Comparisons of glucose,sucrose, and dimethyl sulfoxide as cryoprotective agents for Babesia rodhaini, with estimates of survival rates

Comparisons were made between glucose, sucrose, and dimethyl sulfoxide (DMSO) as cryoprotective agents for the hemoprotozoan parasite, Babesia rodhaini, using infectivity for mice as the criterion of survival. Concentrations of the cryoprotectants tested were from 0.1 to 0.5 M for the sugars, and 1.5 to 2.5 M for DMSO. Glucose and sucrose were comparable as cryoprotectants, although glucose reduced infectivity of the parasites slightly more than did sucrose at above-freezing temperatures. When sucrose and DMSO were compared for cryoprotection during cooling to ?196 °C at nominal rates of 5, 100, and 500 °C/min, parasite survival varied with the type and concentration of cryoprotectant, but was higher in blood containing DMSO at all three cooling rates. The percentages of parasites that survived cooling at 100 °C/min and frozen storage in the presence of DMSO ranged from 20 to 36%.     

Comparative analysis of transcriptional responses to the cryoprotectants, dimethyl sulfoxide and trehalose, which confer tolerance to freeze–thaw stress in Saccharomyces cerevisiae

We have used microarray analysis to monitor the gene expression profile of Saccharomyces cerevisiae BY4743 in the presence of the cryoprotectants, dimethyl sulfoxide (Me₂SO) and trehalose. Analysis of these profiles suggests that both cryoprotectants increased the expression of genes involved in protein synthesis, ribosomal biogenesis, fatty acid biosynthesis, ergosterol biosynthesis, cell wall biosynthesis, and cellular accumulation of low molecular compounds such as glycerol, arginine and proline. Cryoprotectant treatment reduced the expression of genes involved in the β-oxidation of fatty acids. In addition, Me₂SO increased the expression of genes involved in protein refolding and trehalose increased the expression of genes involved in spore formation. This study supported that exposure to cryoprotectants prior to freezing not only reduce the freeze–thaw damage but also provide various process to the recovery from freeze–thaw damage.     

Permeability of cultured megakaryocytopoietic cells of the rat to dimethyl sulfoxide

The permeability of the membrane of the rat megakaryocytopoietic cell to dimethyl sulfoxide was measured to assess its availability to the intracellular compartment. The method used was osmotic, and measured the initial loss of cell water followed by a reswelling to isotonic volume when cells were placed in culture media containing 0.6 M DMSO. Values for the hydraulic coefficient, Lp, the permeability of the membrane to DMSO, wRT , and the reflection coefficient were calculated from the equations of Kedem and Katchalsky . The average value at 25 degrees C for Lp was 0.46 micron min-1 atm1 ; wRT was 9.3 micron min-1, and the reflection coefficient was 0.65. At these cell volumes, 50% equilibration occurred in 5 sec. Cells equilibrated in 0.6 M DMSO increased their volume of osmotically inactive water. Coupled with this phenomenon of stabilization of water was a reduction in the hydraulic coefficient by 50%. These findings are discussed in the context of current hypotheses about cellular viability during freezing and thawing in the presence and absence of cryoprotectants.     

Growth inhibitory effects of dimethyl sulfoxide and dimethyl sulfone on vascular smooth muscle and endothelial cells in vitro

Summary The growth of bovine aortic smooth muscle and endothelial cells was studied after exposure to dimethyl sulfoxide (DMSO) or its major metabolite, dimethyl sulfone (DMSO₂). Both compounds caused a dose-dependent inhibition of cell growth as determined by [³H]thymidine incorporation and by counting the number of cells with time of exposure in culture. The IC₅₀ of DMSO (concentration which produces 50% inhibition of growth) was 1% for smooth muscle cells and 2.9% for endothelial cells. Similarly, the IC₅₀ of DMSO₂ was also 1% for smooth muscle cells, but was 1.8% for endothelial cells. After a 4-d exposure to either compound, the growth inhibition of smooth muscle cells was completely reversible at 1%, partially reversible at 2 to 3% and completely irreversible at 4%. By comparison, inhibition of endothelial cell growth was completely reversible up to 4% of either compound. It is concluded that the growth of smooth muscle cells was similarly inhibited by DMSO, and DMSO₂, but that smooth muscle cells were more susceptible than endothelial cells to the growth inhibitory effects of these compounds. In addition, DMSO₂ was a more potent inhibitor of cell growth than DMSO and its growth inhibition was less reversible than that produced by DMSO.     

