Rabbit and pig ear skin sample cryobanking: effects of storage time and temperature of the whole ear extirpated immediately after death

The post-mortem temporal and thermal limits within which there will be ample guarantees of rescuing living skin cells from dead specimens of two species, rabbit and pig, were studied. Post-mortem extirpated whole ears were stored (in non-aseptic conditions) either at 4 degrees C or at room temperature (from 22 to 25 degrees C) or at 35 degrees C for different time lapses after animal death. In both species, the post-mortem maximum time lapses where cell viability was not significantly reduced were 240, 72, and 24 h post-mortem (hpm) for 4, 22-25 and 35 degrees C, respectively. Once the post-mortem temporal limits for each tested thermal level at which cells from skin samples are able to grow in culture were defined, the survival ability of skin samples submitted to these temporal limits and cryopreserved were tested. In the pig, skin samples stored at the three tested thermal levels survived after vitrification-warming, reaching confluence in culture. In rabbit, only tissue samples from ears stored at 35 degrees C for 24 hpm did not survive after vitrification-warming. In conclusion, we should remark that cell survival rates obtained according to the assayed post-mortem time lapses and thermal levels are sufficient to collect and to cryopreserve skin samples from the majority of dead specimens.     

Stabilization of frozen Lactobacillus delbrueckii subsp. bulgaricus in glycerol suspensions: Freezing kinetics and storage temperature effects

The interactions between freezing kinetics and subsequent storage temperatures and their effects on the biological activity of lactic acid bacteria have not been examined in studies to date. This paper investigates the effects of three freezing protocols and two storage temperatures on the viability and acidification activity of Lactobacillus delbrueckii subsp. bulgaricus CFL1 in the presence of glycerol. Samples were examined at -196 degrees C and -20 degrees C by freeze fracture and freeze substitution electron microscopy. Differential scanning calorimetry was used to measure proportions of ice and glass transition temperatures for each freezing condition tested. Following storage at low temperatures (-196 degrees C and -80 degrees C), the viability and acidification activity of L. delbrueckii subsp. bulgaricus decreased after freezing and were strongly dependent on freezing kinetics. High cooling rates obtained by direct immersion in liquid nitrogen resulted in the minimum loss of acidification activity and viability. The amount of ice formed in the freeze-concentrated matrix was determined by the freezing protocol, but no intracellular ice was observed in cells suspended in glycerol at any cooling rate. For samples stored at -20 degrees C, the maximum loss of viability and acidification activity was observed with rapidly cooled cells. By scanning electron microscopy, these cells were not observed to contain intracellular ice, and they were observed to be plasmolyzed. It is suggested that the cell damage which occurs in rapidly cooled cells during storage at high subzero temperatures is caused by an osmotic imbalance during warming, not the formation of intracellular ice.     

Fluid storage of thrombocytes. In vitro viability of thrombocytes at 12 degrees C storage temperature

Platelet concentrates from ACD-blood were stored with and without agitation at +12 degrees C for 3 days. pH-values, hypotonic shock response, serotonin uptake and adenine nucleotides were investigated. In addition platelet shape was morphologically differentiated in discs with and without pseudopods, in spheres and irregular platelets. In accordance with platelet storage at +22 degrees C an optimum platelet number and gentle agitation were essential to maintain in vitro vitality at +12 degrees C for 3 days. The number of discoid platelets declined from 76% in fresh platelet concentrates to 25% after 3 days at +12 degrees C although pH-values did not fall below 6.8 in the agitated concentrates. A diminished post-transfusion survival could result from this, unless the shape change reverses in the circulation.     

Mechanical effects on endothelial cell morphology: In vitro assessment

Summary Endothelial cells are subjected to fluid mechanical forces which accompany blood flow. These cells become elongated and orient their long axes parallel to the direction of shear stress when the cultured cells are subjected to flow in an in vitro circulatory system. When the substrate is compliant and cyclically deformed, to simulate effects of pressure in the vasculature, the cells elongate an orient perpendicular to the axis of deformation. Cell shape changes are reflected in the alignment of microtubule networks. The systems described provide tools for assessing the individual roles of shear stress, pressure, and mechanical strain on vascular cell structure and function. This work was partially supported by grants HL 17437, HL 18072, and HL 23016 from the National Institutes of Health, Bethesda, MD, and grant C-938 from the Robert A. Welch Foundation.     

