Low-temperature preservation of mammalian cells in tissue culture with polyvinylpyrrolidone (PVP), dextrans, and hydroxyethyl starch (HES)

The effects of freezing and thawing on Chinese hamster cells in tissue culture in the presence of PVP, HES, and various dextrans have been investigated. Cooling and thawing rates within a limited range (20–260 °C per min for cooling and 6–115 °C per min for thawing) were studied and best results were achieved with a cooling rate of 20 °C per min and a thawing rate of 115 °C per min in both 10% PVP, and 10% HES. Experiments demonstrated HES to be as good as, and possibly better than PVP. A number of dextrans of average molecular weight (10,000–500,000 daltons) were shown to be poor as cryoprotective agents in contrast to results obtained with this polymer with red cells and bacteria. The presence of 10% serum during all freezing and thawing procedures decreased day-to-day variability and with dextrans increased their limited effectiveness.     

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 destruction of Salmonella typhimurium in chicken exudate by different freeze-thaw treatments

Antibacterial treatments for frozen poultry, including holding at -5°C and slow thawing at 4°C to which exponential phase cells of Salmonella typhimurium were susceptible, were found to be relatively ineffective against stationary phase cells. Exposure of the latter, however, to a pre-freezing triple stress treatment of cold-shock exposure at 5°C to a solution containing 5 mg/l of free available chlorine in 1% succinic acid (pH 2.5) for 20 min substantially lowered the resistance of the cells to subsequent freezing, storage and thawing in poultry flesh exudate. Cell survival was further decreased by storage of exudate at - 18°C for 28 d and this reduced the proportion of stationary phase cells to less than 1% of initial numbers, with a concomitant increase in sensitivity to deoxycholate. Such a combined pre-treatment may have practical potential for salmonella decontamination in the production of frozen poultry.     

Evaluation of freezing injury and freeze-thaw injury to membranes of Saskatoon serviceberry twigs by measuring HCN release

The influence of thawing on freeze-injured Saskatoon serviceberry ( Amelanchier alnifolia Nutt.) twigs was evaluated by refreezing freeze-thawed twigs and comparing the HCN release at -5°C fro these twigs to the HCN release at -5°C from twigs that had not been thawed. An effect of thawing depended on the physiological state of the twigs, on the degree of freezing stress, or on both. Manifestation of membrane injury does not have an absolute dependence on thawing. Post-thaw temperature influences manifestation of injury, since twigs warmed to 30°C released more HCN than twigs warmed to 1°C when refrozen to -5°C. Although thawing and post-thaw conditions can influence the magnitude of membrane injury, the critical event leading to injury occurs while plants are frozen.     

The origin and development of somatic embryos following cryopreservation of an embryogenic suspension culture ofPicea sitchensis

Summary The development of somatic embryos in an embryogenic suspension culture ofPicea sitchensis was followed every day for two weeks after thawing from liquid nitrogen (LN₂). Only a few cells, primarily located at the periphery of the embryonic region of the embryos, survived cryopreservation in LN₂. Surviving cells were classified into two groups: embryogenic cells (EC) and non-embryogenic cells (NEC), based on their morphology and embryogenic competence. The dense cytoplasmic EC underwent organized growth and differentiation with first divisions occurring after 24 h, and embryo formation 6–8 days after thawing from LN₂. No evidence of asymmetrical divisions or free-nuclear stages was found during somatic embryo formation. NEC had less dense cytoplasm with numerous small vacuoles. One to five days after thawing the NEC became progressively more vacuolated and elongated. Histological examination revealed no mitotic activity in NEC, and six days after thawing NECs were seen as single cells or unorganized cell aggregates. Two weeks after thawing the appearance of the cryopreserved cultures was comparable to that of the untreated cultures.Abbreviations EC embryogenic cells - ECC embryogenic cell clusters - FDA fluorescein diacetate - GMA glycol methacrylate - LN₂ liquid nitrogen (–196°C) - NEC non-embryogenic cells     

