Growth of B cell colonies independent of T cell contact

One of the methods of obtaining B cell colonies involves physically admixing B cells in growth medium containing mitogens with accessory T cells treated with mitomycin C or low dose irradiation to prevent T cell colony formation. However, under these conditions T cells still have the capacity to form colonies in methylcellulose, which is most frequently used for colony assays, and B cells recovered from the colonies are contaminated by the large number of added T cells. Therefore, we have developed a method of growing B cell colonies by physically separating the enriched B cells from the T cells by using millicell-HA inserts (Millipore). The requirement for mitogens, culture conditions, development, and number of B cell colonies formed were similar to those observed with admixture of T and B cells. The advantage of this system is that it permits growth of B cell colonies without the confounding influence of T cell proliferation or treatment of T cells with mitomycin or irradiation, and relatively pure B cell colonies can be recovered for further characterization or functional studies.