In vitro treatment of individual chemotherapeutic agents and cell viability assays

For evaluation of the cellular function and recovery potential of ASCs after exposure to chemotherapeutic agents, ASCs isolated from female patients during reconstructive procedures were treated with 5-FU, DXR, and Cytoxan to determine whether the ASC viability diminished in the presence of these three commonly utilized clinical chemotherapeutic agents. 5-FU, DXR, and Cytoxan (Sigma-Aldrich) was dissolved to make a stock concentration solution, aliquoted and kept in -20 °C refrigerator for freshly using each time. ASCs were plated at a density of 1 × 10⁴ cells per well in a volume of 1ml in 24 well plates and then treated with 5-FU (0.1, 1, and 10 mg/mL), DXR (0.1, 1, and 10 µmol/L), and Cytoxan (0.1, 1, and 10 mmol/L) at the various concentrations. At 24, 48, and 72 h, MTT assay (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide) was used to evaluate the effect of 5-FU, DXR, and Cytoxan on ASCs cell viabilities. At various time points, the culture medium was replaced with a medium containing MTT solution (5 mg/mL) and incubate at 37 °C with 5% CO₂ for 3 h. The wells were then decanted and the purple formazan crystals formed were dissolved in 200 µL dimethyl sulfoxide solution (Sigma-Aldrich). The absorbance of the plate was read on a microplate reader at 570 nm. Fresh cells were used as controls. All assays were done in triplicate.