Investigation of cryoprotectant toxicity to porcine embryos

The objective of this study was to examine the potential toxicity of sucrose (Experiment 1) and of various cryoprotectants (Experiment 2) to porcine preimplantation embryos. In Experiment 1, 65 embryos, ranging from compact morulae to hatched blastocysts, were allocated within donor female across 5 concentrations of sucrose (0, 0.25, 0.50, 1.0, 2.0 M) to determine the highest concentration that would not inhibit subsequent embryo development. After a 48-h post-treatment culture period, the embryos were stained and cell nuclei were counted. The concentration of sucrose affected embryo development (P < 0.001) and embryo quality (P < 0.001). Embryos placed into 2.0 M sucrose exhibited poorer development and quality than embryos at the lower 4 concentrations, which were not different from one another. In Experiment 2, 182 embryos of the same developmental stages as in Experiment 1 were collected from 16 donors. Embryos were allotted within donor female to 2 of the 5 concentrations (10, 20, 30, 40, or 50%) of each of 3 cryoprotectants (ethylene glycol, propylene glycol, glycerol). After a 30-sec exposure to a cryoprotectant, the embryos were cultured and stained as in Experiment 1. As the concentration of an individual cryoprotectant increased beyond 30%, embryo development decreased. Embryos exposed to glycerol or propylene glycol exhibited poorer development than did embryos placed into ethylene glycol, especially at concentrations of 40% or higher.     

Methanol as a cryoprotectant for equine embryos

Equine embryos (n=43) were recovered nonsurgically 7-8 days after ovulation and randomly assigned to be cryopreserved in one of two cryoprotectants: 48% (15M) methanol (n=22) or 10% (136 M) glycerol (n=21). Embryos (300-1000 microm) were measured at five intervals after exposure to glycerol (0, 2, 5, 10 and 15 min) or methanol (0, 15, 35, 75 and 10 min) to determine changes (%) in diameter over time (+/-S.D.). Embryos were loaded into 0.25-ml plastic straws, sealed, placed in a programmable cell freezer and cooled from room temperature (22 degrees C) to -6 degrees C. Straws were then seeded, held at -6 degrees C for 10 min and then cooled to -33 degrees C before being plunged into liquid nitrogen. Two or three embryos within a treatment group were thawed and assigned to be either cultured for 12 h prior to transfer or immediately nonsurgically transferred to a single mare. Embryo diameter decreased in all embryos upon initial exposure to cryoprotectant. Embryos in methanol shrank and recovered slightly to 76+/-8 % of their original diameter; however, embryos in glycerol continued to shrink, reaching 57+/-6 % of their original diameter prior to cryopreservation. Survival rates of embryos through Day 16 of pregnancy were 38 and 23%, respectively (P>0.05) for embryos cryopreserved in the presence of glycerol or methanol. There was no difference in pregnancy rates of mares receiving embryos that were cultured prior to transfer or not cultured (P>0.05). Preliminary experiments indicated that 48% methanol was not toxic to fresh equine embryos but methanol provided no advantage over glycerol as a cryoprotectant for equine blastocysts.     

Glycine betaine as a cryoprotectant for prokaryotes

Osmoprotectants are low molecular weight, hydrophilic, nontoxic molecules that assist a cell under osmotic stress to stabilize its concentration of internal solutes. These properties are similar to compounds used as cryoprotectants for the preservation of prokaryotic cells during freezing. This study tested the ability of a common compatible solute, glycine betaine (GB), to act as a cryoprotectant. In a series of freeze-drying studies using a variety of prokaryotes, GB performed as well, or better than, two commonly used cryoprotectants, sucrose/bovine serum albumin (S/BSA) and trehalose/dextran (T/D). GB did especially well maintaining cell viability after long-term storage (simulated equivalent of 20 years) for microorganisms like Neisseria gonorrhoeae and Streptococcus pneumoniae. GB was tested for its ability to preserve members of the genus Acidothiobacillus, a difficult genus to preserve. For two strains of Acidithiobacillus ferrooxidans that were preserved using liquid drying, GB performed as well as S/BSA. Results were more mixed for two strains of Acidithiobacillus thiooxidans; one strain could be preserved with S/BSA but not GB, the other strain gave low recoveries with both cryoprotectants. GB also proved to be a useful cryoprotectant for liquid nitrogen preservation yielding equivalent results to the cryopreservative, glycerol for halophilic archaea, and neutrophilic Fe-oxidizing bacteria. These results indicate that GB is a simple and useful cryoprotectant that works for a wide range of prokaryotic organisms under different cryopreservation regimens.     

