Addition Of Dimethyl Sulfoxide Research Articles

The Assessment of Cryopreservation Conditions for Human Umbilical Cord Stroma-Derived Mesenchymal Stem Cells towards a Potential Use for Stem Cell Banking

Human umbilical cord stroma-derived mesenchymal stem cells (hUCS-MSCs) are considered as a remarkable and promising stem cell source to be potentially used in cellular therapies. While no graft rejection has been reported in the recipient organism even in xeno-transplantation studies, attenuate tumor cell growth and gene transfers have been experimentally shown. In this study, we have demonstrated a reliable, reproducible and efficient cryopreservation method of hUCS-MSCs resulting in one of the highest cell survival rates reported so far. Conventional, computer-controlled multistep slow freezing (MSSF), and vitrification methods were comparatively tested using cell permeable [dimethylsulfoxide (DMSO), ethylene glycol] and impermeable [trehalose, sucrose, hydroxyethyl starch (HES), human serum albumin] cryoprotectant agents (CPAs). After determining the ice nucleation point for each solution, latent heat evolution was suppressed during freezing, followed by a cooling process to -40°C at 1°C/min or 0.3°C/min. The efficiency of the cryopreservation techniques used was determined by cell viability and proliferation assays, the expression of cell surface markers, cytoskeletal proteins and chromosome alignments. The cell survival rate was found to be highest (87 ± 5%) by MSSF with sucrose (0.1 M) +DMSO (10%) at 1°C/min freezing rate. In this group, no significant difference was noted before and after the cryopreservation in cell morphology, cytokeratin, vimentin, and α-smooth muscle actin profiles and the expressions of CD105, CD90, CD73, CD29 and HLA-DR. Second highest cell survival ratio (85 ± 6%) was obtained in DMSO (10%) alone at 1°C/min freezing rate. Interestingly, poor (18 ± 15%) cell survival rates were obtained after vitrification. Cumulatively, results indicated that MSSF favors the other freezing protocols with an addition of sucrose or DMSO alone depending on the freezing rate used.

Enhancement of the thermoelectric properties of PEDOT:PSS thin films by post-treatment

In this work, the thermoelectric (TE) properties of poly(3,4-ethylenedioxylthiophene):poly(styrene sulfonate) (PEDOT:PSS) thin films at room temperature are studied. Different methods have been applied for tuning the TE properties: 1st addition of polar solvent, dimethyl sulfoxide (DMSO), into the PEDOT:PSS solution; 2nd post-treatment of thin films with a mixture of DMSO and ionic liquid, 1-ethyl-3-methylimidazolium tetrafluoroborate (EMIMBF4). It is verified that DMSO post-treatment is more efficient than DMSO addition in improving the electrical conductivity with a trivial change in the Seebeck coefficient. The power factor is increased up to 30.1 μW mK−2 for the film with DMSO post-treatment, while the optimized power factor by DMSO addition is 18.2 μW mK−2. It is shown that both DMSO addition and post-treatment induce morphological changes: an interconnected network of elongated PEDOT grains is generated, leading to higher electrical conductivity. In contrast, for those films post-treated in the presence of EMIMBF4, an interconnected network of short and circular PEDOT grains with increased polaron density is created, resulting in the improvement in the Seebeck coefficient and a concomitant compromise in the electrical conductivity. An optimized power factor of 38.46 μW mK−2 is achieved at 50 vol% of EMIMBF4, which is the highest reported so far for PEDOT:PSS thin films to our knowledge. Assuming a thermal conductivity of 0.17 W mK−1, the corresponding ZT is 0.068 at 300 K. These results demonstrate that post-treatment is a promising approach to enhance the TE properties of PEDOT:PSS thin films. Furthermore, ionic liquid, EMIMBF4, shows the potential for tuning the TE properties of PEDOT:PSS thin films via a more environmentally benign process.