Freeze Preservation of Cultured Plant Cells

A basic technique for successful freeze preservation and storage at -196°C of cultured plant cells and an assay of percentage survival following the freezing-storage-thawing procedure are described. These techniques have been applied to suspension cultures of carrot (3 cell lines), belladonna and sycamore. Dimethyl sulfoxide (DMSO) and glycerol, when appropriately applied, were the most effective cryoprotectants tested. Although these cryoprotectants were of low toxicity and did not cause alterations in the cytology and growth potential of the recovered cells, the cell lines differed in their sensitivity to the toxicity of these cryoprotectants. Small meristematic cells survived the freezing-thawing procedure better than larger more highly vacuolated cells. Specific differences in survival are in part explained in terms of differences in cell morphology.     

Effect of glycerol and dimethyl sulfoxide on cryopreservation of rhesus monkey (Macaca mulatta) sperm

Glycerol and dimethyl sulfoxide (DMSO) are widely used as penetrating cryoprotectants in the freezing of sperm, and various concentrations are applied in different species and laboratories. The present study aimed to examine the effect of these two cryoprotectants at different concentrations (2%, 5%, 10%, and 15% glycerol or DMSO) on rhesus monkey sperm cryopreservation. The results showed that the highest recovery of post-thaw sperm motility, and plasma membrane and acrosome integrity was achieved when the sperm was frozen with 5% glycerol. Spermatozoa cryopreserved with 15% DMSO showed the lowest post-thaw sperm motility, and spermatozoa cryopreserved with 15% glycerol and 15% DMSO showed the lowest plasma membrane integrity among the eight groups. The results achieved with 5% glycerol were significantly better for all parameters than those obtained with 5% DMSO. The functional cryosurvival of sperm frozen with 5% glycerol was further assessed by in vitro fertilization (IVF). Overall, 85.7% of the oocytes were successfully fertilized, and 51.4% and 5.7% of the resulting zygotes developed into morulae and blastocysts, respectively. The results indicate that the type and concentration of the penetrating cryoprotectant used can greatly affect the survival of rhesus monkey sperm after it is frozen and thawed. The suitable glycerol level for rhesus monkey sperm freezing is 5%, and DMSO is not suitable for rhesus monkey sperm cryopreservation.     

Nucleation and growth of ice crystals inside cultured hepatocytes during freezing in the presence of dimethyl sulfoxide.

A three-part, coupled model of cell dehydration, nucleation, and crystal growth was used to study intracellular ice formation (IIF) in cultured hepatocytes frozen in the presence of dimethyl sulfoxide (DMSO). Heterogeneous nucleation temperatures were predicted as a function of DMSO concentration and were in good agreement with experimental data. Simulated freezing protocols correctly predicted and explained experimentally observed effects of cooling rate, warming rate, and storage temperature on hepatocyte function. For cells cooled to -40 degrees C, no IIF occurred for cooling rates less than 10 degrees C/min. IIF did occur at faster cooling rates, and the predicted volume of intracellular ice increased with increasing cooling rate. Cells cooled at 5 degrees C/min to -80 degrees C were shown to undergo nucleation at -46.8 degrees C, with the consequence that storage temperatures above this value resulted in high viability independent of warming rate, whereas colder storage temperatures resulted in cell injury for slow warming rates. Cell damage correlated positively with predicted intracellular ice volume, and an upper limit for the critical ice content was estimated to be 3.7% of the isotonic water content. The power of the model was limited by difficulties in estimating the cytosol viscosity and membrane permeability as functions of DMSO concentration at low temperatures.     

