Intracellular Ice Crystal Formation Research Articles

Ultrastructural Changes Arising from Freezing of Leaf Blade Cells of Cold Acclimated Rye (Secale cereale)

The survival at sub-zero temperatures of leaf blade cells of rye ( Secale cereale cv. Voima) that had been cold-acclimated at 5 °C for 4 weeks was determined by measuring the efflux of ninhydrin-positive substances: 50 % of the cells were dead at - 8 °C (LT 50 ) and none survived at -16 °C or below. Specimens for transmission electron microscopy were prepared from leaf blade segments by fast freezing followed by freeze-substitution in acetone with O S O 4 fixation. In specimens from plants maintained at 5 °C, chloroplasts and mitochondria were well-structured and the endoplasmic reticulum and dictyosomes easily discernible. Many membrane bound vesicles, ranging in cross sectional diameter from ~ 60 nm upwards were seen in the cytoplasm. Sometimes stacks of membrane strands running parallel to the plasmalemma for up to 2 µm were also observed. Upon freezing to -16 °C, the cells were severely dehydrated and distorted, the vacuoles severely shrunken, and the cytoplasm and mitochondria disorganized; cytoplasmic vesicles and stacks of membrane strands seen in the controls were absent. Upon freezing to - 8 °C, some cells were as disorganized as those at -16 °C, although the cell wall did not always adhere to the collapsed protoplast; others were relatively intact and some showed evidence of intracellular ice crystal formation.

Ultrastructural changes arising from freezing of leaf blade cells of rye (Secale cereale): an investigation using freeze‐substitution

The survival at sub‐zero temperatures of leaf blade cells of rye (Secale cereale L. cv. Voima), which had not been cold acclimated, was determined by measuring the efflux of ninhydrin‐positive substances: 50% of the cells were dead at −4°C (LT50) and none survived at −12°C or below. Examination of ultrastructural changes during cold hardening and freezing injury requires frozen tissues prepared for transmission electron microscopy without thawing. Specimens were prepared from leaf blade segments at room temperature, −4°C or −12°C by plunge freezing at 3 m s−1 into a cooling medium at −170°C followed by freeze‐substitution in acetone with OsO4 fixation. Comparisons of room temperature specimens were made with those prepared by chemical fixation using glutaraldehyde/paraformaldehyde/tannic acid. On freezing to −12°C, the cells were severely dehydrated and distorted, the vacuoles severely shrunken and the cytoplasm and mitochondria disorganized whereas the chloroplasts were little affected. On freezing to −4°C, some cells were as disorganized as those at −12°C, others were relatively intact, and some showed evidence of intracellular ice crystal formation.

The effect of various cooling rates on the membrane ultrastructure of frozen human erythrocytes and its relation to the extent of haemolysis after thawing.

Human erythrocytes suspended in buffered isotonic saline were frozen to the temperature of liquid nitrogen at various cooling rates of 3, 140, 700, 1800, 3500, 8000 and 11 500 deg. C/min. The membrane ultrastructure in the frozen state and the extent of haemolysis after thawing were examined at each cooling rate. As the cooling rates increased from 3 to 3500 deg. C/min, the extent of lysis gradually decreased, but further increase in cooling rates in excess of 8000 deg. C/min resulted in an abrupt increase of lysis. Membrane-associated vesicles devoid of intramembrane particles (IMPs) were formed in the erythrocyte membranes frozen at cooling rates slower than 1800 deg. C/min. The frequency and size of these vesicles were highly cooling-rate-dependent and they were no longer formed in the erythrocyte membranes frozen at cooling rates faster than 3500 deg. C/min. Another membrane ultrastructural change associated closely with the formation of intracellular ice crystals appeared at cooling rates faster than 8000 deg. C/min. The membrane regions in direct contact with intracellular ice crystals were physically damaged and had an appearance resembling worm-eaten spots. The erythrocytes frozen at a cooling rate of 3500 deg. C/min exhibited ultrastructural integrity of the membrane by avoiding the membrane changes caused by either slow or fast freezing. It is suggested, from the close relation between membrane ultrastructure and the extent of haemolysis, that the ultrastructural integrity of membrane in the frozen state is important for avoiding haemolysis after thawing, and that the membrane ultrastructural changes caused by both slow and fast freezing were responsible for the lysis after thawing.