Effect of Processing Additives on PCDTBT:PC60BM Based Organic Photovoltaic Cells

We investigated the effect of processing additives on the morphology, charge transport and device performance of poly[N-9″-heptadecanyl-2,7-carbazole-alt-5,5-(4′,7′-di-2-thienyl-2′,1′,3′-benzothiadiazole)] (PCDTBT) and [6,6]-phenyl C61 butyric acid methyl ester (PC60BM) based bulk heterojunction (BHJ) photovoltaic cells. To carry out this investigation, dimethylsulfoxide (DMSO) and dimethylformamide (DMF) were separately added as processing additives in ortho dichlorobenzene (o-DCB) solution of PCDTBT. A progressive aggregation of PCDTBT in both solutions and films were resulted through the addition of both DMSO and DMF additives. The optimization of the additives resulted in fine tuning the morphology of PCDTBT/PC60BM based solar cells, leading to improved domain structure and hole mobility on the active layer, and significantly enhanced photovoltaic performance.

Phase 1 and Phase 2 Drug Metabolism and Bile Acid Production of HepaRG Cells in a Bioartificial Liver in Absence of Dimethyl Sulfoxide

The human liver cell line HepaRG has been recognized as a promising source for in vitro testing of metabolism and toxicity of compounds. However, currently the hepatic differentiation of these cells relies on exposure to dimethylsulfoxide (DMSO), which, as a side effect, has a cytotoxic effect and represses an all-round hepatic functionality. The AMC-bioartificial liver (AMC-BAL) is a three-dimensional bioreactor that has previously been shown to upregulate various liver functions of cultured cells. We therefore cultured HepaRG cells in the AMC-BAL without DMSO and characterized the drug metabolism. Within 14 days of culture, the HepaRG-AMC-BALs contained highly polarized viable liver-like tissue with heterogeneous expression of CYP3A4. We found a substantial metabolism of the tested substrates, ranging from 26% (UDP-glucuronosyltransferase 1A1), 47% (CYP3A4), to 240% (CYP2C9) of primary human hepatocytes. The CYP3A4 activity could be induced 2-fold by rifampicin, whereas CYP2C9 activity remained equally high. The HepaRG-AMC-BAL secreted bile acids at 43% the rate of primary human hepatocytes and demonstrated hydroxylation, conjugation, and transport of bile salts. Concluding, culturing HepaRG cells in the AMC-BAL yields substantial phase 1 and phase 2 drug metabolism, while maintaining high viability, rendering DMSO addition superfluous for the promotion of drug metabolism. Therefore, AMC-BAL culturing makes the HepaRG cells more suitable for testing metabolism and toxicity of drugs.

Denaturation and Preservation of Globular Proteins: The Role of DMSO

The thermal denaturation of hen egg white lysozyme (HEWL) in D(2)O was followed by IR absorption after addition of dimethyl sulfoxide (DMSO) at different molar fractions. Amide I intensity and position revealed that DMSO reduces the thermal stability of the native protein and favors the formation of ordered aggregates. The comparison with ethanol/water solutions evidenced that ethanol (partially deuterated ethanol EtOD) has a stronger effect on the thermal stability of HEWL: the same down-shift of melting temperature was measured at 0.18 and 0.30 molar fraction of ethanol and DMSO, respectively. This is probably due to lower polarity of EtOD/D(2)O with respect to DMSO/D(2)O solutions. A kinetic study of protein assembling at 0.30 DMSO molar fraction, was also performed at different temperatures. The high viscosity of the solvent was observed to cause a sensitive slowing down of aggregation rate in comparison to that of water/alcohol solutions. The evidence of a retarded self-assembling put forward a possible explanation for the use of dimethyl sulfoxide as a protectant of protein structure. In fact, for both organic solvents a nonspecific interaction with the protein and a water-mediated action is deduced, but the addition of DMSO reduces the irreversible denaturation due to kinetic effects and this can be exploited for lessening one of the main degradation routes of globular proteins during freezing-thawing cycles.

Cardioprotective Properties of Dimethyl Sulfoxide during Global Ischemia–Reperfusion of Isolated Rat Heart

Cardioprotective properties of dimethyl sulfoxide (DMSO) were studied in the isolated rat heart model. Intraperitoneal administration of DMSO to animals for 3 days before the experiment, but not addition of DMSO to the perfusate, reduced infarction size. Both intraperitoneal and intracoronary administration of DMSO reduced the severity of postischemic left-ventricular dysfunction.