In vitro pollen competitive ability in Viola tricolor: temperature and pollen donor effects

In this study on Viola tricolor pollen, the competitive ability of 16 pollen donors originating from a wild population was analysed in a set of greenhouse and germination temperatures. The aim was to examine the consistency in donor pollen performance across temperatures and to see whether variation in performance was random or due to individual differences in the plastic response to temperature. Pollen tube growth rate in vitro was investigated in two greenhouse temperatures (on the day pollen was collected) and in four germination temperatures. In addition, pollen tube growth rate was assessed in vivo (in one temperature) to examine the relationship between in vivo and in vitro growth. A temperature difference of 5 K - corresponding to natural variation in time and space detected in the field - affected pollen tube growth rate. For both temperature components, significant pollen donor by temperature interactions were found and rank order of pollen donors changed across treatments. Although pollen competitive ability in violets was strongly influenced by both temperature components, the occurrence of pollen donor by temperature interactions indicates that donor siring ability varies with temperature. This, in turn, may suggest a means to maintain pollen competitive ability despite selection for this trait.     

More on effects of storage time and temperature on urinary enzymes: a 1-year study.

Results of our conclusive study on urinary enzyme stability during sample storage are reported. We measured alanine aminopeptidase (AAP) and N-acetyl-beta-D-glucosaminidase (NAG) in morning urines from 9 healthy normal subjects immediately after collection and throughout a 1-year storage at -70 and -20 degrees C. AAP proved to be quite stable at -70 degrees C (99.2% of the basal value at the end of the year). NAG is partially preserved (84.1% of the basal value) at -70 degrees C, but significantly decreased (50.4%) at -20 degrees C.     

Long term t in vitro storage of lily: effects of temperature and concentration of nutrients and sucrose

Methods for long-term preservation of lily germplasm were examined. t In vitro regenerated bulblets of 10 lily (t Lilium L.) genotypes (Asiatic hybrids, Oriental hybrids, t L. longiflorum and t L. henryi) were stored for 28 months at -2 °C and 25 °C on four different media: 1/4 or full strength Murashige and Skoog nutrients with 9% (w/v) or 6% sucrose. Sprout growth, bulb growth, and viability were determined. The combination of 1/4 strength MS nutrients and 9% sucrose gave the highest reduction in sprout and bulb growth, the highest viability and the highest percentage of regrowth after 28 months of storage. At 25 °C, all lily genotypes survived 28 months of storage under these conditions. At -2 °C, Asiatic and Oriental hybrids survived 28 months of storage, whereas genotypes of t L. longiflorum and t L. henryi survived 6 months of storage, but died during prolonged storage. This revised version was published online in June 2006 with corrections to the Cover Date.     

A convenient on-line device for reagent addition, sample mixing, and temperature control of cell suspensions in flow cytometry

We describe a simple and inexpensive device which permits the addition of up to three different solutions into a cell suspension which is on-line in a flow cytometer. The mixing chamber houses a disposable plastic cuvette stirred with a magnetic stirrer. The sample chamber is attached to a circulating water bath, hence accurate temperature control is achieved. Because the system is prepressurized and the sample line is very short, the delay time-between the point of sample modification and the point of analysis is reduced to a few seconds. Thus reagents may be added rapidly, and kinetic measurements of high temporal resolution are possible. Because the temperature of the sample chamber is regulated, binding can be observed over longer time periods than was previously possible. We demonstrate the usefulness of this device in determining the binding of fluoresceinated hexapeptide to human neutrophils under conditions where the stimulus is infused into the cell suspension while on-line in the cytometer.     

In vitro synthesis of barley storage proteins

Membrane-bound polysomes were isolated from developing endosperms of barley (Hordeum vulgare L.) and shown to support the synthesis of trichloroacetic acid-insoluble material by an in vitro wheat germ protein synthesis system. The mRNA associated with the polysomes was separated from the ribosomes by affinity chromatography on oligo-dT cellulose and was also shown to support in vitro protein synthesis. The poly-A⁺ RNA isolated contained material of between 0.55 and 2.55 kilobases in length with about 6% poly A. The products of in vitro protein synthesis resembled hordeins (the prolamin storage proteins of the barley endosperm) in that they were predominantly soluble in 55% propan-2-ol, contained a low proportion of lysine as compared with leucine and had similar, but not identical, electrophoretic properties. The differences in the electrophoretic behaviour between the products of poly-A⁺ RNA translation and authentic hordeins is suggested to be due to the presence of an extra (leader?) sequence on the former.     