RELATIONSHIP OF FREEZING PRESERVATION PARAMETERS TO TEXTURE-RELATED STRUCTURAL DAMAGE TO THERMALLY PROCESSED CRUSTACEAN MUSCLE

Selected freezing preservation parameters applied to thermally processed blue crabs and shrimp were evaluated by means of scanning electron microscopy to determine the relationship of the parameters to texture-related structural damage brought about by ice crystal formation and growth. Ice crystal growth and tissue disruption occurs largely during post-freezing frozen storage, the rate of freezing merely setting the pattern of subsequent crystal growth. Damage is more severe with slow (sharp) freezing due to growth of large extracellular ice crystals, causing tissue dehydration and shrinkage and compaction. This also leads to greater drip loss upon thawing. Rapid freezing in “Freon” food freezant or in liquid nitrogen (LN2) results in less disruptive intracellular crystal formation and growth, the water tending to remain in the tissue upon thawing. Immersion in LN2 caused noticeable stress-cracking of the tissue, but there was no evidence of significant ice crystal growth during subsequent warming to freezer storage temperatures. Ice crystal growth during — 29 °C storage was at least one-third slower than at — 10 °C, underscoring the overriding importance of good holding conditions following rapid freezing. It can be concluded that the various fast freezing methods are essentially equally suitable as long as the freezing rate is well within the limit for promoting formation of tiny intracellular ice crystals (i.e. on the order of a few to several minutes). Double freezing of shrimp, a common commercial practice, doesn't appear to result in significant damage to tissue over and above that which develops during post-thermal processing frozen storage. It is advisable to upgrade frozen storage systems in the distribution channels to the level of sophistication of modern freezing systems, and to hold product for as short a time at as low a subfreezing temperature as is practical.

The structure of mitochondrial membranes in frozen sections

Cryomicrotomy under conditions that would involve reduced risks for protein denaturation was applied in an analysis of the structure of the inner mitochondrial membranes of rat heart muscle tissue. In order to prevent extensive conformational changes, the tissue was cross-linked for a short time in glutaraldehyde. To minimize the effects of intracellular ice crystal formation during freezing, the tissue was partially dehydrated by infusion of sucrose according to Tokuyasu (J. Cell Biol. 57, 551 (1973)) or by immersion in 30% glycerol. Caution was taken to reduce the denaturation of proteins through surface tension during transfer of the sections to the grids. The sections were analyzed under conditions close to minimum beam damage. The inner membranes measured about 260 A in thickness, were closely apposed, were lacking any intracristal space, and showed a particulate substructure with the particles measuring from 30 to 100 A in diameter. Prolonged fixation in glutaraldehyde at high concentration (2.5%), elimination of partial dehydration before freezing, collecting the sections on DMSO, and drying the sections before negative staining resulted in the inner mitochondrial membranes appearing as consisting of two 50–70 A thick membrane elements separated by an intracristal space and no particulate substructure. A similar appearance of the membranes was observed when the tissue was dehydrated in ethanol after fixation for 1 hr in 2.5% glutaraldehyde and rehydrated before cryomicrotomy. A similar appearance was also characteristic for osmium-fixed and Epon-embedded material. It is concluded that this appearance of the inner mitochondrial membranes is due to extensive denaturation of the membrane proteins. The first-mentioned appearance with 260 A thick inner membranes is considered to represent a situation close to the native state of these membranes.

Frost Damage Resulting from Ice Crystal Formation in Seedlings of Spruce and Pine

AbstractThe influence of ice crystal formation in tissues of unhardened and hardened seedlings of Pinus silvestris L. and Picea abies (L.) Karst. was determined. An apparatus for this purpose was constructed.Unhardened seedlings of spruce collapsed completely as a result of ice crystal formation in their shoots, while unhardened seedlings of pine survived ice crystal formation with or without injury to the needles. After a hardening period of 4–5 weeks, seedlings of spruce and pine survived ice formation but injuries occurred in spruce. For pine, on the contrary, the temperature after ice formation had a decisive effect on the extent of the injuries. Observed injuries are discussed in terms of inter‐ and intracellular ice crystal formation.

