Culture Conditions that Optimize Outgrowth of Hybridomas

Efforts to refine the methods of producing monoclonal antibodies (MAbs) of known specificity (1 ) have revealed there are many variables that affect the growth of hybridomas generated by the fusion of myeloma cell lines with spleen cells (reviewed in refs. 2 and 3 ). These include the cell-cycle status of B-cells in immunized mice at the time of cell isolation, the myeloma or hybridoma used as a fusion partner, the composition of the reagent used for cell fusion, the presence of contaminating fibroblasts and macrophages in the primary culture, the concentration of fused and unfused cells present in primary cultures, the presence or absence of essential growth factors in fetal bovine serum, coculturing of fresh hybrids with thymocytes, spleen cells, or peritoneal macrophages, the presence or absence of 2-mercaptoethanol, and importantly, the presence or absence of inhibitory substances in culture medium. Efforts to detail which factors exert the most prominent effect on hybridoma survival and outgrowth have verified the importance of culture medium and culture medium supplements, and in addition, have shown that B-cell mitogens have a profound effect on the outgrowth of hybridomas in primary cultures (2 ). The studies have shown that it is possible, on a routine basis, to obtain 2000–3000 hybridomas from 5 � 10⁷ spleen cells when B-cell mitogens are used as one of the culture supplements (see Notes 1 and 2 for further comment). Equally good results have been obtained with fusions of mouse (inter-species) and rat (cross-species) spleen cells with a mouse myeloma fusion partner (2 ). The following is a description of the procedures that we have developed to optimize the yield of hybridomas. The process, starting with immunized mice and ending with newly fused cells plated in 96-well plates, should take about 4 h.