for intracellular vitrification makes this a process of fundamental ecological significance for free-living microbes. It is only where extracellular ice is not present that cells can continue to metabolise below these temperatures, and water droplets in clouds provide an important example of such a habitat. In multicellular organisms the cells are isolated from ice in the environment, and the major factor dictating how they respond to low temperature is the physical state of the extracellular fluid. Where this fluid freezes, then the cells will dehydrate and vitrify in a manner analogous to free-living microbes. Where the extracellular fluid undercools then cells can continue to metabolise, albeit slowly, to temperatures below the vitrification temperature of free-living microbes. Evidence suggests that these cells do also eventually vitrify, but at lower temperatures that may be below −50°C. Since cells must return to a fluid state to resume metabolism and complete their life cycle, and ice is almost universally present in environments at sub-zero temperatures, we propose that the vitrification temperature represents a general lower thermal limit to life on Earth, though its precise value differs between unicellular (typically above −20°C) and multicellular organisms (typically below −20°C). Few multicellular organisms can, however, complete their life cycle at temperatures below ∼−2°C.

Basic Study on Visualization of a Cell During Freezing Using Electrical Impedance Tomography

Visualization of a cell during freezing was tried as a first trial using electrical impedance tomography (EIT). During freezing of a grain of Citrus Sudachi, we found good agreement between video and EIT images. These results indicated that EIT has possibility to visualize a cell in freezing process. I. INTRODUCTION The cryopreservation technology enables long term storage of cells and biological tissues, since theoretically their metabolism is stopped due to freezing. However, there is difficulty in using this technique for complex structure of biological tissues because it may cause an injury, such as denaturation, to the cell membrane by concentrating of the intracellular solution with the extracellular freezing. The destruction of the cell membrane and the intracellular microstructure can happen due to nucleation of ice crystals (intracellular freezing), which depends on the cooling rate of the freezing process (1). If we can visualize a cell or a tissue structure during freezing in real time, an optimal freezing method can be developed. In this study, report the results of a preliminary trial to visualize a cell during freezing process using electrical impedance tomography (EIT) (2).

The ability of the Antarctic nematode Panagrolaimus davidi to survive intracellular freezing is dependent upon nutritional status

The Antarctic nematode Panagrolaimus davidi is the best documented example of an animal surviving intracellular freezing and the only animal so far shown to survive such freezing throughout its tissues. However, a recent study found that after exposure to a freezing stress that produced intracellular freezing in a proportion of nematodes, the resulting survival levels could be explained if those nematodes that froze intracellularly had died. We have thus re-examined the survival of intracellular freezing in this nematode. The ability to survive a freezing exposure that is likely to produce intracellular freezing (freezing at -10 °C) declines with culture age. In cultures that are fed regularly, the ability to survive freezing at -10 °C increases, but in starved cultures freezing survival declines. Survival of intracellular freezing in fed cultures was confirmed using cryomicroscopy, staining of cells with vital dyes and by freeze substitution and transmission electron microscopy. We have thus confirmed that P. davidi can survive intracellular freezing and shown that this ability is dependent upon them being well fed. The effect of culture conditions on the nutrient status of the nematodes should thus be an important factor in the design of experiments.

Freezing pattern and frost killing temperature of apple (Malus domestica) wood under controlled conditions and in nature

The freezing pattern and frost killing temperatures of apple (Malus domestica Borkh.) xylem were determined by differential thermal analysis and infrared differential thermal analysis (IDTA). Results from detached or attached twigs in controlled freezing experiments and during natural field freezing of trees were compared. Non-lethal freezing of apoplastic water in apple xylem as monitored during natural winter frosts in the field occurred at -1.9 ± 0.4 °C and did not change seasonally. The pattern of whole tree freezing was variable and specific to the environmental conditions. On detached twigs high-temperature freezing exotherms (HTEs) occurred 2.8 K below the temperature observed under natural frosts in the field with a seasonal mean of -4.7 ± 0.5 °C. Microporous apple xylem showed freezing without a specific pattern within a few seconds in IDTA images during HTEs, which is in contrast to macroporous xylem where a 2D freezing pattern mirrors anatomical structures. The pith tissue always remained unfrozen. Increasing twig length increased ice nucleation temperature; for increased twig diameter the effect was not significant. In attached twigs frozen in field portable freezing chambers, HTEs were recorded at a similar mean temperature (-4.6 ± 1.0 °C) to those for detached twigs. Upon lethal intracellular freezing of apple xylem parenchyma cells (XPCs) low-temperature freezing exotherms (LTEs) can be recorded. Low-temperature freezing exotherms determined on detached twigs varied significantly between a winter minimum of -36.9 °C and a summer maximum -12.7 °C. Within the temperature range wherein LTEs were recorded by IDTA in summer (-12.7 ± 0.5 to -20.3 ± 1.1 °C) various tiny clearly separated discontinuous freezing events could be detected similar to that in other species with contrasting XPC anatomy. These freezing events appeared to be initially located in the primary and only later in the secondary xylem. During the LTE no freezing events in the bark and central pith tissue were recorded. Attached twigs were exposed to various freezing temperatures at which LTEs occur. Even if 60% of XPCs were frost-damaged twigs were able to recuperate and showed full re-growth indicating a high regeneration capacity even after severe frost damage to XPCs.

