Use Of Cryoprotectants Research Articles

Use of Cryoprotectants to Stabilize Functional Properties of Prerigor Salted Beef During Frozen Storage

ABSTRACTChanges in functional properties (gelforming ability, water‐holding capacity, and protein solubility) of salted prerigor beef and postrigor beef were measured over 6 months frozen storage at −28°C, as affected by addition of 5.6% cryoprotectants [Polydextrose® or a mixture (1:1) of sucrose and sorbitol]. Addition of NaCl to comminuted muscle prior to freezing accelerated destabilization of muscle proteins with respect to functional properties. This effect was reduced by cryoprotectants; most effectively by sucrose/sorbitol and only slightly less effectively by polydextrose. As evidenced by the parameters investigated, quality of prerigor salted muscle treated with cryoprotectants and stored 6 months was approximately equal to that of untreated postrigor meat prior to freezing.

Crystallization and vitrification in aqueous systems

SUMMARYSome key ideas and experimental findings concerning the probability that crystallization of a liquid or its binary solutions will occur at moderate cooling rates are discussed. The use of cryoprotectants and of pressure to favourably influence these probabilities is rationalized, and some of the newer findings on small sample supercooling phenomena are reviewed.

Studies on the cryopreservation of Dictyocaulus viviparus (Nematoda) third-stage larvae.

Various cooling (0.1-5,100 degrees C min-1) and warming (20-6,800 degrees C min-1) rates, stepped cooling schedules and four cryoprotective additives (dimethyl sulphoxide, methanol, ethanediol and glycerol) were investigated in cryopreservation studies with Dictyocaulus viviparus third-stage larvae. Exsheathment with sodium hypochlorite was essential to achieve significant survival. With uninterrupted cooling, highest survival (30% normally motile) was achieved with rates of 10-70 degrees C min-1. Survival was higher (50-75%) using 1 degree C min-1 to -10 degrees C followed by plunging into liquid nitrogen. The optimum warming rate was 6,800 degrees C min-1. The use of cryoprotectants led to marginally lower survival while varying the suspending media had no significant effect on survival. X-irradiated, exsheathed third-stage larvae cryopreserved by the optimum protocol yielded 38.3 +/- 4.2% survival. Two calves each infected with 45,000 (15,000 viable) exsheathed, unirradiated, cryopreserved third-stage larvae harboured 494 worms (1.1% infectivity) and 355 worms (0.8%) at necropsy. Numbers of first-stage larvae in the faeces reached 420/g and 105/g respectively 27 days after infection.

Application of X-ray microanalysis to cell suspensions of oil palm (Elaeis guineensis Jacq.)

X-ray microanalysis has been used to determine the elemental composition of oil-palm (Elaeis guineesis) cell suspensions without the use of cryoprotectants. Results based on individual cells were gathered over a typical growth cycle of 14 d. During the log phase (5-7 d) there is an increase in the number of cells containing high concentrations of both K (400 mmol kg(-1) dry weight) and P (400 mmol kg(-1) dry weight). Morphologically these cells had thin cell walls and were frequently joined to other cells (two to five cells per clump).

Quantitation of the intramembrane particle distribution in C3H/10T½ cells treated with TPA and retinyl acetate.

The mouse embryo fibroblast C3H/10T½ cells are used as a model system for the study of ultrastructural changes caused by tumor promoter and “anti-promoter” treatments. Noninitiated fibroblasts treated with tumor promoter undergo phenotypic transformation. On the other hand, the retinoid, retinyl acetate (RA), is effective in suppression of the malignant phenotype in the 10T½ cells (1). We report on the rapid freezing and freeze fracture electron microscopy study of the plasma membrane ultrastructures of noninitiated C3H/10T½ fibroblasts after treatments of retinyl acetate or a tumor promoter, 12-0-tetradecanoyl-phorbol-13-acetate (TPA).Rapid freezing of cells enables structure to be preserved without the use of cryoprotectants and chemical fixatives, which may alter membrane structural organization. For rapid freezing, 10T½ cells were grown as monolayers on glass specimen supports. The glass-copper specimen holder sandwiches were rapidly frozen by automatic plunging into liquid propane. Freeze fracture was carried out in a Polaron-Ultek ion pump apparatus with Cressington electron guns.

Scanning electron microscopy of cytoskeletal components in Aucuba japonica leaves

A technique for fracturing plant tissues to permit the examination of internal features of cells by scanning electron microscopy is described. Specimens were fractured fresh, without the use of cryoprotectants or chemical fixatives, macerated with a dilute solution of osmium tetroxide, and then critical-point-dried. Cytoskeletal components were observed in mesophyll cells of Japan Laurel, Aucuba japonica (Cornaceae), although they were absent in preparations using fixatives or cryoprotectants. They consisted of radiating arrays of fibrils associated with chloroplasts, plasma membranes, or aggregations of spherical bodies. In places the fibrils were seen to be bundles made up of finer strands of dimensions similar to microtubules.

