Concentrations Of Me2SO Research Articles

Open-source hardware- and software-based cryomicroscopy system for investigation of phase transitions in cryobiological research.

The development of inexpensive equipment adapted for the study of a specific biological object is very important for cryobiology. In the presented work, we have proposed a simple system for microscopy utilising open-source platform Arduino. Testing this system showed that it had sufficient sensitivity to determine the physical processes occurring in a cryopreserved sample such as intra- and extracellular water crystallisation and salt eutectic. Utilising this system, we investigated the mechanisms of cryoprotection and cryodamage of testis interstitial cells (ICs) in cryoprotective media, which included cryoprotective agents such as dimethyl sulphoxide (Me2 SO), as well as foetal bovine serum or polymers (dextran, hydroxyethyl starch and polyethylene glycol). It was shown that a serum-/xeno-free medium that included 0.7M Me2 SO and 100mg/mL dextran was able to reduce intracellular water crystallisation in cells, change the structure of extracellular ice, and reduce salt eutectic and recrystallisation. All these effects correlated with better IC survival after cryopreservation in the medium. This medium is potentially less toxic as it has lower concentrations of Me2 SO compared to serum-containing media developed for cryopreservation of testicular cells. This would pave a way for the creation of nontoxic serum-free compositions that does not require removal before use of cryopreserved living cells for laboratory practice or in clinics.

Cryopreservation of human adipose derived mesenchymal stromal cells in varying Me2SO concentrations in a liquid nitrogen-free system under a quality management system

Cryopreservation of human adipose derived mesenchymal stromal cells in varying Me2SO concentrations in a liquid nitrogen-free system under a quality management system

Effect of cryoprotectant concentration on bovine oocyte permeability and comparison of two membrane permeability modelling approaches

The plasma membrane permeability to water and cryoprotectant (CPA) significantly impacts vitrification efficiency of bovine oocytes. Our study was designed to determine the concentration-dependent permeability characteristics for immature (GV) and mature (MII) bovine oocytes in the presence of ethylene glycol (EG) and dimethyl sulphoxide (Me2SO), and to compare two different modeling approaches: the two parameter (2P) model and a nondilute transport model. Membrane permeability parameters were determined by consecutively exposing oocytes to increasing concentrations of Me2SO or EG. Higher water permeability was observed for MII oocytes than GV oocytes in the presence of both Me2SO and EG, and in all cases the water permeability was observed to decrease as CPA concentration increased. At high CPA concentrations, the CPA permeability was similar for Me2SO and EG, for both MII and GV oocytes, but at low concentrations the EG permeability of GV oocytes was substantially higher. Predictions of cell volume changes during CPA addition and removal indicate that accounting for the concentration dependence of permeability only has a modest effect, but there were substantial differences between the 2P model and the nondilute model during CPA removal, which may have implications for design of improved methods for bovine oocyte vitrification.

Dimethyl sulfoxide maintains structure and function of cryopreserved equine endometrial explants

Availability of viable frozen-thawed endometrial tissues could facilitate detailed studies into physiologic and disease processes influencing the endometrium. This study was designed to investigate the cryosurvival of equine endometrial tissue. Previous studies in the human and horse have focused on cryopreservation of dissociated endometrial cells. To our knowledge, there are no studies on cryopreservation of endometrial explants. Our objectives were to 1) determine the influence of differing concentrations of the permeating cryoprotectant dimethyl sulfoxide (Me2SO) on viability, structural integrity, and gene expression of cryopreserved equine endometrial tissues prior to and following a 5-day explant culture in vitro and 2) examine the influence of low (1000 mg/L dextrose) vs high (4500 mg/L dextrose) glucose medium during in vitro culture. Both 10% and 20% (v/v) concentrations of Me2SO maintained viability following cryopreservation and in vitro culture. In addition, gene expression remained unaltered following cryopreservation with either 10% or 20% Me2SO. However, tissue structural integrity was slightly reduced compared to the fresh control. Furthermore, there was no difference in structural integrity, cell viability, or gene expression between low and high glucose medium during in vitro culture. Although E-cadherin and Ki67 gene expression was not different among fresh, 10% Me2SO, and 20% Me2SO treatments prior to or following tissue culture, estrogen receptor-α and progesterone receptor gene expression were reduced in all groups after explant culture. This is the first report of successful cryopreservation of equine endometrial explants.

