Cryopreservation with sucrose maintains normal physical and biological properties of human plasma low density lipoproteins.

The ability to preserve low density lipoprotein (LDL) preparations frozen for weeks and months without changes in structure or biological properties is of potential use in long-term comparative studies of LDL. We demonstrate that freeze-thawing of LDL causes marked alterations in its structure and biological behavior, and that such changes can be prevented by the addition of sucrose to the LDL solution prior to freezing. Freezing LDL at -70 degrees C in the absence of sucrose resulted in aggregation and fusion of particles as measured by electron microscopy, spectrophotometric absorption, and column gel filtration. This was associated with increased binding affinity of monoclonal antibodies at epitopes distant from the receptor binding region. Functional changes induced by freezing included 3- to 10-fold increases in binding at 4 degrees C and 37 degrees C, and uptake of LDL in fibroblasts, attributable mainly to increases in nonspecific binding processes. Cryopreservation of LDL in 10% sucrose (w/v) completely prevented the structural and functional changes incurred after short-term freezing, and LDL cryopreserved in sucrose for as long as 18 months displayed cell binding, uptake, and degradation very similar to that of freshly obtained LDL.     

Effect of Multiple Freezing/Thawing Cycles on the Structural and Functional Properties of Waxy Rice Starch

The structural and functional properties of non-gelatinized waxy rice starch were investigated after 1, 3, 7, and 10 freezing/thawing cycles. Freezing caused an increasing damaged starch from 1.36% in native waxy rice starch to 5.77% in 10 freezing/thawing-treated starch (FTS), as evidenced by the cracking surface on starch granules. More dry matter concentration was leached, which was characterized by high amylopectin concentration (4.34 mg/mL). The leaching was accompanied by a decrease in relative crystallinity from 35.19% in native starch to 31.34% in 10 FTS. Freezing treatment also led to significant deviations in the functional characteristics, for instance decreased gelatinization temperature range, enthalpy, and pasting viscosities. The resistant starch content of 10FTS significantly decreased from 58.9% to 19%, whereas the slowly digested starch content greatly increased from 23.8% in native starch to 50.3%. The increase in susceptibility to enzyme hydrolysis may be attributed to porous granular surface, amylopectin leaching, and the decrease in the relative crystallinity caused by freezing water.     

Effect of oxygen on freezing damage. II. Physical-chemical effects

The biochemical effects of freezing in the presence or absence of oxygen were investigated in two E. coli B species. Oxygen-dependent freezing damage included generation of a free radical with characteristic power-saturation properties and increased leakage of certain amino acids from frozen and thawed cells. These findings offer a possible explanation for the previously described biological effects of freezing in the presence of oxygen. Freezing also caused oxygen-independent single-strand breaks in DNA and leakage of other intracellular materials.     

Biophysical characterization of the Lactobacillus delbrueckii subsp. bulgaricus membrane during cold and osmotic stress and its relevance for cryopreservation

Freezing lactic acid bacteria often leads to cell death and loss of technological properties. Our objective was to provide an in-depth characterization of the biophysical properties of the Lactobacillus delbrueckii subsp. bulgaricus membrane in relation to its freeze resistance. Freezing was represented as a combination of cold and osmotic stress. This work investigated the relative incidence of increasing sucrose concentrations coupled or not with subzero temperatures without ice nucleation on the biological and biophysical responses of two strains with different membrane fatty acid compositions and freeze resistances. Following exposure of bacterial cells to the highest sucrose concentration, the sensitive strain exhibited a survival rate of less than 10 % and 5 h of acidifying activity loss. Similar biological activity losses were observed upon freeze-thawing and after osmotic treatment for each strain thus highlighting osmotic stress as the main source of cryoinjury. The direct measurement of membrane fluidity by fluorescence anisotropy was linked to membrane lipid organization characterized by FTIR spectroscopy. Both approaches made it possible to investigate the specific contributions of the membrane core and the bilayer external surface to cell degradation caused by cold and osmotic stress. Cold-induced membrane rigidification had no significant implication on bacterial freeze-thaw resistance. Interactions between extracellular sucrose and membrane phospholipid headgroups under osmotic stress were also observed. Such interactions were more evident in the sensitive strain and when increasing sucrose concentration, thus suggesting membrane permeabilization. The relevance of biophysical properties for elucidating mechanisms of cryoinjury and cryoprotection is discussed.

