Affiliations: [a] Laboratory of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, Meibergdreef, Amsterdam, The Netherlands | [b] E.C. Slater Institute for Biochemical Research, Academic Medical Center, University of Amsterdam, Meibergdreef, Amsterdam, The Netherlands | [c] Institute of Medical Biochemistry and Chemistry, University of Bari, Piazza G. Gesare, Bari, Italy
Note: [] Address reprint requests to Dr. J. Kwekkeboom, Laboratory of Cell Biology and Histology, Academic Medical Center, University of Amsterdam. Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.
Abstract: In this study we investigated whether electrofusion would enable efficient hybridization of human B cells to occur without the need of prior activation in vitro. Two cell lines, the murine myeloma SP 2/0 and murine-human heteromyeloma F3B6, were used as fusion partners, and hybridization of human B cells purified either from peripheral blood or from solid lymphoid tissue (tonsil) was studied. Optimal hybridization frequencies were obtained at a poration field strength of 3 to 4 kV/cm. Electrofusion appeared to be a very efficient method to hydridize tonsillar B lymphocytes, resulting on the average in a hydridoma outgrowth of 1 per 1400 lymphocytes in fusions with SP 2/0 and 1 per 3300 in fusions with F3B6. Hybridization frequency in polyethylene glycol-induced fusions was only 1 per 13,000 tonsillar lymphocytes. Seventy-five percent of the resulting hybridomas secreted human immunoglobulin, either IgM or IgG. Electric field-induced hybridization of peripheral blood B lymphocytes resulted in immortalization of 1 per 17,500 lymphocytes when using Sp 2/0 as a fusion partner, and 1 per 15,000 with F3B6 as a fusion partner. Polyethylene glycol fusions yielded only 1 hybridoma per 160,000 blood lymphocytes. On average, 60% of the hybridomas obtained by fusing peripheral blood B cells secreted human immunoglobulin. Both IgM and IgG secretors were found. Furthermore, by the use of a fusion chamber with a small volume (35 μl), hybridomas could be generated from small numbers (300,000 to 55,000) of human B cells. In conclusion, human B cells can be hybridized by electrofusion with myeloma cells with efficiencies comparable to those found for murine splenocytes, without the need for prior activation in vitro.
Keywords: electrofusion, human B lymphocytes, fusion frequency, human-mouse hydridomas
