Cytotoxicity of dimethyl sulphoxide on ocular cells in vitro

Dimethyl sulfoxide(DMSO) is a commonly used adjuvant to promote testing drug solubility to prepare multi-levels testing drug concentrations.DMSO is cell type-dependent cytotoxic and its toxicity can interfere the testing drug evaluation.Determining its safe concentration on commonly used cell types is important for ocular drug development.

This study was to determine the minimal toxic concentration of DMSO for in vitro ocular cell lines in a simulated drug screening setting.

Retinal pigment epithelial (RPE) cells were isolated from one pigmented rabbit and primarily cultured.Human RPE cell strain (ARPE19), scleral fibroblasts line (S75-Fron), human Müller cell line (MIO-M1), human lens epithelial cell line (HLEC), human choroidal melanoma cell line (OCM-1), human umbilical endothelial cell (HUVEC) and human HeLa cell line (HELA) were cultured.Different concentrations of DMSO (1.6%, 1.0%, 0.8%, 0.4%, 0.2% and 0.1%) were prepared with 160 μl DMSO solution and 9.84 ml RPMI1640 (or DMEM/F12 or DMEM) containing 2% fetal bovine serum.Different concentrations of DMSO were added in medium for 96 hours, and the and viability (absorbance) of the cells was detected using MTS to evaluate the cytotoxicity of DMSO.

Rabbit primary RPE cells showed the yellow-green fluorescence for cytokeratin(CK) and HMB45 red fluorescence for S100.The viability of the cells was gradually declined as the increase of DMSO dose, showing significant differences in ARPE19, S75-Fron, HLEC, OCM-1, HUVEC and primary RPE cells (all at P<0.05), and when DMSO concentrations were ≥0.8%, the cell viabilities were significantly lower.But no significant difference was found in MIO-M1 cells among different doses of DMSO (F=0.830, P=0.547). The minimal toxic concentration of DMSO for ARPE19, HUVEC, HELA, HLEC, MIO-M1, OCM-1, primary RPE cells and S75-Fron was 0.8%, 0.1%, 0.8%, >1.6%, >1.6%, 0.2%, 0.2%, 0.2%, respectively, and HUVEC was more sensitive to the cytotoxicity of DMSO (P=0.02), and MIO-M1 was the least sensitive to DMSO (P=0.39). The viability of HUVEC and primary RPE cells went down with the increase of DMSO dose, and S75-Fron viability started to decline in 0.1% DMSO and then stabilize with the higher concentrations until 1.6% DMSO at which the viability showed further decline.

The tolerability of ocular cells in vitro to DMSO varies depending on the cell types.The minimal toxic concentration ranged from 0.1% to 1.6%.The result suggests that a concurrent DMSO control should be set up along with the testing compound.