     

Effective cryoprotection of thylakoid membranes by ATP

Freezing of isolated spinach thylakoids in the presence of NaCl uncoupled photophosphorylation from electron flow and increased the permeability of the membranes to protons. Addition of ATP prior to freezing diminished membrane inactivation. On a molar basis, ATP was at least 100 times more effective in protecting thylakoids from freezing damage than low-molecularweight carbohydrates such as sucrose and glucose. The cryoprotective effectiveness of ATP was increased by Mg²⁺. In the absence of carbohydrates, preservation of thylakoids during freezing in 100 mM NaCl was saturated at about 1–2 mM ATP, but under these conditions membranes were not fully protected. However, in the presence of small amounts of sugars which did not significantly prevent thylakoid inactivation during freezing, ATP concentrations considerably lower than 0.5 mM caused nearly complete membrane protection. Neither ADP nor AMP could substitute for ATP. These findings indicate that cryoprotection by ATP cannot be explained by a colligative mechanism. It is suggested that ATP acts on the chloroplast coupling factor, either by modifying its conformation or by preventing its release from the membranes. The results are discussed in regard to freezing injury and resistance in vivo.Abbreviations CF₁ chloroplast coupling factor - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - PMS phenazine methosulfate - Tris 2-amino-2-(hydroxymethyl)-1,3-propandiol     

Polypeptide markers for low temperature stress during seed germination in sunflower

Sunflower seeds behaved as chilling and freezing sensitive and also exhibited acclimation under low seed moisture content (< 1 %). At high seed moisture content (approx. 22 %) they tolerated chilling stress but failed to acclimate under freezing temperatures. Pre-imbibitional chilling (5 °C) or freezing (−5 or −10 °C) stress significantly enhanced total soluble protein (TSP) content. Chilling treatment after imbibition (in contrast to pre-imbibition) enhanced germination and this was accompanied by increase in 30, 24 and 21.9 kDa TSPs content (3 d after germination). Freezing at −5 and −10 °C suppressed seed germination and increased content of 78 and 56.2 kDa wall bound proteins. Chilling acclimation decreased 35.4, 33.9, 29.5, 23.4 and 21.4 kDa TSPs.     

Solid�CLiquid Transitions and Stability of HPKO-in-Water Systems Emulsified by Dairy Proteins

Stability of oil-in-water emulsions during freezing and thawing is regulated by the phase transitions occurring in the continuous and dispersed phases upon thermal treatments and by the composition of the interfacial membrane. In the present study, the impact of the water phase formulation (0–2.5–5–10–20–30–40% w/w sucrose), the interfacial composition [whey protein isolates (WPI) or sodium caseinate (NaCas) used at different concentrations], and the particle size on the stability of hydrogenated palm kernel oil (30% w/w)-in-water systems was investigated. Phase/state behaviour of the continuous and dispersed phases and emulsion destabilisation were studied by differential scanning calorimetry. System morphology was observed by particle size analysis and optical microscopy. The presence of sucrose in the aqueous phase and reduced particle size distribution significantly improved emulsion stability. WPI showed better stabilising properties than NaCas at lipid to protein ratios of 10:1, 7.5:1, 5:1 and 4:1. Increased WPI concentration significantly improved emulsion resistance to breakdown during freeze–thaw cycling. NaCas showed poor stabilising properties and was ineffective in reducing emulsion destabilisation at 0% sucrose at all the lipid to protein ratios.     

Freezing injury and uncoupling of phosphorylation from electron transport in chloroplasts

Freezing of chloroplast membranes uncouples photophosphorylation from electron transport and inactivates the light-dependent and thiol-requiring ATPase, conformational changes and the light-dependent proton uptake. All of these energy requiring activities can be protected against inactivation by addition of sucrose prior to freezing. The direct relation to photophosphorylation is demonstrated by the quantitatively similar response of photophosphorylation and the other activities to sucrose protection. Salts interfere with the protection afforded by sucrose.

In contrast to the light-dependent ATPase, the ATPase activities which are unmasked by digestion with trypsin show no significant response to freezing. Similarly, the chloroplast coupling factor, which is released from the membranes by ethylenediamine tetraacetic acid treatment, survives freezing. The membranes, which are depleted of the factor, are damaged by freezing.