Enhanced release of rosmarinic acid from Coleus blumei permeabilized by dimethyl sulfoxide (DMSO) while preserving cell viability and growth

Continuous permeabilization of preconditioned Coleus blumei cells with dimethyl sulfoxide (DMSO) is shown to be an effective strategy for the enhanced release of rosmarinic acid (RA) while preserving cell viability. When nonpreconditioned cells were permeabilized with DMSO, they lost their viability at DMSO concentrations higher than a critical value located between 0.1% and 0.5% DMSO. Product release was low [0.49 g RA/100 g dry cell weight (DCW)] at 0.1% DMSO. Preconditioning cells at 0.1% DMSO ensured high viability at DMSO concentrations of 0.5%, 1.0%, and 1.5%. Product release reached a maximum of 2.85 g RA/100 g DCW at 0.5% DMSO, which was 66.4% of the total rosmarinic acid produced. (c) 1992 John Wiley & Sons, Inc.     

Cryopreservation of leucocytes of channel catfish for subsequent cytogenetic analysis

Channel catfish leucocytes cryopreserved with glycerol or dimethyl sulphoxide (DMSO) had significantly higher ( P <0.05) viability and recovery rates than did cells cryopreserved with methanol. After 7 days of frozen storage, a 24 to 27% reduction of viability was observed for cells cryopreserved with glycerol; a 25 to 43% reduction for cells frozen with DMSO, and a 67 to 100% reduction for cells frozen with methanol. The concentration of cryoprotectants affected the viability of cryopreserved cells significantly ( p <0.05). The viability reduction was 36% for cells frozen with 5% of cryoprotectants, 30% for cells frozen with 10% of cryoprotectants, and 49% for cells frozen with 15% of cryoprotectants. The viability of cells frozen at the slower rate (-2.7°C min⁻¹) was significantly higher ( p <0.05) than that of cells frozen at the faster rate (-45°C min⁻¹). Best results were obtained for cells cryopreserved with 10% of glycerol or DMSO and frozen at the slower rate. The chromosomes prepared from cells cryopreserved using this procedure were identical to those prepared from fresh cells, and to those reported in the literature for channel Catfish.     

Highly efficient cryopreservation of human induced pluripotent stem cells using a dimethyl sulfoxide-free solution

Human induced pluripotent stem (hiPS) cells have great potential for regenerative medicine and drug discovery. It is essential to establish highly efficient and reliable methods for hiPS cell cryopreservation. We examined cryopreservation of hiPS cells by the vitrification method using a dimethyl sulfoxide Me2SO-free and serum-free medium, VS2E, that uses Euro-Collins solution as a base with 40% (v/v) ethylene glycol and 10% (w/v) polyethylene glycol as cryoprotectants. This combination of vitrification and cryoprotectants resulted in a higher recovery rate of hiPS cells than with a commercially-available vitrification solution, DAP213, which contained Me2SO and serum components. After vitrification and warming, hiPS cells were cultured easily. Even after several subculturing steps, cells expressed undifferentiated cell markers, such as Oct-3/4 and SSEA-4, and also exhibited alkaline phosphatase activity. The pluripotency of hiPS cells was maintained, as demonstrated by teratoma formation upon hiPS cell transplantation into severe combined immunodeficient mice. Thus, we successfully preserved hiPS cells under liquid nitrogen with high efficiency using Me2SO-free vitrification solution and rapid cooling.     

Effects of dimethyl sulfoxide on Tetrahymena pyriformis GL. Fine structural changes and their reversibility.

Fine-structural changes are induced in Tetrahymena by exposure to 7.5% dimethyl sulfoxide (DMSO) in the presence of growth medium. Some of these changes (nucleolar, mitochondrial, peroxisomal) resemble those seen during starvation, in agreement with the previously reported inhibitory effect of DMSO on food-vacuole formation; however, changes such as helical formations of polyribosomes indicate additional internal actions of the reagent. The effects vary to some extent within the same group of cells, suggesting that sensitivity to the reagent may differ with the stage in the cell cycle. The structural changes induced by a 1-hr exposure to DMSO are reversible, but recovery of the cells after removal of the reagent is slower than that seen after starvation. The observations suggest that the recovery is associated with renewed synthesis.     

