Vitrification of mouse embryos in two cryoprotectant solutions

The objective of this study was to compare the efficiency of 2 media on the vitrification of mouse compacted morulae, early blastocysts and expanded blastocysts after equilibration at room temperature of 4 degrees C. Embryos were equilibrated for 10 min in either 25% VS3 (Rall Equilibration Medium, REM) or 10% glycerol + 20% propylene glycol (Massip Equilibration Medium, MEM) in DPBS at 20 degrees C or 4 degrees C. For vitrification either 100% VS3 (Rall Vitrification Medium, RVM) or 25% glycerol + 25% propylene glycol (Massip Vitrification Medium, MVM) in DPBS was used. Embryos equilibrated at room temperature were loaded in 20 microL of vitrification media into 250 microL straws and then immediately (30 sec) plunged into liquid nitrogen (LN2). After equilibration at 4 degrees C the embryos were put into straws with 20 microL of precooled vitrification medium, and after 20 min at 4 degrees C they were plunged into LN2. Embryos from both groups were thawed in a 20 degrees C water bath for 20 sec, transferred to 1.0 M sucrose in DPBS for 5 min and then cultured for 24 to 48 h in Whitten's medium at 37 degrees C in 5% CO2 in air. In the groups of embryos prepared for vitrification at room temperature the survival rate of compact morulae vitrified in RVM was higher than those vitrified in MVM (65/70, 93% vs 49/74, 66%; P < 0.01). No difference was found in the survival rate of early blastocysts and expanded blastocysts vitrified in RVM or MVM (30/83, 36% vs 25/75, 33% and 4/66, 6% vs 4/76, 5%). No difference was found between the survival rate of compact morulae after equilibration with RVM or MVM at 4 degrees C (62/75, 83% vs 52/74, 70%). Both the early blastocysts and expanded blastocysts equilibrated at 4 degrees C MVM yielded a higher survival rate than RVM (28/74, 38% and 40/70, 57% vs 4/75, 5% and 4/77, 5%; P < 0.01). We conclude that, of the 3 developmental stages, compact morulae withstand the vitrification process best, and reduction of the temperature prior to plunging into LN2 is not required. A 10-fold increase in the survival rate of expanded blastocysts can be achieved using low temperature equilibration (4 degrees C) and MVM.     

Acoustic emission during cooling and heating of aqueous solutions of polyethylene glycols of molecular masses from 300 to 3000

The object of this research is to study acoustic emissions (AE) in aqueous solutions of polyethylene glycols (PEGs) of molecular masses from 300 to 3000 during cooling at 100 degrees C/min and heating at 70 degrees C/min in the temperature range from 0 to -196 degrees C. The dependence of AE intensity on PEG concentration and molecular mass is analysed. The AE intensity correlated with the crystalline to amorphous phase ratio in the frozen system.     

The effect of heating by microwave irradiation and by conventional heating on the aldehyde concentration in aqueous glutaraldehyde solutions

Summary The effect of short time heating of aqueous solutions of glutaraldehyde (GA) on relative aldehyde concentration was determined using spectrophotometric analysis. Because free monomeric GA absorbs U. V. light at 280 nm, whereas the alpha, beta polymeric forms absorb at 235 nm, the purity of GA solutions can be expressed as the ratio: A 235 nm/A 280 nm (purification index, P.I.).Heating of 4 ml aliquots of 0.85% distilled aqueous GA solution resulted in an increase of the absorption at 280 nm which is correlated positively with temperature. No increase of absorption at 235 nm was found when solutions were kept at 40°C for several hours. The increase of absorption at 280 nm is caused by a rapid decyclization of hemiacetals producing an increase in free aldehyde concentration.No major differences in absorption were found between the solutions heated by microwave and by conventional heating. However, because microwave irradiation is known to produce an homogeneous rise in temperature, especially in bulky samples, it is expected that the results of fixation procedures will improve by the combined effect of higher temperature and enhanced diffusion rates of the fixating species.     

The effects of aqueous neutral-salt solutions on the melting temperatures of deoxyribonucleic acids

The helical stability of a variety of DNA samples, ranging in base composition from 0 to 72 mole-% GC, has been studied by heat denaturation at neutral pH in increasing concentrations of LiCl, NaCl, KCl, CsCl, Li₂SO₄, and K₂SO₄. The variation of melting temperature with average base composition, dT_m/dX_GC, was found to decrease drastically in the concentrated salt media, e.g., from 41°C in 0.006M LiCl to 29°C in 3.2M LiCl, and from 39°C in 0.003M Li₂SO₄ to 18°C in 1.6M Li₂SO₄. At the same time, the thermal transition is much more cooperative in the concentrated salt solutions than at low ionic strength. Indeed, at limiting salt concentrations, the transition breadth seems to reach a minimum value irrespective of the compositional heterogeneity of the DNA samples. Attempts to correlate the observed decrease of dT_m/dX_GC with predicted changes in the enthalpy of melting, deduced from a simple theoretical treatment, experimental data on the binding of counterions and water to DNA, and experimental data on thermal denaturation, were unsuccessful. However, the strongly reduced composition dependence of the melting temperature can be understood in terms of a destabilizing effect of the concentrated salt media on GC-base pairs. It is suggested, though not proven, that the destabilization involves the displacement of water molecules from the DNA helix.     

