Role of culture conditions and exposure duration in determining sensitivity of human bone marrow progenitor cells to methotrexate

The effect of drug concentration, exposure duration, and culture conditions on the cytotoxic activity of methotrexate (MTX) on normal granulocyte-macrophage colony-forming units in culture (GM-CFUC) was studied using a bilayer soft agar system with nucleoside-free medium. The degree of inhibition of colony formation depended on the type of serum supplementation. A 1 h or 2 h pulse treatment with 2×10^-4 M (100 μg/ml) MTX failed to kill GM-CFUC, when the cells were subsequently plated in a system containing 15% undialyzed fetal bovine serum (FBS). For continuous exposure the observed LD₅₀ of MTX in the agar system was higher than 10^-4 M for 15% undialyzed FBS, 10^-5 M for 15% dialyzed FBS plus 0.25% undialyzed FBS, 10^-6 M for 15% dialyzed FBS, and 10^-8 M for 15% undialyzed horse serum. The difference for dialyzed FBS versus horse serum can be explained by differences in nucleoside concentrations. The difference for dialyzed FBS versus horse serum may be secondary to an enhancer of MTX in horse serum. For studying MTX sensitivity of human tumor cells in vitro, we suggest testing conditions that lie within the dose survival curve of GM-CFUC.