Freezing stress and membrane injury of Norway spruce (Picea abies) tissues

Effects of sub-zero temperatures (−5 to −35°C) on the tissues of needles, buds and shoots of Norway spruce [ Picea abies (L.) Karst.] were studied. The freezing caused increased efflux of cellular electrolytes. Freezing injury of the primordial shoots and 1-year-old shoots was the result of the spontaneous freezing of a deep supercooled cellular water. The crystallization injures the cellular membranes leading to the loss of semipermeability and to the drastic efflux of K⁺. In the needles there was no deep supercooling of water and two patterns of changes in the membranes, depending upon the range of the applied temperatures, could be distinguished. At 0 to – 25°C, which do not kill the cells, we observed a disturbance in the membrane semipermeability as monitored by electrolytes efflux within a few hours after thawing of the needles. At lower temperatures (−35°C) we observed irreversible loss of the membrane semipermeability, and death of the tissue. Those changes occurred 10 h after thawing and were probably caused by the released lytic enzymes and some toxic compounds, which acted on the cellular membranes.     

The lethal effect of carrot on Listeria species

When shredded or sliced carrots were inoculated with Listeria monocytogenes the number of viable listerias decreased rapidly. On carrot slices stored at 8°C there was a decrease after 3 d followed by an increase, after 7 d, to numbers similar to those present initially. The numbers of spoilage micro-organisms increased throughout storage at 8°C. Carrots macerated in a Stomacher Lab Blender also showed an antilisterial activity which resulted in a decrease in number of viable bacteria and in sublethal damage. The effect was shown by five carrot cultivars and acted on nine strains of L. monocytogenes and single strains of L. innocua, L. ivanovii, L. seeligeri, L. welshimeri . This antilisterial effect was heat-labile, was inactivated after a few hours at 4°C or at 30°C and was active over the pH range 5.8 to 7.0. Maceration of carrots in an Atomix blender for 1 min or in liquid nitrogen destroyed the antilisterial activity.     

Temperature inducible protochlorophyllide reduction in darkness in a pigment mutant of Scenedesmus obliquus

Accumulation of chlorophyll and protochlorophyllide (PChlide) was followed during beterotrophic growth of the pigment mutant C-2A' of Scenedesmus obliquus L. in the darkness at 30 and 20°C. At 30°C the cells remained yellow with accumulation of protochlorophyllide, whereas they became green at 20°C with only traces of protochlorophyllide. The capacity of mutant cells to reduce PChlide to chlorophyllide (Chlide) in the dark with or without addition of 5-aminolevulinic acid as measured in isolated membranes, was high in cells grown at 20°C but negligible at 30°C. The high capacity to reduce PChlide created in cells growing at 20°C was only slightly diminished by exposure of cells to 38°C for 3 h. Mechanisms of temperature-sensitive chlorosis in algae and higher plants are discussed in relation to the results with pigment mutant C-2A' of Scenedesmus obliquus . It is assumed that either an activator of NADPH protochlorophyllide oxidoreductase (EC 1.6.99.1) or a different enzyme system can be activated by lower temperature as by light.     

Relationship between variations in pathogenicity and lag phase at 37°C of Listeria monocytogenes previously stored at 4°C

s. BUNCIC AND S.M. AVERY. 1996. Three haemolytic, pathogenic strains of Listeria monocytogenes (a reference strain, a food-derived strain and a human strain) were held at 4°C for 4 weeks in phosphate-buffered saline pH 5.5 or 7.0, with and without 0.2% potassium sorbate or 0.3% sodium acetate. The number of viable cells did not change significantly during this storage. Pathogenicity of non-growing L. monocytogenes cells for 14-d-old chick embryos was determined before and after storage. Storage at 4°C resulted in decreased pathogenicity, but effects were strain-, pH- and substrate-dependent. After 4 weeks storage at 4°C non-growing bacterial cells were transferred to Brain Heart Infusion broth and growth characteristics were determined during incubation at 37°C. Strains that showed decreased pathogenicity had significantly longer lag phases at 37°C than strains that maintained pathogenicity. It is concluded that decreased pathogenicity of L. monocytogenes stored without growth at 4°C for 4 weeks and subsequent long lag phase at 37°C are correlated.     

