RESEARCH ARTICLE Cytotoxicity of TSP in 3D Agarose Gel

Purpose A reference reagent, 3-(trimethylsilyl) propionic-2, 2, 3, 3-d4 acid sodium (TSP), has been used frequently in nuclear magnetic resonance (NMR) and magnetic resonance spectros-copy (MRS) as an internal reference to identify cell and tissue metabolites, and determine chemical and protein structures. This reference material has been exploited for the quantita-tive and dynamic analyses of metabolite spectra acquired from cells. The aim of this study was to evaluate the cytotoxicity of TSP on three-dimensionally, agarose gel, cultured cells. Materials and Methods A human osteosarcoma cell line (MG-63) was selected, and cells were three dimensionally cultured for two weeks in an agarose gel. The culture system contained a mixture of conven-tional culture medium and various concentrations (0, 1, 3, 5, 7, 10, 20 30 mM) of TSP. A DNA quantification assay was conducted to assess cell proliferation using Quant-iT Pico-Green dsDNA reagent and kit, and cell viability was determined using a LIVE/DEAD Viabili-ty/Cytotoxicity kit. Both examinations were performed simultaneously at 1, 3, 7 and 14 days from cell seeding. Results In this study, the cytotoxicity of TSP in the 3D culture of MG-63 cells was evaluated by quan-tifying DNA (cell proliferation) and cell viability. High concentrations of TSP (from 10 to 30 mM) reduced both cell proliferation and viability (to 30 % of the control after one week of ex-posure), but no such effects were found using low concentrations of TSP (0–10mM). Conclusions This study shows that low concentrations of TSP in 3D cell culture medium can be used for quantitative NMR or MRS examinations for up to two weeks post exposure