Velocity and pattern of ice propagation and deep supercooling in woody stems of Castanea sativa, Morus nigra and Quercus robur measured by IDTA

Infrared differential thermal analysis (IDTA) was used to monitor the velocity and pattern of ice propagation and deep supercooling of xylem parenchyma cells (XPCs) during freezing of stems of Castanea sativa L., Morus nigra L. and Quercus robur L. that exhibit a macro- and ring-porous xylem. Measurements were conducted on the surface of cross- and longitudinal stem sections. During high-temperature freezing exotherms (HTEs; -2.8 to -9.4°C), initial freezing was mainly observed in the youngest year ring of the sapwood (94%), but occasionally elsewhere (older year rings: 4%; bark: 2%). Initially, ice propagated rapidly in the largest xylem conduits. This resulted in a distinct freezing pattern of concentric circles in C. sativa and M. nigra. During HTEs, supercooling of XPCs became visible in Q. robur stems, but not in the other species that have narrower pith rays. Intracellular freezing of supercooled XPCs of Q. robur became visible by IDTA during low-temperature freezing exotherms (<-17.4 °C). Infrared differential thermal analysis revealed the progress and the two-dimensional pattern of XPC freezing. XPCs did not freeze at once, but rather small cell groups appeared to freeze at random anywhere in the xylem. By IDTA, ice propagation and deep supercooling in stems can be monitored at meaningful spatial and temporal resolutions.

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.

69. Innocuous intracellular freezing in human dental stem cells

69. Innocuous intracellular freezing in human dental stem cells

Freezing in non-acclimated oats. II: Thermal response and histology of recovery in gradual and rapidly frozen plants

Freezing in winter cereals is a complex phenomenon that can affect various plant tissues differently. To better understand how freezing affects specific tissue in the over wintering organ (crown) of winter cereal crops, non-acclimated oats ( Avena sativa L .) were gradually frozen to −3 °C and tissue damage during recovery was compared to plants that had been supercooled to −3 °C and then frozen suddenly. Percentage of total water frozen, was the same whether crowns were frozen suddenly or gradually although the rate of freezing was considerably different. For example, all available water froze within 3 h in suddenly frozen crowns but it took more than 15 h for all available water to freeze in gradually frozen crowns. When plants were suddenly frozen, cells in the apical meristem were disrupted and apparently killed. In these plants re-growth was limited or non-existent. In contrast, the apical region of plants that were slowly frozen appeared undamaged but extensive vessel plugging was observed in cells of the lower crown, possibly from accumulation of phenolics or from microbial proliferation. These histological observations along with the calorimetric analysis suggested that the apical region was killed by intracellular freezing when frozen suddenly while the crown core was damaged by a process, which either induced production of putative phenolic compounds by the plant and/or permitted what appeared to be microbial proliferation in metaxylem vessels.

Kinetics and activation energy of recrystallization of intracellular ice in mouse oocytes subjected to interrupted rapid cooling

Intracellular ice formation (IIF) is almost invariably lethal. In most cases, it results from the too rapid cooling of cells to below −40 °C, but in some cases it is manifested, not during cooling, but during warming when cell water that vitrified during cooling first devitrifies and then recrystallizes during warming. Recently, Mazur et al. [P. Mazur, I.L. Pinn, F.W. Kleinhans, Intracellular ice formation in mouse oocytes subjected to interrupted rapid cooling, Cryobiology 55 (2007) 158–166] dealt with one such case in mouse oocytes. It involved rapidly cooling the oocytes to −25 °C, holding them 10 min, rapidly cooling them to −70 °C, and warming them slowly until thawed. No IIF occurred during cooling but intracellular freezing, as evidenced by blackening of the cells, became detectable at −56 °C during warming and was complete by −46 °C. The present study differs in that the oocytes were warmed rapidly from −70 °C to temperatures between −65 and −50 °C and held for 3–60 min. This permitted us to determine the rate of blackening as function of temperature. That in turn allowed us to calculate the activation energy ( E a) for the blackening process; namely, 27.5 kcal/mol. This translates to about a quadrupling of the blackening rate for every 5 °C rise in temperature. These data then allowed us to compute the degree of blackening as a function of temperature for oocytes warmed at rates ranging from 10 to 10,000 °C/min. A 10-fold increase in warming rate increased the temperature at which a given degree of blackening occurred by 8 °C. These findings have significant implications both for cryobiology and cryo-electron microscopy.

19. Intracellular freezing behavior under conditions of altered water permeability and initial cell volume

19. Intracellular freezing behavior under conditions of altered water permeability and initial cell volume

Cold tolerance of an Antarctic nematode that survives intracellular freezing: comparisons with other nematode species

Panagrolaimus davidi is an Antarctic nematode with very high levels of cold tolerance. Its survival was compared with that of some other nematodes (P. rigidus, Rhabditophanes sp., Steinernema carpocapsae, Panagrellus redivivus and Ditylenchus dipsaci) in both unacclimated samples and those acclimated at 5 degrees C. Levels of recrystallization inhibition in homogenates were also compared, using the splat-cooling assay. The survival of P. davidi after the freezing of samples was notably higher than that of the other species tested, suggesting that its survival ability is atypical compared to other nematodes. In general, acclimation improved survival. Levels of recrystallization inhibition were not associated with survival but such a relationship may exist for those species that are freezing tolerant.