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.     

Trehalose as cryoprotectant for preservation of yeast strains

Preservation of genetic banks of yeast strains as well as of any kind of eukaryotic cells during dehydration and subsequent rehydration depends upon the maintenance of the integrity of the cell membrane. Trehalose has been successfully used as a non-toxic cryoprotectant for plant cells (Bhandal et al., 1985), as well as for lobster sarcoplasmic vesicles (Rudolph and Crowe, 1985). The hypothesis underlying these observations is that the disaccharide avoids fusion of membranes by replacing water molecules in the bilayer (Crowe et al., 1984). The viability of yeast strains submitted to different drying techniques is reported in this paper. Mutant strains with defects in the regulation of the trehalose-6-phosphate synthase complex were compared. Yeast strains dried in layers at 37°C for 6 h did not lose their viability, however, they died thereafter at 5°C, unless trehalose was used for resuspending the cells before drying. It should be noted that no trehalose accumulation was seen during drying at 37°C under our experimental conditions. In experiments in which cells were frozen at −120°C, addition of 10% trehalose to the suspending buffer had a significant protective effect. On the other hand, a mutant strain with an extremely high trehalose-6-phosphate synthase activity showed an intrinsic capacity for survival which did not depend upon addition of exogenous trehalose. This raises the question of the location of the internal trehalose pool and whether it could replace the externally added cryoprotectant.     

The relevance of cryoprotectant "toxicity" to cryobiology

Cryoprotective agents are essential for the cryopreservation of almost all biological systems. These additives, however, do not usually permit 100% survival after freezing and thawing, though from a theoretical point of view they should be able to fully suppress all known types of freezing injury. In view of the known biological and physicochemical effects of cryoprotectants, it is suggested that the toxicity of these agents is a key limiting factor in cryobiology. Not only does this toxicity prevent the use of fully protective levels of additive, but it may also be manifested in the form of cryoinjury over and beyond the cryoinjury due to classical causes. Evidence for this extra injury ("cryoprotectant-associated freezing injury") is reviewed. It is suggested that better suppression of toxicity is possible and will lead to advances in cryopreservation.     

Preliminary report of novel technique for cryopreservation--vacuum-assisted cryoprotectant infiltration

To date, cryopreservation of large soft tissues has not been successfully achieved because of limitation of cryoprotective agent (CPA) infiltration into the tissue. This study aimed to investigate the effects of a vacuum on the tissue-infiltration of a CPA. An instant pickle-maker was modified for use as a vacuum apparatus, and glycerol was selected as the CPA. Twenty-six rats were used, and their thighs were divided into three treatment groups. Group 1: fresh control; Group 2: cryopreserved control, i.e., immersed in the CPA for 1h under atmospheric pressure and cryopreserved; Group 3: vacuum-assisted CPA infiltration, i.e., immersed in the CPA under negative pressure (20, 40 and 60 cmHg, for durations of 10, 20 and 30 min at each) and cryopreserved. The Groups 2 and 3 specimens were thawed after 3 weeks of cryopreservation at -80 °C and histologically examined, in comparison with Group 1. Skin: in Groups 2 and 3, the skin was well preserved. Muscle: in Group 2, both extracellular and intracellular ice crystal formation was widely distributed throughout the muscle tissue. In Group 3, under an adequate vacuum, the muscle tissue was well preserved, with no ice crystal formation. However, when the treatment was conducted under excessive vacuum conditions, the muscle tissue showed focal necrosis. Blood vessels: in Group 3, both the arteries and veins were well preserved up to the tunica intima. The method described in this paper may be a useful technique for achieving cryopreservation of large soft tissues.     

