Mitomycin C as an alternative to irradiation to inhibit the feeder layer growth in long-term culture assays

Background Mitomycin C (MMC), an antitumoral antibiotic, has deem described inhibiting the proliferation of different cell types in vitro. Since irrad iation is commonly used to stop the cell growth of adherent cells in severa l experimental models, we aimed to define the optimal dose and incubation t ime of MMC capable of inhibiting the growth of murine fibroblasts, used as an adherent feeder layer in long-term hematopoietic culture assay. Methods M2 10B4 (both parental and engineered to produce human IL-3 and G-CSF) and Sl/Sl (engineered to produce human IL-3 and steel factor) murine fibroblast cell-lines, frequently used in LTC-IC assay were incubated with increasing doses of MMC for either a short (3 h) or a long (16 h) period. The efficie ncy of MMC in stopping the cell growth was evaluated for 5 days following M MC removal. The effects of MMC treatment on human hematopoietic cells were studied using both LTC-IC and limiting dilution (CAFC) assays. Results The growth of M2 10B4 cells was stopped at 3 and 16 h in the presence of 20 mug/mL and 2 mug/mL of MMC, respectively while Sl/Sl fibroblasts required a lower dose of drug (2 and 0.2 mug/mL, respectively). No significant diffe rence was found between the number of LTC-IC or CAFC obtained from cultures containing irradiated or MMC-treated feeder cells. Discussion MMC inhibits the growth of murine fibroblasts used as adherent feeder cells in long-term culture assays, without interfering with the subsequent growt h of co-cultured hemopoietic cells. Different cell types might present a di fferent sensitivity to MMC and therefore a dose-response curve to MMC has t o be obtained for each cell type of interest.