The effect of a low-frequency electromagnetic field on DNA molecules in aqueous solutions

A chemiluminescence study showed that hepatitis B virus (HBV) and hepatitis C virus (HCV) DNA amplicons are capable of induced radiation when exposed to electromagnetic fields (EMFs) that range from 7.5 to 30 Hz in frequency and from 24 to 40 A/m in field strength. An EMF with a frequency of 9 Hz was shown to exert the greatest effect on aqueous solutions of the hepatitis virus DNA amplicons. The hydration shell of the DNA amplicons was observed to change. The change in the DNA hydration shell on exposure to a low-frequency EMF was presumed to restore hydrogen bonds, to induce crosslinks, and to facilitate DNA repair.     

Calorimetric study of aqueous solutions of histone H1 and poly-L-proline II at low temperatures

Low temperature differential scanning microcalorimetric investigation of water-histone H1 and water-poly-L-proline investigation was carried out. The concentrational dependence of the thermodynamic parameters (delta H(C), Tmax(C), delta S(T, C] for "bulk" water layers were studied. It was shown that the influence of these macromolecules on the structure and properties of surrounding water layers at the same degree of hydration is different.     

Mechanical testing of spider silk at cryogenic temperatures

A method and results for mechanical testing of spider silk in extreme environments is presented. In particular, silk from the spider Steatoda triangulosa is harvested, and samples are subjected to cryogenic temperatures by means of liquid nitrogen submersion. Samples are destructively tested while immersed in liquid nitrogen, and the stress-strain characteristics are compared to those of silk at room temperature. The strength, elasticity, and toughness of the cryogenically submersed silk are determined. It is found that on average, silk is 64% stronger while immersed in liquid nitrogen (i.e., at -196°C). The testing method could also be used for testing of silk in chemically hostile environments.     

Flow cytometric analysis from fish samples stored at low,ultra-low and cryogenic temperatures

Flow cytometry is a valuable tool in biomedical and animal sciences. However, equipment used for such analysis presents limitations at field conditions, suggesting then preservation procedures for future analysis at laboratory conditions. In this study, freezing at low (−20 °C), ultra-low (−80 °C) and cryogenic temperatures (−196 °C, i.e. liquid nitrogen) were used as preservation procedures of fish tissue. Samples were maintained in 0.9% NaCl or lysing solution, and stored at the temperatures above for 0 (fresh control), 60, 120 and 180 days of storage. After storage, the samples were thawed and proceeded to flow cytometric analysis. Storage at low temperatures (−20 °C), both in lysing and 0.9% NaCl, exhibited poor results when analyzed after 60, 120 and 180 days, showing noisy peaks, deviation in the DNA content and absence of peaks. Ultralow (−80 °C) and cryogenic (−196 °C) temperatures, both in lysing solution and 0.9% NaCl, showed good results and high quality of histograms. Both storage procedures gave similar histograms and DNA content in comparison with control group (fresh) even after 60, 120 and 180 days of storage, exhibiting the main peak at 2C content from diploid cells and a secondary peak at 4C derived from dividing cells. In conclusion, samples may be stored for 180 days at −80 °C and −196 °C in both, 0.9% NaCl or lysing solution. As cryogenic temperatures in liquid nitrogen permits indefinite storage, this procedure should be used for long-term preservation.     

Survival of tissue balls from the coral Pocillopora damicornis L. exposed to cryoprotectant solutions

In this study, the tolerance of tissue balls (TBs, 100–300 μm in diameter) from the coral Pocillopora damicornis produced using mechanical excision to exposure to cryoprotectant (CPA) solutions was tested. TBs were treated for 20 min at room temperature with solutions of ethylene glycol (EG), methanol (Met), glycerol (Gly) or dimethyl sulfoxide (Me₂SO) at concentrations between 1.0 and 4.5 M. Two parameters were used to evaluate the survival of TBs following CPA treatment. The Undamaged Duration of Tissue Balls (expressed in h) corresponded to the time period during which the membrane surface of TBs remained smooth and their motility was preserved. Tissue Ball Regression (expressed in μm/h) corresponded to the size reduction of TBs over time. TBs tolerated exposure to all CPAs tested at the three lower concentrations employed (1.0 M, 1.5 M and 2.0 M). No survival was achieved following exposure to a 4.5 M CPA solution. At concentrations of 3.0 and 4.0 M, higher Undamaged Duration of Tissue Balls and lower Tissue Ball Regression were obtained following treatment with EG compared to the other three CPAs. Our experiments show that TBs constitute a good experimental material to evaluate CPA toxicity on corals using large numbers of samples. Performing preliminary experiments with TBs may allow reducing the number of tests carried out with less easily available coral forms such as planulae, thereby preserving larval stocks.     

Rapid MR imaging of cryoprotectant permeation in an engineered dermal replacement

Magnetic resonance (MR) imaging is a powerful technique for monitoring the permeation of cryoprotective agents (CPAs) inside tissues. However, the techniques published until now suffer from inherently long imaging times, limiting the application of these techniques to slow diffusion processes and large CPA concentrations. In this study, we present a rapid MR imaging technique based on a CHESS-FLASH scheme combined with Keyhole image acquisition. This technique can image the fast permeation of Me(2)SO solutions into freeze-dried artificial dermal replacements for concentrations down to 10% v/v. Special attention is given to evaluating the technique for quantitative analysis.