Mitomycin C-treated 3T3/B (3T3/A31) cell feeder layers in hybridoma technology

Several methods were compared for their efficiency at supporting the growth of hybridoma cells at low cell densities. Soluble growth factors from different sources gave poor results, whereas actively metabolising syngeneic cells proved to be effective feeder systems. Of the latter, the transformed subline of BALB/c embryo fibroblast 3T3/B cells - 3T3/A31 - were best. Macrophage (peritoneal exudate) feeder layers could be as effective as the 3T3/A31 cells, but were much more variable, probably reflecting the physiological state of the donor mice, the degree of sensitisation of the cells in vitro and the rate of recovery after isolation from the peritoneal cavity. Since the 3T3/A31 cells could replicate more rapidly than the hybridoma cells, division of the feeder cell chromosomes was inhibited using mitomycin C; 1-2 X 10(4) 3T3/A31 cells/ml were treated with 1 microgram/ml mitomycin C for 8-16 h at 37 degrees C, washed, and incubated for 3-7 days at 37 degrees C. The latter incubation was to permit the metabolism and breakdown of unreacted but intracytoplasmic drug, and the establishment of an active feeder layer before use with the hybridoma cells.