Cytotoxicity Assessment in Cultures of Differentiating Rodent Embryonic Cells

Several techniques for assessing the cytotoxicity of chemical agents in the micromass cell culture systems for rat embryo midbrain (CNS) and limb bud (LB) cells were compared. Cultures were treated with the antitubulin agent alben-dazole (ABZ) and its sulfoxide derivative (SOABZ) and cultured in Primaria 35 mm dishes, Primaria 96-well plates, or collagen-coated 96-well plates. Three assays for cytotoxicity were employed: staining with the vital dye neutral red (NR), mitochondrial reduction of MTT, and total culture protein levels. Comparison of the cytotoxicity IC₅₀ values was also made to the IC₅₀ value for differentiation. The IC₅₀ values for cytotoxicity and differentiation in CNS cultures were statistically equivalent regardless of the procedure employed. However, in LB cultures, the IC₅₀'s for cytotoxicity generated by NR staining of fixed cells in Primaria 96-well plates were significantly different from those for differentiation and total culture protein content. With a single exception (NR staining of unfixed ABZ-treated LBs in collagen-coated 96-well plates), no other cytotoxicity procedures yielded IC₅₀ values that differed significantly from those for differentiation. We conclude that the NR cytotoxicity procedure should be applied to micromass culture with caution due to the sensitivity of this assay to cell density, cell type, and the surface on which the cells are plated. Assaying for total culture protein content yields important supplementary information.