Reciprocal regulation of adult rat hepatocyte growth and functional activities in culture by dimethyl sulfoxide

Hepatocyte DNA synthesis, initiated by epidermal growth factor (EGF), is reversibly inhibited by 2% dimethyl sulfoxide (DMSO). At that concentration, both the survival of the cells in culture and the expression of differentiated functions are prolonged. DMSO does not affect thymidine uptake or EGF receptor binding. Moreover, EGF receptor binding is maintained at 84% of initial 12 hr binding when cells are cultured for several days in the presence of DMSO, whereas specific receptor binding declines to 49% of initial binding under standard culture conditions without DMSO. Studies of hepatocyte functional activity indicate that, during early culture, total cellular export protein synthesis, specific albumin synthesis, and glycogen synthesis are enhanced in the presence of DMSO. Dexamethasone is required for the effect of DMSO on survival, and although dexamethasone alone enhances hepatocyte DNA synthesis in the presence of EGF, it does not reverse the inhibitory effect of 2% DMSO on DNA replication. The correlation of prolonged survival with growth inhibition supports the hypothesis that hepatic growth and differentiated functional activity may be reciprocally regulated.     

Cryopreservation of erythropoietin-responsive cells in murine hematopoietic tissue

The optimal conditions were determined under which maximum survival of murine hematopoietic erythropoietin-responsive cells (ERC) could be ensured during manipulations required for cryopreservation. Cell survival was similar over freezing rates between 2 and 10 °C/min. Optimal cryoprotectants were 10% dimethyl sulfoxide (DMSO) and 20% fetal calf serum; the DMSO was removed by centrifugation after stepwise dilution with 20 vol of medium over a 10-min period. Differing thawing rates for the cell suspensions had minimal effects on survival. “Seeding” the cell suspensions with ice crystals had no effect on ERC recovery. Overall ERC survival varied between 20 and 40%. These results confirm earlier reports that certain ERC populations are more sensitive to damage during cryopreservation than are other hematopoietic progenitor cells.     

Combination of intracellular cryoprotectants preserves the structure and the cells proliferative capacity potential of adult collared peccary testicular tissue subjected to solid surface vitrification

The objective was to evaluate different permeating cryoprotectants to vitrify testicular tissue biopsies from adult collared peccaries. Five pairs of testicles were dissected into fragments (9 mm³) that were allocated to non-vitrified (control) and vitrified groups using a solid-surface method following exposure to different cryoprotectants (3.0 M dimethyl sulfoxide (DMSO), 3.0 M ethylene glycol (EG) or 1.5 M DMSO + 1.5 M EG). After warming, samples were evaluated for histomorphology, ultrastructure, viability, and proliferative capacity potential. The appropriate conservation of the ultrastructural organization of the seminiferous tubule in terms of lumen presence and cell junctions was only observed at the use of DMSO/EG combination. Regardless of the cryoprotectant, the vitrification effectively preserved cell nuclear visualization and condensation similarly as observed at the non-vitrified group. Moreover, DMSO/EG combination provided a better preservation of basal membranes of seminiferous tubules than DMSO (P < 0.05). The occurrence of cell swelling was more evident in the use of DMSO than EG (P < 0.05), but both isolate cryoprotectants were similar to the DMSO/EG combination. Only the DMSO/EG combination maintained the proliferative capacity potential for spermatogonia (3.69 NORs/cell) and Sertoli cell (3.19 NORs/cell) similar to controls (3.46 and 3.31 NORS/cell, respectively). Moreover, ~40% cell viability was found after vitrification independent of cryoprotectant. In conclusion, DMSO/EG in combination is better than DMSO or EG alone for SSV of testicular tissue biopsies from adult collared peccaries.     

Effect of endocellular cryoprotectant upon polymorphonuclear neutrophil function during storage at low temperature.