Modeling the addition/removal of dimethyl sulfoxide into/from articular cartilage treated with the liquidus-tracking method

The successful preservation of articular cartilage (AC) is of great clinical and scientific significance. The vitreous cryopreservation of AC through the liquidus-tracking (LT) method has achieved satisfactory levels of post-warming chondrocyte survival, but the design of the entire procedure could be improved further. Mathematical modeling would help to better understand the nature and features of the LT process as experienced by the AC sample, possibly providing additional instructive data useful for guiding experimental design. In this paper, the addition/removal of the cryoprotectant dimethyl sulfoxide (Me2SO) into/from the LT-treated AC was modeled and studied numerically. The cryoprotectant permeation model and the cell membrane transport model were combined, and the spatiotemporal concentrations of Me2SO in the AC matrix and the chondrocytes were profiled. In addition, the possible injuries to the cell were predicted based on the biophysical properties of chondrocytes under both the stepwise and the continuous LT protocols. Based on these results, improvements to the stepwise LT method were proposed. Compared to the existing protocol, these improvements might eliminate two processing steps and reduce the protocol duration by 35%. Decreasing the number of processing steps and protocol duration not only saves labor and material resources, but also lowers the risk that chondrocytes will incur toxicity- and chilling-associated injury.

Cryopreservation and transplantation of common carp spermatogonia.

Common carp (Cyprinus carpio) is one of the most cultured fish species over the world with many different breeds and plenty of published protocols for sperm cryopreservation. However, data regarding preservation of gonadal tissue and surrogate production is still missing. A protocol for freezing common carp spermatogonia was developed through varying different factors along a set of serial subsequent experiments. Among the six cryoprotectants tested, the best survival was achieved with dimethyl sulfoxide (Me2SO). In the next experiment, a wide range of cooling rates (0.5–10°C/min) and different concentrations of Me2SO were tested resulting in the highest survival achieved using 2 M Me2SO and cooling rate of -1°C/min. When testing different tissue sizes and incubation times in the cryomedia, the highest viability was observed when incubating 100 mg tissue fragments for 30 min. Finally, sugar supplementation did not yield significant differences. When testing different equilibration (ES) and vitrification solutions (VS) used for needle-immersed vitrification, no significant differences were observed between the tested groups. Additionally, varied exposure time to VS did not improve the vitrification outcome where the viability was 4-fold lower than that of freezing. The functionality of cryopreserved cells was tested by interspecific transplantation into sterilized goldfish recipients. The exogenous origin of the germ cells in gonads of goldfish recipient was confirmed by molecular markers and incorporation rate was over 40% at 3 months post-transplantation. Results of this study can serve for long-term preservation of germplasm in carp which can be recovered in a surrogate recipient.

Negative air pressure treatment accelerates the penetration of permeable cryoprotectants into bovine ovarian tissue in vitrification protocol and improves cell density after vitrification

Effects of additional physical treatments during vitrification of the bovine ovarian tissue were examined for increasing of permeability of ethylene glycol (EG) and dimethyl sulfoxide (Me2SO). The concentrations of EG and Me2SO and histological changes in the ovarian tissue were evaluated. In the first equilibration step (7.5% EG and 7.5% Me2SO), all the 10-min physical treatments, i.e., negative (679 hPa) or positive (1347 hPa) air pressure applied with a disposable syringe, and shaking (60 rpm) applied with a laboratory shaker, were comparable to 25-min non-physical treatment (plain) vitrification. When effects of the negative air pressure were examined in the second equilibration step (20% EG and 20% Me2SO), its 10-min treatment was equivalent to 15-min plain vitrification (140–170 mg/g tissue). It was thus indicated that the negative air pressure treatment accelerates the penetration of permeable cryoprotectants into the ovarian tissue slices. Histological examination showed that the cell density and the amount of pan-cadherin in the tunica albuginea of the ovary was reduced by the vitrification, but was improved by the negative air pressure treatment. The amount of pan-cadherin in the tunica albuginea was recommended as a biomarker for evaluation of effectiveness of protocol for cryopreservation of bovine ovarian tissue and considered to be a candidate biomarker for human ovarian tissue cryopreservation.