Isolation and characterization of nisin-producing Lactococcus lactis subsp. lactis from bean-sprouts

Bacterial isolates from bean-sprouts were screened for anti- Listeria monocytogenes bacteriocins using a well diffusion method. Thirty-four of 72 isolates inhibited the growth of L.monocytogenes Scott A. One, HPB 1688, which had the biggest inhibition zone against L.monocytogenes Scott A, was selected for subsequent analysis. Both ribotyping and DNAsequencing of 16S ribosomal RNA gene demonstrated that the isolate was Lactococcus lactis subsp. lactis . Polymerase chain reaction and nucleotide sequencing revealed that thegenomic DNA of the bean-sprout isolates contained a nisin Z structural gene. In MRS broth,bean-sprout isolate HPB 1688 survived at 3–4·5°C for at least 20 d, grew at 4°Cand produced anti-listerial compoundsat 5°C. When co-cultured with L. monocytogenes in MRS broth, the isolate inhibited thegrowth of L. monocytogenes at 4°C after 14d and at 10°C after 2 d. When co-inoculatedwith 10²cells g⁻¹ of L.monocytogenes on fresh-cut ready-to-eat Caesar salad, L. lactis subsp. lactis (10⁸cells g⁻¹) was able to reduce the number of L. monocytogenes by 1–1·4 logs after storage for 10 d at 7° and 10°C. A bacteriocin-producing Enterococcusfaecium was also able to reduce the numbers of L. monocytogenes onCaesar salad, butdid not act synergistically when co-inoculated with L. lactis subsp. lactis .     

The regulation of the rate of ATP hydrolysis by H-meromyosin

The effect of N-ethylmaleimide on the ATPase activity and ADP binding of tryptic H-meromyosin was studied at 6 and 23 °C temperatures. The affinity constant of H-meromyosin for ADP with Mg as activator was increased by small concentrations of N-ethylmaleimide (2.25 moles per mole of enzyme) at both temperatures, accompanied by activation of ATP hydrolysis at 25 °C and inhibition at 6 °C. With higher N-ethylmaleimide concentrations, the ATPase activity was inhibited at both temperatures, without comparable inhibition of ADP binding. Rapid kinetic analysis of the rate of development of difference spectrum after the addition of ATP or ADP to H-meromyosin indicates, that blocking of the S₁ and S₂ SH groups of H-meromyosin decreases both the formation (k₁) and the dissociation (k₂) rate constants of H-meromyosin substrate complex. At 6 °C, in the presence of Mg, the value of k₂ for ADP is similar to the turnover number of ATP hydrolysis, suggesting that dissociation of ADP from the active site may be the rate-limiting step of ATP hydrolysis. At 23 °C, the turnover number of Mg-moderated ATP hydrolysis is much smaller than k₂, indicating that the rate limitation shifted so another, so far unidentified, step.     

A simplified method for screening acetylcholinesterase inhibitors by the flesh fly (Dipt., Sarcophagidae) cell culture system

A simplified economical method for assaying acetylcholinesterase inhibitors was devised. A flesh fly, Sarcophaga peregrina Robineau-Desvoidy, cell line, NIH-SaPe-4, was cultured in 96-well microplates at 25°C. After 2 days culture, the culture media was removed and phosphate buffer was introduced. The cells were disintegrated by freezing–thawing, and a reaction mixture containing substrate, inhibitor, and colour developer was added. The change of absorbance at 405 nm at 25°C was measured with a microplate photometer and the concentration of the test substance required to inhibit the enzyme reaction by 50% (I₅₀) was calculated.     

Influence of the sporulation temperature upon the heat resistance of Bacillus subtilis

The influence of different sporulation temperatures (30, 37, 44 and 52°C) upon heat resistance of Bacillus subtilis was investigated.
Heat resistance was greater after higher sporulation temperatures. Relation of heat resistance and temperature of sporulation was not linear over all the range of temperatures tested. Heat resistance increased about tenfold in the range of 30–44°C. Sporulation at 52°C did not show any further increase in heat resistance.
This effect was constant over all the range of heating temperatures tested (100–120°C). z value remained constant ( z = 9°C).
Greater heat resistances at higher temperatures of sporulation were not due to selection of more heat resistant cells by a higher sporulation temperature. Spores obtained from cells incubated at 32 or 52°C always possessed heat resistances that corresponded to the sporulation temperature regardless of the incubation temperature of their vegetative cells.     