The effect of cryoprotective agents dimethyl sulfoxide (DMSO), glycerol and ethylene glycol upon the function of polymorphonuclear neutrophils (PMNs) during storage between 0 ° and 4 °C was investigated.Increasing concentration of each cryoprotectant caused an increasing inhibition of chemotaxis with complete inhibition at 16.7%. At this concentration most PMNs were still able to exclude trypan blue dye. Chemotaxis was not inhibited if PMNs were exposed to 4.2 or 8.3% concentrations of cryoprotectants for 1 hr, and washed subsequently. However, the recovery of chemotaxis was not observed at 16.7% after 1 hr exposure to cryoprotectants. Moreover, a considerable number of PMNs could not exclude the dye. This would indicate that cells become fragile with cryoprotectants at a high concentration and the PMNs are easily damaged by washing. With 20 hr exposure PMNs, the inhibitory effect on chemotaxis was removed by washing when a 4.2% agent was used, but using an 8.3% agent, chemotaxis was not restored but PMNs exposed to DMSO displayed almost the same chemotaxis as a control. On the other hand, the ability of PMNs to ingest bacteria was not so markedly inhibited as the chemotaxis. With 1 hr exposure to cryoprotectants, the ingestive ability was hardly affected within 8.3%. As for 20 hr exposure, the same ingestive ability as that of a control was observed in all cases using a 4.2% concentration. However, using an 8.3% concentration, the DMSO-exposed PMNs retained a good ingestive ability.Judging from the above findings, DMSO would be suitable as a crypotrotective agent although the problem on toxicity remains to be resolved.     

Incorporation of penetrating cryoprotectants in diluents for pellet-freezing ram spermatozoa

Glycerol may be toxic to frozen-thawed ram spermatozoa and reduce their fertilizing capacity. This study examined the cryoprotective effects of dimethyl sulphoxide (DMSO), ethylene glycol, glycerol and propanediol alone and in combinations with each other in Triscitrate-glucose diluents on the post-thaw motility and acrosome integrity of pellet-frozen ram spermatozoa. The 4 cryoprotectants were examined in diluents at 5 concentrations (0, 1.5, 3.0, 6.0, 12.0% v/v). Post-thaw motility of spermatozoa was higher in diluents containing ethylene glycol (1.5 to 6.0% v/v), glycerol (at all levels tested) and propanediol (1.5 and 3.0% v/v) than in diluents without cryoprotectant (P<0.001), but there was no effect of DMSO on post-thaw motility. Motility of spermatozoa was higher in diluents containing ethylene glycol or glycerol than DMSO or propanediol (P<0.001). In diluents containing the 4 cryoprotectants at 3 concentrations (1.5, 3.0, 6.0% v/v), better recovery of spermatozoa was found with the addition of 18.0 than 4.5% v/v egg yolk. Combinations of ethylene glycol and/or propanediol (0 to 6.0% v/v) with glycerol (0 to 6.0% v/v) in diluents were also examined. In the presence of glycerol at all levels tested, increasing levels of ethylene glycol and/or propanediol decreased motility and acrosome integrity of spermatozoa (P<0.001). We conclude that the compounds examined exert a cryoprotective effect on pellet-frozen ram spermatozoa, except for DMSO which had no effect. In this study, glycerol remained the single most effective cryoprotectant, and there was no enhancement of this cryoprotection by addition of the other compounds.     

Wheat proteins improve cryopreservation of rat hepatocytes

Hepatocytes are an important physiological model for in vitro studies of drug metabolism and toxicity. However, fresh hepatocytes are not always available and hence cyopreservation is needed to preserve large quantities until they are needed for these applications. Hepatocytes are extremely sensitive to damage induced by the freeze–thaw process, even after addition of traditional cryoprotectants such as dimethyl sulfoxide (DMSO). Furthermore, they do not proliferate in culture. We previously demonstrated that a crude wheat extract protects rat hepatocytes during cryopreservation and could provide a promising alternative to DMSO. We have considerably improved this novel cryopreservation procedure by using wheat extracts that are partially purified by either ammonium sulphate or acetone precipitation, or by using recombinant wheat freezing tolerance‐associated proteins such as WCS120, TaTIL, WCS19, and TaIRI‐2. These improved procedures enhance long‐term storage (2–12 months) and recovery of large quantities of healthy cells after cryopreservation, and maintain the differentiated functions of rat hepatocytes, compared to freshly isolated cells, as judged by viability (77–93%), adherence (77%) and metabolic functions of major cytochrome P450 isoforms CYP1A1/2, CYP2C6, CYP2D2, and CYP3A1/2. The advantage of using wheat proteins as cryopreservants is that they are non‐toxic, natural products that do not require animal serum, and are economical and easy to prepare. Biotechnol. Bioeng. 2009;103: 582–591. © 2009 Wiley Periodicals, Inc.     