Impact of Ionic Liquid Pretreatment Conditions on Cellulose Crystalline Structure Using 1-Ethyl-3-methylimidazolium Acetate

Ionic liquids (ILs) have been shown to affect cellulose crystalline structure in lignocellulosic biomass during pretreatment. A systematic investigation of the swelling and dissolution processes associated with IL pretreatment is needed to better understand cellulose structural transformation. In this work, 3-20 wt % microcrystalline cellulose (Avicel) solutions were treated with 1-ethyl-3-methylimidazolium acetate ([C(2)mim][OAc]) and a mixture of [C(2)mim][OAc] with the nonsolvent dimethyl sulfoxide (DMSO) at different temperatures. The dissolution process was slowed by decreasing the temperature and increasing cellulose loading, and was further retarded by addition of DMSO, enabling in-depth examination of the intermediate stages of dissolution. Results show that the cellulose I lattice expands and distorts prior to full dissolution in [C(2)mim][OAc] and that upon precipitation the former structure leads to a less ordered intermediate structure, whereas fully dissolved cellulose leads to a mixture of cellulose II and amorphous cellulose. Enzymatic hydrolysis was more rapid for the intermediate structure (crystallinity = 0.34) than for cellulose II (crystallinity = 0.54).

Pressure dependence on the single-particle dynamics and hydrogen-bond structural relaxation of water–DMSO mixtures under ambient and cold conditions

The effects of pressure on the single-particle translational and rotational dynamics and the hydrogen-bond structural relaxation of water–dimethyl sulfoxide (DMSO) mixtures were investigated under ambient and cold conditions using classical molecular dynamics simulations. We considered five different concentrations of DMSO with mole fractions X DMSO = 0.012, 0.09, 0.20, 0.35, and 0.50 and seven different pressures ranging from 0.1 to 500 MPa. It was found that the addition of DMSO to water leads to slow dynamics for both water and DMSO molecules under pressure. At very low concentrations of DMSO, water exhibits anomalous behavior with the application of pressure at 258 K, whereas DMSO molecules exhibit average pressure dependencies. The formation of strong hydrogen bonds between water and DMSO molecules occurs when DMSO is added to water, but the extended network is altered due to the absence of any hydrogen-bond donating ability of DMSO. Our calculated water–water and DMSO–water hydrogen-bond structural relaxation times show an initial decrease with the application of pressure at 258 K for solutions with low DMSO concentrations. However, at higher concentrations of DMSO, the relaxation times increase, on average, and no anomalous behavior of the dynamical properties is found on the application of pressure.

Structural studies of the effect that dimethyl sulfoxide (DMSO) has on cisplatin and carboplatin binding to histidine in a protein

The anticancer complexes cisplatin and carboplatin target the DNA major groove, forming intrastrand and interstrand cross-links between guanine bases through their N7 atoms, causing distortion of the DNA helix and apoptotic cell death. A major side effect of these drugs is toxicity, which is caused via binding to many proteins in the body. A range of crystallographic studies have been carried out involving the cocrystallization of hen egg-white lysozyme (HEWL) as a test protein with cisplatin and carboplatin in aqueous and dimethyl sulfoxide (DMSO) conditions. Different cryoprotectants, glycerol and Paratone, were used for each of the cisplatin and carboplatin cocrystallization cases, while silicone oil was used for studies involving N-acetylglucosamine (NAG). Both cisplatin and carboplatin do not bind to HEWL in aqueous media on the timescales of the conditions used here, but upon addition of DMSO two molecules of cisplatin or carboplatin bind either side of His15, which is the only His residue in lysozyme and is assumed to be an imidazolyl anion or a chemical resonance moiety, i.e. both imidazole N atoms are chemically reactive. To identify the platinum-peak positions in the 'with DMSO conditions', anomalous scattering maps were calculated as a cross-check with the F(o) - F(c) OMIT maps. Platinum-occupancy σ values were established using three different software programs in each case. The use of EVAL15 to process all of the diffraction data sets provided a consistent platform for a large ensemble of data sets for the various protein and platinum-compound model refinements with REFMAC5 and then SHELXTL. Overall, this extensive set of crystallization and cryoprotectant conditions allowed a systematic evaluation of cisplatin and carboplatin binding to lysozyme as a test protein via detailed X-ray crystal structure characterizations. DMSO is used as a super-solvent for drug delivery as it is deemed to cause no effect upon drug binding. However, these results show that addition of DMSO causes the platinum anticancer drugs to bind to HEWL. This effect should be considered in toxicity assessments of these drugs and perhaps more widely.

