Dimethylsulfoxide cell culture

Demirbas, S. and Stavchansky, S., Effects of citicholine and dimethylsulfoxide on transepithe-lial transport of passively diffused drugs in the Caco-2 cell culture model, Int.. Pharm., 251, 107, 2003. 

Da Violante, G., Zerrouk, N., Richard, I., Provot, G., Chaumeil, J.C., and Arnaud, P., Evaluation of the cytotoxicity effect of dimethylsulfoxide (DMSO) on Caco2/TC7 colon tumor cell cultures, Biol. Pharm. Bull., 25,1600, 2002. 

Secondary metabolites produced by plant cell culture are commonly accumulated in the cells. With few exceptions such as Capsicum frutescens, Thalictrum minus (9) and Vanilla planifolia (Knorr, D. and Romagnoli, L., Univ. of Delaware, unpublished data) cultures, which release valuable compounds such as capsaicin, berberine and vanillin, respectively, into the medium, procedures to induce product release are required to develop continuous production processes. Reported permeabilization methods include treatment with dimethylsulfoxide (DMSO), isopropanol, toluene, phenethyl alcohol or chloroform (9, 28). But as Fontanel and Tabata (9) pointed out, such treatments with organic solvents are severe and other methods of permeabilization need to be developed. 

When the production of the secondary metabolites coincides with the death and general lysis of the cells, the recovery of the product is simply a matter of separation from the spent production solution downstream of the reactor. An example of this type of operation was initially used in Japan during the production of shikonin. However, if the secondary metabolites are stored in the vacuole of the cells and the cells remain viable but dormant during the production phase, then a permeabilizing agent such as dimethylsulfoxide (DMSO), detergents, proteins, and antibiotics may be employed in some cases in concentrations that make the cells leak product out but maintain cell viability. Success for this type of product recovery has been reported in C. roseus, Datura innoxia, and Daucus carota cell cultures. 

Drugs can be insoluble or poorly soluble in cell culture medium. Therefore, stock solutions of the drugs can be made in 100% dimethylsulfoxide (DMSO) at 50 mM. The highest testable drug concentration should preferably not contain DMSO concentrations higher than 1% to avoid toxicity of DMSO to the cell cultures. 

Curcumin is not water-soluble, but it is soluble in ethanol or in dimethylsulfoxide. The degradation kinetics of curcumin under various pH conditions and the stability of curcumin in physiological matrices have been established. When curcumin was incubated in O. IM phosphate buffer and semm-free medium (pH 7.2 at 37°C), about 90% decomposed within 30min. A series of pH conditions ranging from 3 to 10 were tested, and the results showed that decomposition was pH-dependent and occurred faster at neutral-basic conditions. It is more stable in cell culture medium containing 10% fetal calf seram and in human blood. Less than 20% of curcumin decomposed within Ih, and after incubation for 8h, about 50% of curcumin still remained. Trans-6-(4 -hydroxy-3 -methoxyphenyl)-2,4-dioxo-5-hexenal was predicted to be the major degradation product, and vanillin, feralic acid, and feraloyl methane were identified as minor degradation products. The amount of vanillin increased with incubation time. 

Retinoids are insoluble in water, but soluble in organic solvents, such as ethanol and dimethylsulfoxide (DMSO), which are the most commoidy used solvents for administration of tRA. Although one always performs control experiments, it is important to be aware of the fact that DMSO acts as a differentiating agent to embryonal carcinoma cell cultures in the same way that tRA does, so perhaps ethanol is a better solvent. The maximum solubility of tRA in DMSO is about 50 mg/mL. 

The strains of Hb. salinarum, S9, were cultured in peptone medium at 37 °C at pH 7.0 for six days [18, 35]. The cells were washed and resuspended in the basal salt solution (4 mol dm-3 NaCl containing 2.5 x 10 2moldm 3 HEPES) at pH 6.8. A 10mm3 of dimethylsulfoxide solution of BCECF/AM at 1.0 x 10 2 mol dm 3 was added to the 10 cm3 cell suspension. The cells were incubated in the dark at 18 °C for three days to load the dye [18,19]. The cell suspension was then centrifuged and the obtained cells were repeatedly washed with the basal salt solution at pH 6.8 until the supernatant showed no fluorescence. The dye-loaded cells were resuspended in the basal salt solution at pH 7.7. 

Cell viability was evaluated using a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) (Sigma Chemical Co.) assay. After culture, cells were treated with 1 mg/ml MTT for 2 h, precipitated dye was solubilized in dimethylsulfoxide, and the absorbance at 570 run was measured. 

A. Preparation of Cells. In order to facilitate the uptake of plasmids into bacteria, cultures are treated to make them more permeable, or competent . Competent cells may be purchased commercially and may be preferable for critical cloning steps as they are prepared using methods that generally give better uptake than the one given here. The method provided here (modified from Sambrook et al. 1989) is cheap and simple, does not use obnoxious chemicals such as dimethylsulfoxide (DMSO), and works with a wide variety of bacterial strains. We use it fbr routine maintenance of our plasmid stocks and for general subcloning. 

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