Dimethyl sulfoxide toxicity kinetics in intact articular cartilage

Osteochondral defects can degenerate into osteoarthritis and currently there are no good treatment alternatives available to most Orthopaedic surgeons. Osteochondral allografting can restore damaged joint surfaces but its clinical use is limited by poor access to high quality tissue. Vitrification of osteochondral tissue would allow the banking of this tissue but requires high concentrations of cryoprotective agents. This study was designed to ascertain dimethyl sulfoxide (DMSO) toxicity kinetics to chondrocytes in situ after exposure to DMSO at different temperatures recorded as a function of time. Porcine osteochondral dowels were exposed to 1, 3, 5, and 6M DMSO at 4, 22, and 37°C for 0.5 min to 120 min. Chondrocyte recovery was determined by membrane integrity (Syto 13 and ethidium bromide) and mitochondrial (WST-1) assays. Results demonstrated that cell recovery was concentration, temperature and time dependent. At higher concentrations and temperatures, significant cell loss occurred within minutes. A rate constant calculated for chondrocyte death was dependent on temperature. 1 M DMSO appeared relatively non-toxic. This experiment established a method to examine systematically toxicity parameters for chondrocytes in situ and this data can be used to tailor vitrification protocols by limiting exposure temperature and time or lowering DMSO concentrations below toxic levels recorded.     

Changes in the erythrocyte membrane-cytoskeleton complex induced by dimethyl sulfoxide, polyethylene glycol, and low temperature

The effect of the cryoprotectants DMSO and PEG-1500 as well as freezing-thawing on the proteins of the canine erythrocyte membrane-cytoskeleton complex was studied using the cross-linking agent diamide. It was shown that the intensity of disturbances in the protein network structure correlated with the increased SH-group accessibility for oxidative bridging by this compound and accordingly, enhanced formation of high-molecular-weight protein aggregates. The maximum level of diamide-induced aggregability was revealed upon freezing of erythrocytes in liquid nitrogen without cryoprotectant. Electrophoretic analysis of the ghosts of erythrocytes incubated with cryoprotectants showed a significant increase in the aggregation level only for the cells in the polymer solution. After the freezing-thawing cycle, the diamide-induced protein aggregability in erythrocytes cryopreserved with PEG-1500 strongly increased; when DMSO was used for cell protection, the aggregation was much less pronounced than in the unprotected cells. One can suppose that the exocellular cryoprotectant PEG-1500, as distinct from the endocellular cryoprotectant DMSO, is unable to provide for preservation of the structure of the membrane-cytoskeleton protein complex at a level necessary for the maintenance of cell integrity after the return to physiological conditions.     

The effects of cryoprotectants on sperm motility of the Chinese pearl oyster,Pinctada fucata martensii

Cryopreservation has been widely employed to preserve genetic material of aquatic animals. Although of common use in bivalves, resulting effects due to the toxicity of the cryoprotectants dimethyl sulfoxide (DMSO), propanediol (PG), methanol (MET) and ethylene glycol (EG), upon sperm motility in the Chinese pearl oyster, Pinctada fucata martensii, has remained undocumented. This study endeavors to identify the least toxic among the effective cryoprotectant agents by observing and comparing their toxic effects on sperm motility under varying concentrations and duration of exposure. Sperm samples were exposed during controlled experiments, for 1, 3, 6, 9, 12 and 15 min durations, to each of the listed cryoprotectants at 5, 10, 15, and 20% (volume:volume) concentrations. Sperm motility was observed to diminish when exposed to all cryoprotectant solutions, and observations demonstrated that toxicity increased relative to both concentration and equilibration time. After 6 min of exposure to the cryoprotectants, sperm motility was seen to have diminished significantly in DMSO at just 5% concentration, and in MET, PG and EG at 10% concentrations, respectively (the values of the lowest observed effect concentrations). The relationship between the quantity of immotile sperm and the cryoprotectant concentration was described using the logarithmic regression equation. MET exhibited the lowest effective concentration required to inhibit sperm motility by 50% (EC₅₀), followed by EG, PG and DMSO, in order. Therefore, MET proved most toxic under the test conditions for sperm of P. fucata martensii, whereas DMSO, PG and EG were observed as comparatively safer, suggesting that DMSO, PG and EG warrant further study in the application of cryopreservation of Chinese P. fucata martensii sperm.