Rheological properties of cellulose/ionic liquid/dimethylsulfoxide (DMSO) solutions

Rheological properties of cellulose dissolved in two ionic liquids (ILs), 1-allyl-3-methylimidazolium chloride (AmimCl) and 1-butyl-3-methylimidazolium chloride (BmimCl), with co-solvent dimethylsulfoxide (DMSO), are studied in the concentration range of cellulose from 0.070 to 6.0 wt%. The viscosities of ILs are exponentially decreased by adding DMSO in the concentration range of 0–100 wt%. The co-solvent DMSO decreases the monomer friction coefficient in cellulose solutions and has no significant change for the entanglement state of cellulose, thus results in the reduced solution viscosity, shortened relaxation time and unchanged moduli of the cross-over point. For cellulose solutions, dilute regime, semidilute unentangled regime and semidilute entangled regime were determined by steady shear experiments. In semidilute entangled regime, the specific viscosities ηsp, relaxation time τ, and plateau modulus GN, exhibit concentration dependences as ηsp ∼ C4.4, τ ∼ C2.2, andGN ∼ C1.9, respectively, in AmimCl-DMSO (80/20 w/w); and ηsp ∼ C4.3, τ ∼ C2.0, and GN ∼ C2.1, respectively, in BmimCl–DMSO (80/20 w/w). Therefore, the rheological properties of cellulose/IL/DMSO solutions are approximately of IL-independence in this study. The dependence of ηsp upon cellulose concentration shows that the IL–DMSO mixture is more like a θ solvent for cellulose, and the thermodynamic properties of IL–DMSO mixtures are similar with those of ILs for cellulose at 25 °C. The conformation of cellulose in ILs would not be changed with the addition of DMSO not only in the dilute regime but also in the entanglement regime.

Effect of DMSO on the sensitivity and diffusion of FPGX gel dosimeter

A stable hydrogel 3D dosimeter was prepared by using Fricke solution, poly (vinyl alcohol) (PVA) and glutaraldehyde (GA). A linear relationship between the dose and the UV absorbency was obtained by determining the color change of the dosimeter in the range of 0–2.5Gy. Dimethyl sulfoxide (DMSO) was used as a free radical scavenger to analyze the thermo-stability and diffusion of FPGX gel dosimeter systems. The results showed that increasing the DMSO concentration could slow down the color change and the Fe3+ diffusion rate in a certain range. With the addition of DMSO, the FPGX gel dosimeter was stable in the storage time before exposure to radiation.

Weak Interactions between Folate and Osmolytes in Solution

Previous osmotic stress studies on the role of solvent in two structurally unrelated dihydrofolate reductases (DHFRs) found weaker binding of dihydrofolate (DHF) to either enzyme in the presence of osmolytes. To explain these unusual results, weak interactions between DHF and osmolytes were proposed, with a competition between osmolyte and DHFR for DHF. High osmolyte concentrations will inhibit binding of the cognate pair. To evaluate this hypothesis, we devised a small molecule approach. Dimerization of folate, monitored by nuclear magnetic resonance, was weakened 2-3-fold upon addition of betaine or dimethyl sulfoxide (DMSO), supporting preferential interaction of either osmolyte with the monomer (as it possesses a larger surface area). Nuclear Overhauser effect (NOE) spectroscopy experiments found a positive NOE for the interaction of the C3'/C5' benzoyl ring protons with the C9 proton in buffer; however, a negative NOE was observed upon addition of betaine or DMSO. This change indicated a decreased tumbling rate, consistent with osmolyte interaction. Osmotic stress experiments also showed that betaine, DMSO, and sucrose preferentially interact with folate. Further, studies with the folate fragments, p-aminobenzoic acid and pterin 6-carboxylate, revealed interactions for both model compounds with betaine and sucrose. In contrast, DMSO was strongly excluded from the pterin ring but preferentially interacted with the p-aminobenzoyl moiety. These interactions are likely to be important in vivo because of the crowded conditions of the cell where weak contacts can more readily compete with specific binding interactions.