Cryopreservation of heart cells from the eastern oyster

Summary Conditions were developed to cryopreserve cells from pronase-dissociated atria and ventricles of eastern oysters (Crassostrea virginica). The effect of three concentrations (5, 10, 15%) of the cryoprotectants (dimethyl sulfoxide, glycerol, and propylene glycol), three thawing temperatures (25, 45, 75°C), and three cooling rates (slow, medium, fast) were compared. Cells were frozen at −80°C and plunged in liquid nitrogen. Thawed cells were seeded in 96-well plates and primary cultures were evaluated after 3 d by measuring the metabolic activity using a tetrazolium compound, 3-(4,5-dimethylthiazol-2-yl)-5-( 3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, and by comparing the relative spreading of cells between treatments. The best conditions for freezing and thawing of cells for each cryoprotectant were selected and a final study was performed to compare cryoprotectants. For this final study, we measured the number of cells and their viability 3 d after thawing, in addition to determining cell metabolic activity and cell spreading. Primary cultures of cells frozen without cryoprotectant and of nonfrozen cells were used as controls in all studies. Atrial cells were best cryopreserved with glycerol at a concentration of 10%, a medium cooling rate, and thawing at 45°C. After thawing, atrial cells showed 53±5% of the metabolic activity, 84±5% of the number, and 92±2% of the viability of nonfrozen cells. For ventricular cells, 10% glycerol, a medium cooling rate, and thawing at 25°C yielded the best results. The thawed ventricular cells showed 83±5% of the metabolic activity, 91±5% of the number, and 96±2% of the viability of nonfrozen cells.     

GRAZER-INDUCED COLONY FORMATION IN SCENEDESMUS ACUTUS (CHLOROPHYCEAE): ECOMORPH EXPRESSION AT DIFFERENT TEMPERATURES

Scenedesmus acutus Meyen was cultured at four temperatures (9.5°, 16.5°, 24°, and 29° C) in standard medium or in medium with filtered water from a Daphnia culture. Growth was significantly reduced at low temperatures. At 9.5° C it took more than a week before formation of eight-celled coenobia occurred in both the absence and presence of water from a Daphnia culture. At higher temperatures, formation of four- and eight-celled coenobia occurred more rapidly and was already observed in the presence of Daphnia water within 2 days. As cultures aged, also in the absence of Daphnia water, four-celled coenobia became dominant. At cold temperature, cell volume initially was significantly larger but declined after 3–4 weeks. Grazer-induced colony formation had occurred independent of the incubation temperature, but the number of cells per colony was increased with declining temperature. The morphological expression may be interpreted as a cyclomorphosis driven by nutrients, temperature, and chemical cues from grazers.     

Elevation of the heat resistance of Salmonella typhimurium by sublethal heat shock

The survival of Salmonella typhimurium after a standard heat challenge at 55°C for 25 min increased by several orders of magnitude when cells grown at 37°C were pre-incubated at 42°, 45° or 48°C before heating at the higher temperature. Heat resistance increased rapidly after the temperature shift, reaching near maximum levels within 30 min. Elevated heat resistance persisted for at least 10 h. Preincubation of cells at 48°C for 30 min increased their resistance to subsequent heating at 50°, 52°, 55°, 57° or 59°C. Survival curves of resistant cells were curvilinear. Estimated times for a '7D' inactivation increased by 2.6- to 20-fold compared with cells not pre-incubated before heat challenge.     

The autophagy-related protein LC3 is processed in stallion spermatozoa during short-and long-term storage and the related stressful conditions