Intrinsic viscoelasticity of blood cell suspensions: effects of erythrocyte deformability

The intrinsic viscoelasticity of erythrocyte suspensions holds great potential for specifying the deformability of the individual, noninteracting cells in an oscillatory shear flow field. In order to extrapolate to zero cell concentration, the complex viscoelastic modulus was measured as a function of hematocrit using 2 Hertz oscillatory flow and a shear rate of 10/sec. This was done for both normal cells and cells with severely reduced deformability when hardened with glutaraldehyde. Suspension media were blood plasma, isotonic saline, and Dextran solutions. The real parts of the complex intrinsic visco-elasticities were obtained by an extrapolation using a regression fit to Huggins' equation. For normal cells in native plasma the values ranged from 1.7 to 2, increasing to the range 2.4 to 3.1 when the plasma was diluted with isotonic saline solution. For hardened cells the value obtained was near 3.5. These results are compared with theories for suspensions of both rigid and deformable particles. Several theories for deformable particles predict an increase in intrinsic viscoelasticity with increases in the ratio of the viscosity of the interior of the particle to that of the suspending medium. This ratio controls the balance between rotational and deformational response of the cell in the flow field. The trends of these theories were observed in the measurements.     

The effects of storage time and temperature and anticoagulant on laboratory measurements of canine blood progesterone concentrations

The effects of anticoagulant, storage time, storage temperature, and assay method, on laboratory measurements of blood progesterone concentrations of dogs is unclear; these factors have had a dramatic effect on blood progesterone concentrations in other species (particularly cows). In six experiments, we determined the effects of assay technique (chemiluminescence versus radioimmunoassay (RIA)), storage time, and temperature, as well as the use of heparinized plasma versus serum (coagulated blood) on measured progesterone concentrations of bitches. The studies showed that: (a) RIA measured significantly higher serum progesterone concentration (SPC) than chemiluminescence; (b) refrigeration of whole blood during the first 2 h after sample collection significantly decreased measured SPC; (c) progesterone concentration in heparinized plasma was not affected by storage temperature of whole blood for at least 5 h; (d) refrigeration of whole, clotted blood did not affect SPC, provided that samples were held at room temperature for the first 2 h after collection. These findings are of particular importance when blood samples are collected for determination of the initial rise in SPC that is associated with the LH surge in estrous bitches.     

Long term lily scale bulblet storage: effects of temperature and storage in polyethylene bags

Collections of lily genotypes are usually maintained by yearly planting, harvesting and storage of the bulbs. To facilitate this maintenance, a storage method has been developed for a collection of lily genotypes, including Asiatic hybrids, Oriental hybrids, Lilium longiflorum and L. henryi. Scale bulblets were stored either dry, sealed air-tight in polyethylene bags, or in moist vermiculite in open polyethylene bags for a period of 2 yr. The decrease in mass, sprouting proportion and ion leakage or sprouting proportion alone were determined for treatments carried out at -2°C, °C and 17°C. Sealing scale bulblets in polyethylene bags at -2°C resulted in the smallest decrease in mass, the least ion leakage and the highest sprouting proportion after 2 yr of storage.     

Progesterone metabolism during storage of blood samples from Gyr cattle: effects of anticoagulant, time and temperature of incubation

Ten Gyr cows with a functional corpus luteum were used to evaluate the effects of time and temperature of incubation of blood samples on progesterone (P4) concentrations detected in plasma or serum. From each cow, a blood sample was collected into a flask containing no anticoagulant, another into an heparinized flask and a third into a flask containing sodium fluoride. The blood from each flask was divided into 46 aliquots. One of them was centrifuged within 5 min of collection. The remaining 45 aliquots were divided into three groups and kept at three different temperatures: 4 degrees C, 17 degrees C, or 37 degrees C. For each anticoagulant, aliquots from every cow and incubation temperature were centrifuged every 30 min for 6 h, and then at 8, 12 and 24 h. Plasma or serum were separated immediately after centrifugation and were kept frozen at -20 degrees C until assayed for progesterone. The mean initial concentration of P4 in serum (8.3 ng/ml) significantly diminished (P<0.05) to 6.7 ng/ml after 5 h of incubation at 4 degrees C, 3 h at 17 degrees C, or 2 h at 37 degrees C. In plasma from heparinized blood the initial concentration (7.8 ng/ml) declined significantly after 6 h of incubation at 4 degrees C, 2 h at 17 degrees C, or 1 h at 37 degrees C. Sodium fluoride used as anticoagulant prevented the degradation of P4 since the initial concentration of P4 (6.7 ng/ml) never declined during incubation at either 4 degrees C or 37 degrees C; the only significant reduction occurred after 24 h of incubation at 17 degrees C.     