Effect of glycerol and dimethyl sulfoxide on the phase behavior of lysozyme: Theory and experiments

Salt, glycerol, and dimethyl sulfoxide (DMSO) are used to modify the properties of protein solutions. We experimentally determined the effect of these additives on the phase behavior of lysozyme solutions. Upon the addition of glycerol and DMSO, the fluid-solid transition and the gas-liquid coexistence curve (binodal) shift to lower temperatures and the gap between them increases. The experimentally observed trends are consistent with our theoretical predictions based on the thermodynamic perturbation theory and the Derjaguin-Landau-Verwey-Overbeek model for the lysozyme-lysozyme pair interactions. The values of the parameters describing the interactions, namely the refractive indices, dielectric constants, Hamaker constant and cut-off length, are extracted from literature or are experimentally determined by independent experiments, including static light scattering, to determine the second virial coefficient. We observe that both, glycerol and DMSO, render the potential more repulsive, while sodium chloride reduces the repulsion.

Effect of Various Concentrations of Dimethyl Sulfoxide (DMSO) on Cell Recovery and Clonogenic Potential of Cryopreserved Peripheral Blood Hematopoietic Progenitors

Abstract 1930 BACKGROUND:Hematopoietic stem cells mobilized to peripheral blood and collected by leukapheresis are predominantly used for autologous transplantation. Prior to cryopreservation the cells need to be processed including the addition of DMSO (dimethyl sulfoxide), which enhance cell survival, but is potentially toxic to stem cell recipient. The most commonly used concentration of DMSO is 10%. The goal of our study was to test if the concentration may be reduced without negative impact on cell recovery and clonogenicity. METHODS:Samples were prospectively collected from 12 patients with lymphomas mobilized with chemotherapy combined with G-CSF. Small volumes (2–3 ml) of cell suspensions obtained from the leukapheresis product were divided into 4 parts placed in separate small vials, each containing different cryoprotective mixture - with 10%, 7.5%, 5% and 2.5% DMSO. The final volume of cell suspensions equaling 1 ml, the cell concentration (0.8–1 × 108 /ml) and the proportions of human albumin and plasma were the same in all vials. The cells were frozen in IceCube, using a computer controlled cooling program and stored in liquid nitrogen for 2 weeks – 4 months. The quality of cryoprotective mixtures was evaluated by cell recovery and clonogenic potential. The recovery was determined by comparing number of living cells before and after cryopreservation, using trypan blue staining. Clonogenic potential was carried out by colony forming unit (CFU) assays. Depending on CD34+ percentage, 5 or 10 × 103 living cells were plated (in triplicates) in medium MethoCult and cultured for 14 days. RESULTS:The median recovery of nucleated cells for 10% DMSO was 62.4% (range 41.2–86.8) and was significantly higher compared to 7.5% DMSO (54.9%, 41.2–89.1; p=0.04), 5% DMSO (49.2%, 28.1–69.8; p=0.002) and 2.5% DMSO (37.2%, 19.3–54.3; p=0.002). The number of CFUs calculated per 100 000 cryopreserved cells did not differ significantly according to DMSO concentration: 217 (14–1795) for 10% DMSO, 225 (27–2718) for 7.5% DMSO, 196 (26–2761) for 5% DMSO, and 178 (14–2208) for 2.5% DMSO. Neither cell recover nor clonogenic potential correlated with the percentage of CD34+ cells in the leukapheresis product. CONCLUSIONS:Reduction of DMSO concentration to equal or below 7.5% is associated with impaired recovery of nucleated cells after cryopreservation. However, it does not appear to negatively affect clonogenic potential of leukapheresis product, suggesting relative resistance of hematopoietic progenitor cells. Therefore, considering potential toxicity of DMSO to stem cell recipient, its lower concentrations may be clinically beneficial. This hypothesis requires prospective verification in a setting of autologous transplantation. Disclosures:No relevant conflicts of interest to declare.