Use of cooled and frozen semen is becoming increasingly prevalent in the equine industry. However, these procedures cause harmful effects in the sperm cell resulting in reduced cell lifespan and fertility rates. Apoptosis and necrosis-related events are increased during semen cryopreservation. However, a third type of cell death, named autophagy, has not been studied during equine semen storage. Light chain (LC)3 protein is a key component of the autophagy pathway. Under autophagy activation, LC3-I is lipidated and converted to LC3-II. The ratio of LC3-II/LC3-I is widely used as a marker of autophagy activation. The main objective of this study was to investigate whether LC3 is processed during cooling, freezing and the stressful conditions associated with these technologies. A secondary objective was to determine if LC3 processing can be modulated and if that may improve the quality of cryopreserved semen. LC3 processing was studied by Western blot with a specific antibody that recognized both LC3-I and LC3-II. Viability was assessed by flow cytometry. Modulation of LC3-I to LC3-II was studied with known autophagy activators (STF-62247 and rapamycin) or inhibitors (chloroquine and 3-MA) used in somatic cells. The results showed that conversion of LC3-I to LC3-II increased significantly during cooling at 4°C, freezing/thawing and each of the stressful conditions tested (UV radiation, oxidative stress, osmotic stress and changes in temperature). STF-62247 and rapamycin increased the LC3-II/LC3-I ratio and decreased the viability of equine sperm, whereas chloroquine and 3-MA inhibited LC3 processing and maintained the percentage of viable cells after 2 h of incubation at 37°C. Finally, refrigeration at 4°C for 96 h and freezing at −196°C in the presence of chloroquine and 3-MA resulted in higher percentages of viable cells. In conclusion, results showed that an ‘autophagy-like’ mechanism may be involved in the regulation of sperm viability during equine semen cryopreservation. Modulation of autophagy during these reproductive technologies may result in an improvement of semen quality and therefore in higher fertility rates.     

Isolation and properties of LC3 cells, a new cold-resistant L cell subline.

LC3 cells, selected from the L-As cells by repeated exposures to 4 degrees C for 3--6 weeks with intermittent reincubations at 36 degrees C, differ from the initial population by better survival at 4 degrees C, more rapid recovery at 36 degrees C, a higher multiplication at subnormal temperature, a higher sensitivity to supranormal temperature, increased cell size at 36 degrees and 4 degrees C, and higher oxygen consumption at 36 degrees C. These properties are the same as those described in our previously isolated cold-resistant L cell variants and are typical for the resistance to low temperature. The increased activity of alkaline phosphatase, detected in two of our cold-resistant L cell sublines, was not found in the LC3 cells and has thus no relation to decreased cold sensitivity.     

Effect of culture age, pre-incubation at low temperature and pH on the thermal resistance of Aeromonas hydrophila

S. CONDON, M.L. GARCIA, A. OTERO AND F.J. SALA. 1992. The thermal resistance of Aeromonas hydrophila strain NCTC 8049 was determined within the range 48°-65°C with a thermoresistometer TR-SC and McIlvaine buffer. The effects of culture age, pre-incubation at 7°C and the pH of the heating menstruum were evaluated. The pattern of thermal death was dependent on culture age. Cells heated in the late logarithmic growth phase (15 h at 30°C) were twice as resistant as those in the early stage (5 h at 30°C), and the maximum D -value was obtained after 72 h incubation (5.5 total increase). The age of the cells did not affect z -values significantly. The heat resistance of cells incubated for 48 h at 30°C increased (twice) after holding at 7°C for 72 h. Pre-incubation at low temperature of older cultures (72 h, 30°C) did not influence their D -values. Maximum heat resistance was found at pH 6.0 and minimal at pH 4.0. Decreasing the pH from 6.0 4.0 reduced D -values by a factor of 5. Although the strain studied was heat-sensitive ( D _55°C= 0.17 min; z = 5.11°C), survivor curves of cultures older than 50 h showed a significant tailing. Organisms surviving in the tails were only slightly more resistant than were the original population.     

A cryopreservation procedure for the rumen protozoon Entodinium caudatum: estimation of its viability by fluorescence microscopy

AIMS: To study the viability of a culture of the rumen protozoon Entodinium caudatum after a cryopreservation procedure by a fluorescence microscopy staining method. METHODS AND RESULTS: Fluorescence method is based on the different colour of cells depending on their membrane integrity. When the temperature effect was studied either by fluorescence or motility, the techniques were correlated (r = 0.727) and their slopes and intercepts were not different (P > 0.05). However, motility showed a higher variation coefficient (0.40 vs 0.12). There were no differences between cooling rates at cryopreservation (1 and 4 degrees C min-1) at 38, 15 or 5 degrees C, nor after thawing. CONCLUSIONS: Fluorescence staining is more accurate than motility for assessing protozoal viability. Viability after thawing was 0.50, and the number of viable cells per 250 microl straw was 320 and 420 for 1 and 4 degrees C min-1. SIGNIFICANCE AND IMPACT OF THE STUDY: This cryopreservation procedure seems to ensure culture recovery for E. caudatum.