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 irradiation is commonly used to stop the cell growth of adherent cells in several experimental models, we aimed to define the optimal dose and incubation time 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 efficiency of MMC in stopping the cell growth was evaluated for 5 days following MMC 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 difference 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 growth of co-cultured hemopoietic cells. Different cell types might present a different sensitivity to MMC and therefore a dose-response curve to MMC has to be obtained for each cell type of interest.