Improved method for cloning human B-cell lines

Abstract Difficulties in the successful cloning of B-cell lines have prevented widespread establishment of specific antibody-forming human B-cell lines. We have improved on standard methods of cloning by culturing human lymphoid cells suspended in agarose with nonproliferating human fetal lung fibroblasts. Using this method, cloning efficiencies up to 20% were observed, from as few as 50 plated lymphoid cells. These results are significantly better than the efficiencies of up to 1% using 5 to 10 × 10 3 cells with traditional soft agar methods. The number of colonies observed increased in proportion to the number of fibroblasts plated with the lymphoid cells. A threshold number of fibroblasts was found. Microscopically, close association between lymphoid cells and fibroblasts was seen. A growth-enhancing factor appears to be produced by fibroblasts suspended in agarose. This cloning method is applicable to both well-established and newly established lymphoid cell lines and should be useful for growing and cloning B cells.