Cryopreservation: Extending the viability of biological material from sea urchin (Echinometra lucunter) in ecotoxicity tests

The sea urchin, Echinometra lucunter, is widely used in embryo-larval tests for ecotoxicological studies in Brazil and other countries. For each test, sea urchins are collected from the wild and this can cause impact on wild populations and it is limited by the weather and season which in turn limits the ability to carry out the tests. Cryopreservation is a method of live biological material storage at low temperature and can be used for long periods with little decline in viability, reducing the number of animals taken from the wild and enabling testing to be carried out on demand, irrespective of spawning season or location. In this study, 15 combinations of cryoprotective agents (CPAs) were evaluated on spermatozoa, subjected to a rapid cooling curve followed by immersion in liquid nitrogen. Twenty-four CPA combinations were evaluated on eggs subjected to a more gradual cooling curve in nitrogen vapor down to −35 °C and then plunging in liquid nitrogen. Fertilization tests using cryopreserved spermatozoa gave high pluteus larvae yields (≈80%) when concentrations of 10.5% or 13.65% ME2SO or 13.65% ME2SO+15.75% sucrose were used. The higher concentrations of ME2SO plus sucrose were more effective at maintaining the fertilization capacity of spermatozoa post-thawing. Egg cryopreservation was not successful with 0% fertilization observed post-thawing. The results suggest that it is feasible to implement spermatozoa cryopreservation as technological innovation to create a sperm bank for E. lucunter, which can be used in ecotoxicological tests, bringing benefits for researches and contributing to the conservation of the species.

Cryopreservation of rat hepatocytes with disaccharides for cell therapy

Cryopreservation of hepatocytes is a crucial step in the implementation of cell therapy for treating certain liver diseases. In the present study we investigated the effect of the some disaccharides on the cryopreservation of rat hepatocytes. Liver cells were frozen in media in the presence or absence of low concentrations of Me2SO (5% Me2SO) supplemented with varying concentrations of disaccharides (sucrose, glucose and trehalose). After 7 days of cryopreservation, the hepatocytes were thawed and viability was measure by exclusion of trypan blue and by the MTT technique, as well as by determining albumin production. Among the investigated disaccharides and concentrations, 0.2 M trehalose showed the best overall outcome. Compared to the use of Me2SO alone, significant improvement in post-thaw cell viability was observed. The new solution may reduce Me2SO side effects on patients and improve the viability and quality of cryopreserved hepatocytes.

C-2014: Investigation of genetic and epigenetic changes of cryopreserved mesenchymal stem cells

Cryopreservation is routinely used to store different types of cells and tissues. It is used for transportation and cell banking. However, the impact of cryopreservation on the genetic and epigenetic profile of frozen cells is still unclear. Here, we try to explore the alterations caused to cryopreserved mesenchymal stem cells and its possible implications. Our freezing protocol showed that the cells frozen with 5% dimethyl sulfoxide (Me2SO) at all passages (early, mid and late) had the best post thaw survivability in terms of MTT assay and adherence capacity. 1% and 15% Me2SO concentrations proved to be sub-optimal after thawing the cells. Adipogenic differentiation of MSCs showed that cells frozen in all concentrations of Me2SO could differentiate to adipocytes. Cells frozen with 5%, 10% and 15% Me2SO showed comparable oil droplet formation. DNA methylation plays a critical role in health and disease. Changes in DNA methylation has been seen in cancer, ageing and auto immune diseases. Alterations in the 5-mC% content of DNA were observed in the frozen samples as compared to control (unfrozen cells, never exposed to Me2SO). Further studies need to be conducted so as to determine the implication of these alterations. In total, cryopreservation seemed to cause changes in the mRNA expressions, global DNA methylation which could alter the cell fate at a later time point. Therefore, there is a need for comprehensive and orderly studies to determine the possible impact of subtle genomic/epigenomic and proteomic changes resulting from cryopreservation.