Surface Modification of Poly(3,4-ethylene dioxthiophene):Poly(styrene sulfonic acid) (PEDOT:PSS) Films by Atmospheric-Pressure Argon Plasma for Organic Thin-Film Solar Cells

Highly-conductive poly(3,4-ethylene dioxythiophene):poly(styrene sulfonic acid) (PEDOT:PSS) films obtained by the addition of dimethylsulfoxide (DMSO) and the argon plasma exposure were used as a transparent conductive anode (TCA) for copper-phthalocyanine (CuPc)/C60 organic thin-film solar cells (OSCs). The CuPc/C60 OSCs on as-grown DMSO added PEDOT:PSS layer showed a power efficiency of 0.6%, whereas it was improved markedly to 1.34% after the atmospheric-pressure argon plasma exposure, which was comparable to that formed on indium-tin-oxide layer. Effects of the DMSO addition and the argon plasma exposure in the spin-coated PEDOT:PSS films is demonstrated in terms of the in-depth characterization of optical and electrical properties.

Effect of Bcl-xL overexpression on erythropoietin production in recombinant Chinese hamster ovary cells treated with dimethyl sulfoxide

Dimethyl sulfoxide (DMSO) can increase the specific productivity (q) of foreign proteins in mammalian cells, while it can also induce cell death, particularly apoptosis. Bcl-xL is a typical anti-apoptotic protein that inhibits the apoptosis in recombinant Chinese hamster ovary (rCHO) cell culture. To evaluate the potential role of Bcl-xL overexpression on DMSO-mediated erythropoietin (EPO) production, we used EPO-producing rCHO cells with regulated Bcl-xL overexpression (EPO-off-Bcl-xL) by doxycycline. Although DMSO addition enhanced specific EPO productivity (qEPO), it also induced cell death in EPO-off-Bcl-xL cells. Bcl-xL overexpression reduced the DMSO-induced cell death followed by release of various enzymes from plasma membrane-damaged cells as evidenced from LDH assay, resulting in delayed loss of EPO. However, it did not significantly improve the maximum EPO production. In addition, Bcl-xL overexpression suppressed DMSO-induced apoptosis, characterized by DNA fragmentation and Annexin V staining. Taken together, Bcl-xL overexpression could inhibit DMSO-induced apoptosis, thereby delaying the loss of EPO.

DMSO regulates osteoclast development in vitro

Dimethyl sulfoxide (DMSO) is routinely used in the laboratory as a solvent and vehicle for organic molecules. Although it has been used in previous studies involving myeloid cells and macrophages, we are unaware of data demonstrating the effects of DMSO alone on osteoclast development. Recently, we were using DMSO as a vehicle and included a non-vehicle control. Surprisingly, we observed a marked change in osteoclast development, and therefore designed this study to examine the effects of DMSO on osteoclast development. Osteoclasts were generated from two sources: bone marrow macrophages and an osteoclast progenitor cell line. Cells were cultured with DMSO for various durations and at differing concentrations and mature, multinucleated (>3 nuclei) TRAP(+) cells were assessed in terms of cell number, cell surface area, and number of nuclei/cell. Osteoclast surface area increased in 5 μM DMSO to a mean of 156,422 pixels from a mean of 38,510 pixels in control culture, and subsequently decreased in 10 μM DMSO to a mean of 18,994 pixels. With serial addition of DMSO over 5 d, a significant increase in mean surface area, and number of nuclei/cell was also observed, while the opposite was true when DMSO was serially removed from culture. These findings show that DMSO exerts a marked effect on osteoclast differentiation. Since many investigators use DMSO to solubilize compounds for treatment of osteoclasts, caution is warranted as altering DMSO concentrations may have a profound effect on the final data, especially if osteoclast differentiation is being assessed.