Some factors contributing to the survival of rapidly cooled plant cells

The stability of innocuous intracellularly frozen cells was found to be influenced remarkably by the surrounding solutions and the degree of prefreezing. In the cells suspended in isotonic or slightly hypertonic glucose solution, innocuous intracellular freezing readily occurred and these rapidly frozen cells remained stable even at −30 °C for 20 min, while cells prefrozen to −10 °C after suspending in water survived standing at −30 °C only for 5 sec at most. These facts suggest that when the innocuous intracellularly frozen cells are surrounded with concentrated sugar solutions, these cells remain far more stable than those surrounded with ice. When winter hardy cortical cells suspended in water were rapidly cooled, they were killed as a result of prompt formation of intracellular ice crystals immediately after the beginning of freezing of suspending medium. In less hardy cells which resisted freezing to only −10 °C, the innocuous intracellular freezing seldom occurred, even when cooled and rewarmed ultrarapidly without suspending with water, since these less hardy cells were quite apt to cause fatal intracellular freezing during rapid cooling. However, in less hardy cells and winter hardy cortical cells suspended with water, innocuous intracellular freezing by rapid cooling could be readily induced, provided that they were prefrozen to the temperatures below −5 °C or suspended in isotonic or slightly hypertonic solutions of glucose and the related compounds, DMSO, etc., individually or in combinations depending upon the degree of freezing resistance of the samples.

The ultrastructure of small bowel epithelium during freezing

The effectiveness of glycerol, dimethyl sulfoxide (DMSO), and a combination of the two as perfused cryoprotective agents for the survival of segments of dog ileum was evaluated. The results were compared with studies on the effectiveness of these agents in the preservation of ultrastructural detail and the prevention of ice crystal formation in freeze-etched specimens of intestinal epithelium. The evidence obtained supports the concept that intracellular ice crystal formation is compatible with viability of certain tissues specifically in the dog ileum. The size of the crystals was not taken into consideration. The results indicate that the degree of cryoinjury varies from cell to cell and that cryoprotective agents may be somewhat selective at their site of action. Intestinal segments survived in spite of rather severe cellular damage, which appeared primarily as injury to the cell membranes. It is suggested that studies of the ultrastructural features of frozen-thawed cells, as well as replicas of frozen cell surfaces, may lead to an improved knowledge of the factors involved in cryoinjury and its prevention.

Discussion - Formation of intra-cellular ice

When normal tissues are frozen slowly and fixed in absolute alcohol at – 79° C the majority of the nuclei show ice-crystal spaces and may be dead (Heard 1955 a , b ). On the other hand, the same method applied to Craigie’s paramorphic cells (which are ascites tumour cells in a cold-resistant state (Craigie 1954)) produces very few nuclear ice-crystal spaces. This may correlate with their survival. The same tumour grown subcutaneously is less resistant to freezing and similar preparations show abundant nuclear crystals. I suggest tentatively that such an approach to normal tissues may help to decide if some cells are more resistant than others. Several workers since Chambers & Hale (1932) have shown that intracellular ice-crystal formation goes with cell death, and I would suspect that most of the ovarian tissue transplanted by Dr Parkes is not viable. Lack of crystals might be a way of finding out how much of it is ready to grow again. And then again, it might not !

The expressible fluid of fish fillets. I. —Nucleic acid as an index of cell damage in fillets frozen from both sides

AbstractThe appearance of deoxypentosenucleic acid (DNA) in muscle expressible fluid has been taken to indicate rupture of the sarcolemmas, with liberation of nuclear material. When fish fillets are frozen more and more rapidly, the expressible nucleic acid suddenly rises to a maximum, which is thought to correspond with the point of formation of intracellular ice crystals. In ultra‐rapid freezing, there is also a rise in expressible DNA, probably owing to a different kind of cell damage.