The effect of glycerol on aggregation and ultrastructure of human platelets

Cryopreservation of platelets depends on the use of cryoprotectants to reduce freezing damage. However, the cryoprotectants may in themselves be harmful, and it is important to determine the amount of damage caused by these compounds. Platelets were incubated at 37 °C in plasma containing 0, 0.5 and 1.0 mol/liter glycerol. The aggregation response to 10 and 5 μmol/liter ADP was determined after 2, 15, 30, 60, and 120 min of incubation. Samples were prepared for electron microscopy after 30 min at 37 °C. Glycerol at a concentration of 0.5 mol/liter had no effect on the extent of aggregation, whereas 1.0 mol/liter glycerol caused a progressive decline in the response. However, platelet ultrastructure appeared to be undisturbed by 1.0 mol/liter glycerol. The results demonstrated a lack of toxicity of 0.5 mol/liter glycerol and support the use of glycerol at concentrations less than 1.0 mol/liter for cryopreservation.

Improved indirect hemagglutination test for cytomegalovirus using human O erythrocytes in lysine

A modified indirect hemagglutination test for cytomegalovirus antibodies is described in which glutaraldehyde-fixed human O cells, rather than sheep cells, are used. Nonspecific hemagglutination was reduced by use of the optimal tannic acid concentration for each cell batch and the addition of 0.1 M lysine to the phosphate-buffered saline in which the fixed, tanned, sensitized cells were resuspended for use in the test. Three of 349 sera showed nonspecific hemagglutination by this technique. Antigen made by freezing phosphate-buffered saline (pH 7.2) over an infected monolayer can be used at dilutions of 1:8 to 1:15. Fixed, tanned, sensitized cells ready for use in the test can be stored in liquid nitrogen for up to 8 months. Use of cryoprotectants and washing after thawing is unnecessary. Simplification of the assay permits one person to screen 300 sera in 1 day or to determine the immune status of a potential donor or recipient in 45 min after the test is set up. The modified assay compares favorably in sensitivity with the complement fixation test and with previously described methods for performing the indirect hemagglutination test.

Skin Storage in Liquid Nitrogen

The problem of cell survival as well as the biochemical and histological effects on skin of low temperature storage in liquid nitrogen has already received attention. However, little is known of the submicroscopic alterations induced by such treatment. Our study shows that rapid or slow freezing, direct or mediate contact with the liquid nitrogen, pretreatment before freezing, type and concentration of cryoprotectants, all have various effects on the fine morphology of epidermal cells. Glycerol is shown as having better cryoprotective activity than DMSO at concentrations of 15% or less. Our data are examined taking into account all available reports on the problem of cryopreservation and the use of cryoprotectants.

A study of rapid mitochondrial structural changes in vitro by spray-freeze-etching.

The spray-freeze-etching technique has been used to study energy-linked mitochondrial structural changes in rat liver mitochondria incubated in vitro. The technique involves spraying the suspension of mitochondria into liquid propane at -190 degrees C, and does not require the use of cryoprotectants or chemical fixatives. The results confirmed that freshly isolated mitochondria have a condensed matrix and that this expands at the expense of the outer compartment to give the orthodox configuration when the mitochondria are incubated in a K+ medium in the presence of substrate and phosphate. Addition of adenosine diphosphate (ADP) caused a rapid shrinkage of the matrix compartment, and the time-course and extent of this shrinkage has been measured quantitatively by coupling a rapid sampling device to the spray-freezing apparatus. These data show that for orthodox mitochondria the onset of phosphorylation is accompanied by a reduction of 30% in the matrix volume in 20's, and there is no evidence that the decrease in matrical volume affects the phosphorylation efficiency. These results suggest that natural ionophores in the mitochondrial inner membrane make it permeable enough to permit a rapid readjustment of matrix volume after the addition of ADP, and that the associated ion movement does not cause uncoupling of oxidative phosphorylation.

The ultrastructure of rat intestinal epithelium as revealed by cryoultramicrotomy and transmission electron microscopy

Rat jejunal mucosa, rapidly frozen without the use of cryoprotectants, has been sectioned by cryoultramicrotomy and examined by transmission electron microscopy. The quality of the sections varied with their morphological origin, but acceptable preservation of ultrastructure was obtained near the extremities of the villi. The appearance of the brush border indicated that the microvilli were covered with a substantial mucous coating.