Evaluations of bioantioxidants in cryopreservation of umbilical cord blood using natural cryoprotectants and low concentrations of dimethylsulfoxide

Transplantation using hematopoietic stem cells from umbilical cord blood (UCB) is a life-saving treatment option for patients with select oncologic diseases, immunologic diseases, bone marrow failure, and others. Often this transplant modality requires cryopreservation and storage of hematopoietic stem cells (HSC), which need to remain cryopreserved in UCB banks for possible future use. The most widely used cryoprotectant is dimethylsulfoxide (Me 2SO), but at 37 °C, it is toxic to cells and for patients, infusion of cryopreserved HSC with Me 2SO has been associated with side effects. Freezing of cells leads to chemical change of cellular components, which results in physical disruption. Reactive oxygen species (ROS) generation also has been implicated as cause of damage to cells during freezing. We assessed the ability of two bioantioxidants and two disaccharides, to enhance the cryopreservation of UCB. UCB was processed and subjected to cryopreservation in solutions containing different concentrations of Me 2SO, bioantioxidants and disaccharides. Samples were thawed, and then analysed by: flow cytometry analysis, CFU assay and MTT viability assay. In this study, our analyses showed that antioxidants, principally catalase, performed greater preservation of: CD34+ cells, CD123+ cells, colony-forming units and cell viability, all post-thawed, compared with the standard solution of cryopreservation. Our present studies show that the addition of catalase improved the cryopreservation outcome. Catalase may act on reducing levels of ROS, further indicating that accumulation of free radicals indeed leads to death in cryopreserved hematopoietic cells.

Charged Amino Acid Residues 997–1000 of Human Apolipoprotein B100 Are Critical for the Initiation of Lipoprotein Assembly and the Formation of a Stable Lipidated Primordial Particle in McA-RH7777 Cells

We previously demonstrated that a portion, or perhaps all, of the residues between 931 and 1000 of apolipoprotein (apo) B100 are required for the initiation of apoB-containing particle assembly. Based on our structural model of the first 1000 residues of apoB (designated as apoB:1000), we hypothesized that this domain folds into a three-sided lipovitellin-like "lipid pocket" via a hairpin-bridge mechanism. We proposed that salt bridges are formed between four tandem charged residues 717-720 in the turn of the hairpin bridge and four tandem complementary residues 997-1000 located at the C-terminal end of the model. To identify the specific motif within residues 931 and 1000 that is critical for apoB particle assembly, apoB:956 and apoB:986 were produced. To test the hairpin-bridge hypothesis, the following mutations were made: 1) residues 997-1000 deletion (apoB:996), 2) residues 717-720 deletion (apoB:1000Delta717-720), and 3) substitution of charged residues 997-1000 with alanines (apoB:996 + 4Ala). Characterization of particles secreted by stable transformants of McA-RH7777 cells demonstrated the following. 1) ApoB:956 did not form stable particles and was secreted as large lipid-rich aggregates. 2) ApoB:986 formed both a lipidated particle that was denser than HDL(3) and large lipid-rich aggregates. 3) Compared with wild-type apoB:1000, apoB:1000Delta717-720 displayed the following: (i) significantly diminished capacity to form intact lipidated particles and (ii) increased propensity to form large lipid-rich aggregates. 4) In striking contrast to wild-type apoB:1000, (i) apoB:996 and apoB:996 + 4Ala were highly susceptible to intracellular degradation, (ii) only a small proportion of the secreted proteins formed stable HDL(3)-like lipoproteins, and (iii) a majority of the secreted proteins formed large lipid-rich aggregates. We conclude that the first 1000 amino acid residues of human apoB100 are required for the initiation of nascent apoB-containing lipoprotein assembly, and residues 717-720 and 997-1000 play key roles in this process, perhaps via a hairpin-bridge mechanism.

Chronic Treatment with Resveratrol Induces Redox Stress- and Ataxia Telangiectasia-mutated (ATM)-dependent Senescence in p53-positive Cancer Cells

The induction of senescence, an irreversible growth arrest, in cancer cells is regarded as a mean to halt tumor progression. The phytoalexin resveratrol (RV) is known to possess a variety of cancer-preventive, -therapeutic, and -chemosensitizing properties. We report here that chronic treatment with RV in a subapoptotic concentration induces senescence-like growth arrest in tumor cells. In contrast to the widely accepted antioxidant property of RV, we demonstrate that one causative stimulus for senescence induction by chronic RV is an increased level of reactive oxygen species (ROS). The ROS formed upon RV exposure include hydrogen peroxide and superoxide and originate largely from mitochondria. Consistently, co-incubation with the antioxidant N-acetyl cysteine interfered with RV-mediated reactivation of the senescence program. Molecular mediators on the way from increased ROS levels to the observed growth arrest include p38 MAPK, p53, and p21. Moreover, we provide evidence that RV-initiated replication stress, apparent by activation of the ataxia telangiectasia-mutated kinase pathway, is associated with increased ROS levels and senescence induction. This is the first report linking cell cycle effects with a pro-oxidant and pro-senescent effect of RV in cancer cells.