DMSO modulates the pathway of apoptosis triggering

We demonstrate here that distribution of caspase-9 influences the pathway of apoptosis triggering, since caspase-9 is activated efficiently only when it is distributed solely in the cytosol. Caspase-9 moves to the nuclei in a response to cell stress during isolation of primary hepatocytes; this is called preapoptotic cell stress response. The dimethyl sulfoxide (DMSO) treatment cannot prevent the migration of caspase-9 into the nuclei when it is added to primary hepatocytes immediately after isolation; however, it can trigger redistribution of caspase-9 from the nuclei into the cytosol when added 1 day post-isolation. This redistribution is temporary, since caspase-9 returns to the nuclei within 48 hours of DMSO treatment. Thereafter, some caspase-9 is retained in the nuclei of DMSO-treated hepatocytes for longer than in the nuclei of untreated hepatocytes. By measuring caspase activities, we demonstrate that the addition of DMSO to cell culture medium can temporarily normalize the susceptibility of hepatocytes for apoptosis triggering through the intrinsic pathway. DMSO contributes also to the prolonged pathway inactivation, i.e., by extending preapoptotic cell stress response. We propose that DMSO extends the survival of primary hepatocytes by modulating preapoptotic cell stress response, which could be exploited for extending the lifespan of other primary cell cultures.

An Electron Spin Resonance Study for Real-time Detection of Ascorbyl Free Radicals After Addition of Dimethyl Sulfoxide in Murine Hippocampus or Plasma During Kainic Acid-Induced Seizures

Electron spin resonance (ESR)-silent ascorbate solutions generate a detectable, likely concentration-dependent signal of ascorbyl free radicals (AFR) immediately upon addition of a molar excess of dimethyl sulfoxide (DMSO). We aimed to perform quantitative ESR analysis of AFR in real time after addition of DMSO (AFR/DMSO) to evaluate ascorbate concentrations in fresh hippocampus or plasma following systemic administration of kainate in mice. Use of a special tissue-type quartz cell allowed immediate detection of AFR/DMSO ESR spectra in fresh tissues from mice. AFR/DMSO content was increased significantly in fresh hippocampus or plasma obtained during kainate-induced seizures of mice, reaching maximum levels at 90 min after intraperitoneal administration of 50 mg/kg kainic acid. This suggests that oxidative injury of the hippocampus resulted from the accumulation of large amounts of ascorbic acid in the brain after kainic acid administration. AFR/DMSO content measured on an ESR spectrometer can be used for real-time evaluation of ascorbate content in fresh tissue. Due to the simplicity, good performance, low cost and real-time monitoring of ascorbate, this method may be applied to clinical research and treatment in the future.

Exposure to cardiomyogenic stimuli fails to transdifferentiate human umbilical cord blood-derived mesenchymal stem cells

The ability of human umbilical cord blood-derived mesenchymal stem cells (UCBMSCs) to transdifferentiate towards cardiomyocytes remains unclear. The aim of this study was to direct UCBMSCs to the cardiac lineage by exposure to: (1) 5-azacytidine (AZ) or dimethyl sulfoxide (DMSO); (2) a combination of growth factors involved in early cardiomyogenesis (BMP-2 + bFGF + IGF-1); (3) the Wnt signaling activators lithium chloride (LiCl) and phorbol-12-myristate-13-acetate (PMA); and (4) direct contact with neonatal rat cardiomyocytes. Expression of cardiomyocyte-specific proteins and beta-catenin were assessed by quantitative RT-PCR, immunofluorescence and Western blot. Cocultures of human UCBMSCs with neonatal rat cardiomyocytes were also analyzed for the presence of calcium oscillations and changes in electrical potential using Fura Red and di-4-ANEPPS confocal imaging, respectively. Induction of cardiac-specific proteins was not detected in 5-AZ- or DMSO-treated cells. Following DMSO addition, beta-catenin cytoplasmic expression increased, but did not translocate into cell nuclei to promote cardiac gene activation. Likewise, neither co-stimulation with BMP-2 + bFGF + IGF-1, nor exposure to LiCl and PMA resulted in the acquisition of a cardiac phenotype by UCBMSCs. Direct contact with neonatal rat cardiomyocytes promoted neither the expression of cardiomyocyte-specific proteins, nor the presence of calcium rhythmic oscillations and potential-dependent fluorescence emission in UCBMSCs. The cardiomyogenic stimuli investigated in this study failed to transdifferentiate human UCBMSCs. Alternative strategies or regulatory factors and signaling pathways may be better suited to recruit UCBMSCs into cardiac cell lineage.