In vitro effects of calcium phosphate biomaterials on fibroblastic cell behavior

The effect of synthetic granular hydroxyapatite (HAP) on cultured fibroblastic cells (L929, human bone and gingiva cells) was studied. Phagocytosis of HAP particles and resulting morphological cell changes were demonstrated by microscopic examinations. Cell counts and [3H]thymidine uptake indicated significant increases in cell proliferation and DNA synthesis. These results could account for some of the alterations of the fibroblast behavior induced by changes in intracellular levels of calcium ions released from the material.     

Stability of radioiodinated monoclonal antibodies: In vitro storage and plasma analysis

The in vitro stability and immunointegrity of four radioiodinated monoclonal antibodies was evaluated in various storage conditions and also in plasma samples. The monoclonal antibodies studied included T101, B72.3, Lyml, and 16.88. Stabilities of typical monoclonal antibody therapy solutions, with radioactivities ranging from 2220 to 3700 MBq (60–100 mCi) were assessed using conventional instant thin layer chromatography and size exclusion high performance liquid chromatography. Radioimmunoreactivity was assessed using a live cell attenuated cell, or mucin-linked bead assay. Results of the study demonstrated that therapy solutions were stable to degradation, if properly stored in 5 or 10% human serum albumin at 4 °C for the duration of the study (5 days).Minor losses in immunoreactivity were also measured in stabilized therapy solutions. When incubated in plasma samples, radioiodinated monoclonal antibodies generally remained stable for the duration of the study (3 days). However, significant decreases in immunoreactivity were measured for specific radioiodinated monoclonal antibody preparations.     

In vitro effects of dihydrotestosterone on granulosa cell production of estrogen and progesterone

To investigate the direct effects of androgens on follicle development, intact, immature female rats were given 8 IU PMSG (0 hour) and four injections of either vehicle or dihydrotestosterone (DHT), 1 mg/kg body weight, at 0, 12, 24, and 36 hours after PMSG. Granulosa cells from small (less than 200 microns), medium (200 to 400 microns), and large (greater than 400 microns) follicles were isolated and cultured in the presence or absence of 0.5 microM DHT in vitro for 48 hours, and the medium was assayed for progesterone and estrogen. Results show that DHT caused an increase in progesterone accumulation in all granulosa cells, regardless of follicle size. However, DHT inhibited estrogen accumulation in granulosa cells from different-size follicles and the inhibition varied depending on the duration of androgen exposure in vivo. The inhibition of estrogen accumulation was seen in granulosa cells from small follicles without prior exposure to DHT in vivo, while an inhibition of estrogen accumulation was seen in granulosa cells from medium and large follicles exposed to DHT treatment in vivo. Taken together, the results of experiments with in vivo and/or in vitro DHT treatment show that the androgen increases granulosa cell progesterone synthesis regardless of follicle size. However, the estrogen accumulation by granulosa cell is dependent on follicle size and duration of DHT exposure.     

Ethylene production by sunflower cell suspensions : effects of plant growth retardants

From a variety of undifferentiated plant cell suspensions, 2,4-dichlorophenoxyacetic acid-dependent cells of sunflower (Helianthus annuus L. Spanners Allzweck) produced large quantities of ethylene. The maximum rate was about 1 nanomole × gram fresh weight⁻¹ × hour⁻¹ during the exponential growth phase. The action of various compounds known to interfere with ethylene formation in plant tissue was studied in sunflower cell suspensions. The influence on ethylene, 1-aminocyclopropanecarboxylic acid (ACC), and N-malonyl-ACC (MACC) levels suggested that the final steps in ethylene synthesis resemble those of other plant systems. This makes sunflower cells suitable for analyzing the effects of biologically active compounds on cellular ethylene biosynthesis. In particular, plant growth retardants of the norbornenodiazetine and triazole type inhibited ethylene production of sunflower cells. On the other hand, the ACC level was considerably elevated while that of MACC did not change significantly. It is assumed that the conversion of ACC to ethylene catalyzed by the ethylene-forming enzyme was influenced.