Activation of Inhibitors by Sortase Triggers Irreversible Modification of the Active Site

Sortases anchor surface proteins to the cell wall of Gram-positive pathogens through recognition of specific motif sequences. Loss of sortase leads to large reductions in virulence, which identifies sortase as a target for the development of antibacterials. By screening 135,625 small molecules for inhibition, we report here that aryl (beta-amino)ethyl ketones inhibit sortase enzymes from staphylococci and bacilli. Inhibition of sortases occurs through an irreversible, covalent modification of their active site cysteine. Sortases specifically activate this class of molecules via beta-elimination, generating a reactive olefin intermediate that covalently modifies the cysteine thiol. Analysis of the three-dimensional structure of Bacillus anthracis sortase B with and without inhibitor provides insights into the mechanism of inhibition and reveals binding pockets that can be exploited for drug discovery.

Further work on the cryopreservation of articular cartilage with particular reference to the liquidus tracking (LT) method

The cryopreservation of articular cartilage with survival of living cells has been a difficult problem. We have provided evidence that this is due to the formation of ice crystals in the chondrons. We have developed a method in which the concentration of the cryoprotectant dimethyl sulphoxide (Me 2SO) is increased progressively, in steps, as cooling proceeds so that ice is never allowed to form, but the very high concentrations of Me 2SO required at low temperatures are reached only at those low temperatures. In this paper, we describe some new experiments with discs of ovine articular cartilage similar to those used in our previous studies and we show that continuous stirring throughout the process resulted in a significant increase in the rate of 35S sulphate incorporation into glycosoaminoglycans (GAGs), now reaching 87% of the corresponding fresh control values. We confirmed that the method is also effective for human knee joint cartilage, which gave 70% of fresh control ability to synthesise GAGs; continuous stirring was also used in this experiment. We then extended the method to ovine knee joint osteochondral dowels and showed that, again with continuous stirring, the method produced tissue concentrations of Me 2SO that were sufficient to prevent freezing in dowels too, and to permit cell function at 60% of control. The most important mechanical property (instantaneous compressive modulus) was unaffected by the process. Finally, we experimented with some technical variations to facilitate clinical use—a more rapid process for warming and removal of Me 2SO was developed and a method of short-term storage before or after cryopreservation was developed. Finally, pilot experiments were carried out to provide proof of principle for a closed, continuous flow method in which both temperature and Me 2SO concentration were computer-controlled.

Development of Calpain-specific Inactivators by Screening of Positional Scanning Epoxide Libraries

Calpains are calcium-dependent proteases that are required for numerous intracellular processes but also play an important role in the development of pathologies such as ischemic injury and neurodegeneration. Many current small molecule calpain inhibitors also inhibit other cysteine proteases, including cathepsins, and need improved selectivity. The specificity of inhibition of several calpains and papain was profiled using synthetic positional scanning libraries of epoxide-based compounds that target the active-site cysteine. These peptidomimetic libraries probe the P4, P3, and P2 positions, display (S,S)- or (R,R)-epoxide stereochemistries, and incorporate both natural and non-natural amino acids. To facilitate library screening, an SDS-PAGE assay that measures the extent of hydrolysis of an inactive recombinant m-calpain was developed. Individual epoxide inhibitors were synthesized guided by calpain-specific preferences observed from the profiles and tested for inhibition against calpain. The most potent compounds were assayed for specificity against cathepsins B, L, and K. Several compounds demonstrated high inhibition specificity for calpains over cathepsins. The best of these inhibitors, WRH(R,R), irreversibly inactivates m- and mu-calpain rapidly (k(2)/K(i) = 131,000 and 16,500 m(-1) s(-1), respectively) but behaves exclusively as a reversible and less potent inhibitor toward the cathepsins. X-ray crystallography of the proteolytic core of rat mu-calpain inactivated by the epoxide compounds WR gamma-cyano-alpha-aminobutyric acid (S,S) and WR allylglycine (R,R) reveals that the stereochemistry of the epoxide influences positioning and orientation of the P2 residue, facilitating alternate interactions within the S2 pocket. Moreover, the WR gamma-cyano-alpha-aminobutyric acid (S,S)-complexed structure defines a novel hydrogen-bonding site within the S2 pocket of calpains.