The results suggest that uncoupling of phosphorylation from electron transport is caused by interference of freezing with a structure involved in the formation of a non-phosphorylated high energy state of chloroplasts. They are best explained on the basis of Mitchell's theory of phosphorylation. Since freezing alters the permeability properties of chloroplast membranes—frozen membrane vesicles no longer function as osmometers—it may be assumed that freezing uncouples phosphorylation from electron transport by preventing the formation of a pH gradient across the vesicle membranes owing to proton leakage through the membranes. From the results, the basic injury caused by freezing appears to consist in the alteration of permeability properties of biological membranes due to the dehydration which accompanies freezing.

     

Effects of Gastric Freezing on the Isolated Denervated Gastric Pouch in Dogs

The purpose of this study was to investigate the effects of gastric freezing on the chief and parietal cells of the stomach. To carry this out six Heidenhain pouch dogs were prepared. The secretions of these pouches were studied, both for HCl levels and pepsin activity, for 30 days before and 30 days after the freezing of the pouch. Freezing was carried out for one hour at -17 to -20° C. The results show that no essential depression of either the HCl levels or the pepsin activity of the juice was effected by freezing. Therefore, it is deduced that freezing does not affect the functional activity of either the chief or the parietal cells. Complications such as ulceration and fistula formation into adjacent organs occurred as a result of freezing in certain pouch dogs.     

Flash freezing of erythrocyte suspensions

Freezing whole blood in bulk usually results in severe cellular destruction through the action of ice crystals and osmotic effects in the freezing liquid. The potential of flash freezing blood aerosols onto a liquid nitrogen surface as a means of inhibiting cellular damage was studied in this work. Three commercial spraying devices were employed to spray-freeze either whole blood or concentrated erythrocyte suspensions, using hydroxyethyl starch (HES) as a cryoprotectant. The integrity and viability of the processed cells were assessed by measuring gross rheological properties and the extent of hemolysis. Cells were found to be susceptible to the very high shear stresses imposed by some of the spraying devices. Bulk freezing of blood, even in the presence of the cryoprotectant, resulted in complete cellular destruction. Whereas flash freezing was capable of substantially reducing the level of hemolysis to 12.6% and preserving the cellular deformability.     

Optimization of the cryopreservation of African clawed frog (Xenopus laevis) sperm

Cryopreservation of testicular sperm in the African clawed frog, Xenopus laevis, was tested using three penetrating cryoprotectants (DMSO, methanol, and glycerol) and three semen diluents (300 mmol/L glucose, 300 mmol/L sucrose, and a motility inhibiting saline [MIS] solution [150 mmol/L NaCl, 3 mmol/L KCL, 1 mmol/L Mg₂SO₄, 1 mmol/L CaCl₂, and 20 mmol/L Tris, pH 8.0]). Three freezing rates and four thawing rates were also tested, and the best freezing/thawing conditions have been determined. The responses of sperm motility, viability, and fertility were assessed. Incubation of the sperm macerates with penetrating cryoprotectants showed that DMSO was the least toxic and methanol the most toxic. Semen in cryodiluents frozen 10 cm above the surface of liquid nitrogen (freezing rate of 20 to 25 °C/min) and thawed at room temperature for 40 sec had significantly higher percentages of motile and viable sperm than that of semen frozen 5 cm or 8 cm above the surface of liquid nitrogen and thawed at 5, 25, or 30 °C for 10, 15, or 60 sec, respectively. Sperm frozen in MIS containing 5% DMSO had a higher hatching rate than that of sperm frozen in sucrose and glucose diluents containing 5% or 10% DMSO and in MIS containing 10% DMSO. Addition of 73 mmol/L sucrose to the sperm extender MIS + 5% DMSO could improve the postthaw sperm motility and fertility. In conclusion, dilution of collected sperm in MIS solution (to have a final concentration of 6.5 × 10⁶ to 8 × 10⁶/mL) containing 5% DMSO and 73 mmol/L sucrose, freezing in a vapor of liquid nitrogen at 10 cm above the surface, and thawing at room temperature for 40 sec was the best cryopreservation protocol. This protocol gave 70% hatching rate, 80% motility rate, and 75% viability rate of fresh hormonally induced sperm.     

