Murine B cell lymphopoiesis in long term culture

A method is described for establishing continuous cultures of early B lymphocytes from mouse bone marrow. Total bone marrow elements are cultured at 37 degrees C in medium containing 5% fetal calf serum and 10(-5) M 2-mercaptoethanol. Under these conditions, adherent bone marrow cells grow to confluency by 2-3 weeks and provide a feeder layer on which the growth of the B lymphocytes depends. Lymphoid cell growth becomes apparent by 3-5 weeks. Such cultures are a rich source of pre-B cells, B cells and potentially stem cell-like elements capable of immunoglobulin gene rearrangement. The procedures for initiating and maintaining the bone marrow cultures are described in detail as are the phases of growth through which the cultures should progress. Also included are methods for preparing and manipulating low cell-density feeder layers, on which B cell populations can be expanded, and a method for obtaining pre-B and B cell clonal lines. The lymphoid cells which are obtained from these cultures early after establishment (3-8 weeks) are predominantly pre-B cells which can serve as targets for transformation by Abelson murine leukemia virus. Cells obtained from older cultures (3-6 months) frequently are B cells that express both membrane IgM and IgD. The numbers of cells which can be obtained from these mass cultures and clonal lines are sufficient to analyze the molecular processes involved in B cell maturation, virus transformation and antigen activation.