Stromal cell-dependent growth of B-1 B cell progenitors in the absence of direct contact

This protocol describes a transwell culture system in which stromal cells support the growth and differentiation of B cell progenitors in the absence of direct contact. In this system, a confluent layer of S17 stromal cells pre-established in 0.4 microm transwells is placed over wells seeded with purified B cell progenitors. The stromal cell-derived factors and additional cytokines added to the culture medium support the differentiation of the progenitors in the lower chamber. B-1 B cell progenitors seeded in the presence of thymic stromal lymphopoietin undergo significant expansion and differentiation in this culture system. Since the expanded B-1 B lineage cells are not contaminated with stromal cells, no additional purification steps are required before subsequent phenotypic, functional or genetic analyses of these lymphoid cells are performed. Once the transwell cultures and B cell progenitors are available, cultures can be initiated in less than an hour. The overall procedure, however, takes approximately 10 h when the initiation of the S17 transwell cultures and the isolation of the B cell progenitors steps are included.