Cryopreservation of the SB-M photosynthetic soybean [Glycine max (L.) Merr.] suspension culture

Summary The photosynthetic cell suspension culture of soybean [Glycine max (L.) Merr. cv. Corsoy] (SB-M) was successfully cryopreserved in liquid nitrogen using a preculture and controlled freezing to −40° C (two-step) freezing method. The effective method included a preculture treatment with gradually increasing levels of sorbitol added to the 3% sucrose already present in the medium. The cells were then placed in a cryoprotectant solution [10% DMSO (dimethylsulfoxide) and 9.1% sorbitol, or 10% DMSO and 8% sucrose], incubated for 30 min at 0° C, cooled at a rate of 1° C/min to −40° C, held at −40° C for 1 h, and then immersed directly into liquid nitrogen. The cells were thawed at 40° C and then immediately placed in liquid culture medium. The cell viabilities immediately after thawing were 75% or higher in all cases where cell growth resumed. The original growth rate and chlorophyll level of the cells was recovered within 40 to 47 d. If the sorbitol level was not high enough or the preculture period too short, growing cultures could not be recovered. Likewise, survival was not attained with cryoprotectant mixtures consisting of 15% DMSO, 15% glycerol, and 9.1% sucrose or 15% glycerol and 8% sucrose. The successful method was reproducible, thus allowing long-term storage of this and certain other unique photosynthetic suspension cultures in liquid nitrogen.     

The Use of Microwaves to Monitor the Freezing and Thawing of Water in Plants

Harbinson, J. and Woodward, F. I. 1987. The use of microwavesto monitor the freezing and thawing of water in plants.—J.exp. Bot. 38: 1325–1335. The use of radiation with a frequency of 10·687 GHz tomeasure the freezing and thawing of water in plants is discussed.Results showing the freezing and thawing of plant material arepresented. In its simplest form the technique offers a methodfor investigating the freezing and thawing of water in plants. Key words: Freezing, microwaves     

Preservation of the proteolytic activity of a bovine spleen lysosomal-enriched extract using various freezing conditions

The preservation of the proteolytic activity of a bovine spleen lysosomal-enriched (BSLE) extract was investigated. The BSLE extract (pH = 5.8), was subjected to storage under different conditions: refrigeration at 0 degrees C for 60 days; freezing at -20 degrees C -either directly or previously frozen in liquid nitrogen-, -80 degrees C and in liquid nitrogen; freeze-drying and stored at 0 degrees C; and freezing at -20 degrees C or in liquid nitrogen in the presence of glycerol and sorbitol as cryoprotectants. Freezing at low temperatures (-80 degrees C and in liquid nitrogen) was most effective for preserving about 100% of the initial activity of all cathepsins (B, B+L and D), as well as the activity of the extract on myofibrils, for two years. Freezing at -20 degrees C, on the contrary, led to significant (P < 0.01) losses of activity. Freeze-drying was able to preserve cathepsin activity, while it failed to maintain activity on myofibrils. Both cryoprotectants sorbitol and glycerol significantly (P < 0.01) enhanced enzyme preservation, particularly cathepsin D and the activity on myofibrils, even at a freezing temperature of -20 degrees C.     

Effects of freezing on plasma membrane H<Superscript>+</Superscript>-ATPase of the callus from <Emphasis Type="Italic">Chorispora bungeana</Emphasis>

The influence of freezing treatment on plasma membrane (PM) H⁺-ATPase was investigated using plasma membrane vesicles isolated from calluses from Chorispora bungeana Fisch. & C.A. Mey. by the discontinuous sucrose gradient centrifugation. Freezing treatment (−4 °C) for 5 d resulted in significant increases in the ATPase activity and the activity of p-nitrophenyl phosphate (PNPP) hydrolysis, decreases in the K_m for ATP hydrolysis and PNPP hydrolysis, and the shift of optimal pH from 6.5 to 7.0. Also, the activity PNPP hydrolysis was less sensitive to vanadate after freezing treatment compared to control, while the inhibition of ATP hydrolysis by hydroxylamine was more sensitive. In addition, freezing treatment also decreased the activation effects of trypsin on PNPP hydrolysis, but increased the activation effects of lysophosphatidylcholine on ATP hydrolysis. Taken together, these results suggested that PM H⁺-ATPase might play an important role during adaptation to freezing and enhancing the frost hardness in C. bungeana.     

Effects of ice-seeding temperature and intracellular trehalose contents on survival of frozen Saccharomyces cerevisiae cells

Freezing tolerance is an important characteristic for baker’s yeast, Saccharomyces cerevisiae, as it is used to make frozen dough. The ability of yeast cells to survive freezing is thought to depend on various factors. The purpose of this work was to study the viability of yeast cells during the freezing process. We examined factors potentially affecting their survival, including the growth phase, ice-seeding temperature, intracellular trehalose content, freezing period, and duration of supercooling. The results showed that the ice-seeding temperature significantly affected cell viability. In the stationary phase, trehalose accumulation did not affect the viability of yeast cells after brief freezing, although it did significantly affect the viability after prolonged freezing. In the log phase, the ice-seeding temperature was more important for cell survival than the presence of trehalose during prolonged freezing. The importance of increasing the extracellular ice-seeding temperature was verified by comparing frozen yeast survival rates in a freezing test with ice-seeding temperatures of −5 °C and −15 °C. We also found that the cell survival rates began to increase at 3 h of supercooling. The yeast cells may adapt to subzero temperatures and/or acquire tolerance to freezing stress during the supercooling.     

