Improvement in the basic technology of electrofusion for generation of antibody-producing hybridomas

In order to define the optimum conditions of electrofusion technique for the generation of antibody-producing hybridomas, mouse spleen cells or EBV-transformed human B cells were fused with mouse myeloma cells (SP2/0) or human fusion partner cells (KR-4 or KR-12), respectively, by electric field pulse under various conditions. The results confirm reports that the presence of both Ca2+ and Mg2+ in fusion medium and pretreatment of mixed cells with proteases improve hybridoma yield. Moreover, the presence of liposome or hydrophobic protein in the fusion medium greatly enhanced the yield. Under optimum conditions, hybridoma yields of mouse cells and human cells were 2.5 X 10(-4) and 1 X 10(-4), respectively. These efficiencies were about ten times higher than those obtained by the conventional polyethylene glycol technique. Microscopic observation of the fusion-process revealed that in a human cell system 20%-50% of the cells were physically fused, although only one in 5000 physically fused human cells grew as a hybridoma after hypoxanthine-aminopterin-thymidine selection.