52. Changes in intracellular potassium and sodium content of 2-cell mouse embryos induced by exposure to vitrification concentrations of EG and Me 2SO

52. Changes in intracellular potassium and sodium content of 2-cell mouse embryos induced by exposure to vitrification concentrations of EG and Me 2SO

Caliciviruses Differ in Their Functional Requirements for eIF4F Components

Two classes of viruses, namely members of the Potyviridae and Caliciviridae, use a novel mechanism for the initiation of protein synthesis that involves the interaction of translation initiation factors with a viral protein covalently linked to the viral RNA, known as VPg. The calicivirus VPg proteins can interact directly with the initiation factors eIF4E and eIF3. Translation initiation on feline calicivirus (FCV) RNA requires eIF4E because it is inhibited by recombinant 4E-BP1. However, to date, there have been no functional studies carried out with respect to norovirus translation initiation, because of a lack of a suitable source of VPg-linked viral RNA. We have now used the recently identified murine norovirus (MNV) as a model system for norovirus translation and have extended our previous studies with FCV RNA to examine the role of the other eIF4F components in translation initiation. We now demonstrate that, as with FCV, MNV VPg interacts directly with eIF4E, although, unlike FCV RNA, translation of MNV RNA is not sensitive to 4E-BP1, eIF4E depletion, or foot-and-mouth disease virus Lb protease-mediated cleavage of eIF4G. We also demonstrate that both FCV and MNV RNA translation require the RNA helicase component of the eIF4F complex, namely eIF4A, because translation was sensitive (albeit to different degrees) to a dominant negative form and to a small molecule inhibitor of eIF4A (hippuristanol). These results suggest that calicivirus RNAs differ with respect to their requirements for the components of the eIF4F translation initiation complex.

The Two-stage Pathway of Ataxin-3 Fibrillogenesis Involves a Polyglutamine-independent Step

The aggregation of ataxin-3 is associated with spinocerebellar ataxia type 3, which is characterized by the formation of intraneuronal aggregates. However, the mechanism of aggregation is currently not well understood. Ataxin-3 consists of a folded Josephin domain followed by two ubiquitin-interacting motifs and a C-terminal polyglutamine tract, which in the non-pathological form is less than 45 residues in length. We demonstrate that ataxin-3 with 64 glutamines (at(Q64)) undergoes a two-stage aggregation. The first stage involves formation of SDS-soluble aggregates, and the second stage results in formation of SDS-insoluble aggregates via the poly(Q) region. Both these first and second stage aggregates display typical amyloid-like characteristics. Under the same conditions at(Q15) and at(QHQ) undergo a single step aggregation event resulting in SDS-soluble aggregates, which does not involve the polyglutamine tract. These aggregates do not convert to the SDS-insoluble form. These observations demonstrate that ataxin-3 has an inherent capacity to aggregate through its non-polyglutamine domains. However, the presence of a pathological length polyglutamine tract introduces an additional step resulting in formation of a highly stable amyloid-like aggregate.

Dimethylsulfoxide oxidizes glutathione in vitro and in human erythrocytes: Kinetic analysis by 1H NMR

The interaction of dimethylsulfoxide (Me 2SO) with glutathione was investigated under non-equilibrium conditions in solution using 1H NMR and in intact erythrocytes using 1H spin–echo NMR. In solution the reaction was observed to follow second-order kinetics (Rate = k 1[glutathione][Me 2SO]) at 300 K pH 7.4, k sol = 4.7 × 10 −5 mol −1 L 1 s −1. In intact erythrocytes the rate constant for the cellular environment, k cell, was found to be slightly larger at 8.1 × 10 −5 mol −1 L 1 s −1. Furthermore, the reaction of Me 2SO with erythrocyte glutathione showed a biphasic dependence on the Me 2SO concentration, with little oxidation of glutathione occurring until the Me 2SO concentration exceeded 0.5 mol L −1. The results suggest that at lower concentrations, Me 2SO can be effectively removed, most probably by reaction with glutathione, which is regenerated by glutathione reductase, although preferential reaction with other cellular components (e.g., membrane or cellular thiols) cannot be ruled out. Thus the concentrations of Me 2SO that are commonly used in cryopreservation of mammalian cells (∼1.4 mol L −1) can cause oxidation of intracellular glutathione.