Optimization of cryoprotectant loading into murine and human oocytes

Loading of cryoprotectants into oocytes is an important step of the cryopreservation process, in which the cells are exposed to potentially damaging osmotic stresses and chemical toxicity. Thus, we investigated the use of physics-based mathematical optimization to guide design of cryoprotectant loading methods for mouse and human oocytes. We first examined loading of 1.5 M dimethyl sulfoxide (Me₂SO) into mouse oocytes at 23 °C. Conventional one-step loading resulted in rates of fertilization (34%) and embryonic development (60%) that were significantly lower than those of untreated controls (95% and 94%, respectively). In contrast, the mathematically optimized two-step method yielded much higher rates of fertilization (85%) and development (87%). To examine the causes for oocyte damage, we performed experiments to separate the effects of cell shrinkage and Me₂SO exposure time, revealing that neither shrinkage nor Me₂SO exposure single-handedly impairs the fertilization and development rates. Thus, damage during one-step Me₂SO addition appears to result from interactions between the effects of Me₂SO toxicity and osmotic stress. We also investigated Me₂SO loading into mouse oocytes at 30 °C. At this temperature, fertilization rates were again lower after one-step loading (8%) in comparison to mathematically optimized two-step loading (86%) and untreated controls (96%). Furthermore, our computer algorithm generated an effective strategy for reducing Me₂SO exposure time, using hypotonic diluents for cryoprotectant solutions. With this technique, 1.5 M Me₂SO was successfully loaded in only 2.5 min, with 92% fertilizability. Based on these promising results, we propose new methods to load cryoprotectants into human oocytes, designed using our mathematical optimization approach.     

Dog cloning from post-mortem tissue frozen without cryoprotectant

Successful reproductive cloning depends on obtaining intact donor nuclei from viable cells, ideally isolated by tissue biopsy of a living donor. However, owners and veterinarians often freeze deceased animals, which eventually causes damage to cellular micro-organelles due to the formation of intracellular water crystals. In the present study, we have reported the production of viable cloned puppies using donor nuclei of cells obtained from frozen carcasses. Five cases of deceased and frozen canine specimens were presented to be cloned. Skin fibroblast cell lines were successfully established for four specimens. Significant longer time was needed for the cell growth from frozen tissues (4 days) to reach 80% confluency compared to fresh tissue and frozen tissues frozen for 1- or 2-days. Similarly, SA-βgal positive cells (death cells) were significantly higher in frozen cells for 2- or 4- days compared to samples from fresh or frozen (1 day) sources. The cloning efficiency (CE) and the pregnancy rates (PR) of frozen cells were lower than those obtained from fresh or living donors (CE 2.4 ± 1.8% vs. 0.6 ± 0.3%, PR 21.7 ± 16.1% vs. 7.7 ± 5.3% for fresh vs. frozen, respectively). Here we demonstrate is the possibility to produce healthy offspring from cell lines obtained from frozen tissue collected post-mortem.     

Permeation of several cryoprotectant agents into porcine articular cartilage

Objective

Osteochondral allografting is an effective method to treat large osteochondral defects but difficulties in tissue preservation have significantly limited the application of this technique. Successful cryopreservation of articular cartilage (AC) could improve the clinical availability of osteochondral tissue and enhance clinical outcomes but cryopreservation of large tissues is hampered by a lack of knowledge of permeation kinetics within these tissues. This study describes the refinement and extension of a recently published technique to measure the permeation kinetics of cryoprotectant agents (CPAs) within porcine AC.