Freezing solution containing dimethylsulfoxide and fetal calf serum maintains survival and ultrastructure of goat preantral follicles after cryopreservation and in vitro culture of ovarian tissue

Goat ovarian cortex fragments were subjected to slow freezing in the presence of various solutions containing intracellular cryoprotectants, including 1.0 M ethylene glycol (EG), propanediol (PROH), or dimethyl sulfoxide (DMSO), with or without sucrose and/or fetal calf serum (FCS). Histological examination revealed that only the DMSO-containing solutions were able to maintain a follicular ultrastructure similar to the morphology observed in the fresh control. Therefore, fragments previously cryopreserved in DMSO solutions (with and without sucrose and/or FCS) were cultured in vitro for 48 h and then subjected to viability, histological, and ultrastructural analysis. No significant differences were observed among the percentages of morphologically normal follicles in cryopreserved ovarian tissue before in vitro culture (DMSO: 62.5%; DMSO + sucrose: 68.3%; DMSO + FCS: 60.0%; DMSO + sucrose + FCS: 60.0%) and after culture (DMSO: 60.8%; DMSO + sucrose: 64.2%; DMSO + FCS: 70.8%; DMSO + sucrose + FCS: 55.0%). Following in vitro culture, the viability analysis showed that only the freezing solution containing DMSO and FCS (75.6%) maintained a percentage of viable follicles similar to that observed after culture without cryopreservation (89.3%). As determined by ultrastructural analysis, morphologically normal preantral follicles were detected in the fresh control and in fragments cultured before and after cryopreservation with DMSO and FCS. Thus, a freezing solution containing DMSO and FCS, under the experimental conditions tested here, guaranteed the maintenance of viability and follicular ultrastructure after short-term in vitro culture.     

Induction of freezing tolerance in microspore-derived embryos of winter Brassica napus

Freezing tolerance was induced in microspore derived embryos of winter Brassica napus cv. Jet neuf by the addition of ABA or mefluidide to the culture media during embryogenesis. Survival after freezing was estimated by culture of frozen-thawed embryos to plantlets. A higher freezing tolerance (50% survival at –15°C) was induced when 50 M ABA or 3.2 M mefluidide was incorporated initially into the medium during embryogenesis at 25°C followed by culture at 2°C for 3 weeks. When embryos were induced in the absence of ABA or mefluidide and maintained at 2°C for even as long as 12 weeks a lower degree of freezing tolerance (10% survival at –15°C) was obtained. Plants regenerated from embryos hardened maximally by a combination of either ABA or MFD with low temperature did not require further vernalization for flowering.Abbreviations ABA abscisic acid - MFD mefluidide - 2,4-D 2,4-dichlorophenoxyacetic acid - LT₅₀ killing temperature for 50% of the embryos     

Quick freezing of rat embryos

Quick freezing of rat morulae and blastocysts was attempted after they were dehydrated at room temperature. Combined solutions of 2.8 M glycerol and 0.125, 0.25, 0.50 and 1.0 M sucrose in phosphate buffered saline + 20% steer serum were compared. Survival rates (expanding blastocysts 15 h after thawing) were 42.1, 79.4, 87.5 and 16.7%, respectively (P<0.01). Freezing procedures consisted of either a direct plunge into liquid nitrogen (48.8%), holding for 5 min in the neck of a liquid nitrogen container or holding the samples for 60 min at -30 degrees C before insertion into liquid nitrogen. The direct plunge method resulted in a lower survival rate than either the 5- or the 60-min treatments (48.8% vs 76.9% and 77.6%, respectively). After thawing, dilution at room temperature in sucrose solutions of 0.25, 0.50 and 1.0 M gave survival rates of 80.0, 90.6 and 69.4%, respectively (NS). If diluted directly in PBS + 20% steer serum, 86.8% of embryos survived at +37 degrees C vs 0% at 0 degrees C (P<0.01).