Design

Dowels of porcine AC (10 mm diameter) were immersed in solutions containing 6.5 M concentrations of four commonly used CPAs [dimethyl sulfoxide (DMSO), propylene glycol (PG), ethylene glycol (EG) and glycerol] for different times (1 s, 1, 2, 5, 10, 15, 30, 60, 120, 180 min , 24 h) at three different temperatures (4, 22, and 37 °C). The cartilage was isolated and the amount of CPA within the matrix was determined.

Results

Diffusion coefficients (DMSO = 2.4-6.2 × 10⁻⁶ cm²/s; PG = 0.8-2.7 × 10⁻⁶ cm²/s; EG = 1.7-4.2 × 10⁻⁶ cm²/s; and glycerol = 0.8-2.4 × 10⁻⁶ cm²/s) and activation energies (DMSO = 4.33 kcal/mol, PG = 6.29 kcal/mol, EG = 3.77 kcal/mol, and glycerol = 5.56 kcal/mol) were determined for each CPA.

Conclusion

The results of this experiment provide accurate permeation kinetics of four commonly used CPAs in porcine articular cartilage. This information will be useful for developing effective vitrification protocols for cryopreservation of AC.     

Protocols for thawing and cryoprotectant dilution of heart valves

PURPOSE: To reduce the time taken for thawing and removal of cryoprotectant from heart valves. METHODS: Three sets of experiments were carried out using porcine heart valves. The valves in all three experiments were first exposed to 10% (v/v) dimethyl sulphoxide (DMSO) by a 2-step protocol. Outcome was determined after the various experimental treatments by monitoring the outgrowth of cells from valve leaflet explants. Experiment 1-Dilution protocol. Valves exposed to 10% DMSO were subjected to 4-, 2- or 1-step dilution to remove the DMSO. Experiment 2-Warming rate. The rate of warming was increased by reducing the volume of cryoprotectant medium in which the valves were frozen. Valves were exposed to 10% DMSO, frozen in different volumes (100, 50, 25 or 0 ml) of cryoprotectant medium, and warmed in a 37 degrees C water bath. The DMSO was removed by 4-step dilution. Experiment 3-Standard vs. Modified protocol. Valves were either frozen in 100 ml 10% DMSO, thawed, and subjected to 4-step dilution (Standard) or frozen in 50 ml 10% DMSO, thawed, and the DMSO removed by single-step dilution (Modified). RESULTS: Neither the rate of warming nor the rate of dilution of DMSO had any influence on the subsequent outgrowth of valve leaflet fibroblasts. There were no differences in the outgrowth of cells from valve leaflets cryopreserved by the Standard or Modified protocols. CONCLUSION: The time taken for thawing and dilution of heart valves could be reduced from >20 min to <10 min without detriment to the viability of the leaflet fibroblasts. This should have a positive impact on valve replacement surgery as the thawing and dilution of valves are typically carried out while the patients are on cardiopulmonary bypass.     

Dimethyl-acetamide as a cryoprotectant for rainbow trout spermatozoa

Dimethyl-acetamide was evaluated as a cryoprotectant for rainbow trout (Oncorhynchus mykiss ) semen on the basis of motility, percentage of dead spermatozoa as determined by fluorometry, and fertility of frozen-thawed spermatozoa. Dimethyl-acetamide performed significantly better (P<0.05) than the conventional cryoprotectant, dimethyl sulfoxide, by all evaluation methods. An extender comprised of 0.137 M NaCl, 0.011 M KCl, 0.004 M Na(2)HPO(4) 7H(2)O, 7.5 g/l L-alpha-lecithin and 10 % DMA showed promise for cryopreserving rainbow trout spermatozoa. Future evaluation of new extenders should be carried out by several in vitro techniques and fertility measurements to present a complete assessment of an extender's ability to cryopreserve sperm cells.