Serum-free culture of resting, PHA-stimulated, and transformed lymphoid cells, including hybridomas

A chemically-defined medium containing casein, insulin, testosterone, transferrin, and linoleic acid (CITTL) has been developed for the culture of lymphoid cells. Resting human mononuclear leukocytes can be maintained in CITTL for up to 8 weeks with excellent viability. In the absence of mitogen, undifferentiated monocytes become the predominant cell type by 7–11 days as assessed by morphology, population distribution of cell sizes, and fluorescence-activated cell sorter analysis. Phytohemagglutinin (PHA)-induced proliferation is retained in these cultures for 7–10 days and results in a population of 85% T lymphoblasts. T lymphoblasts survive for 2 weeks in CITTL following mitogenic stimulation and proliferate for up to 5 weeks in CITTL if supplemented with 2-mercaptoethanol and conditioned medium containing crude T-cell growth factor. In addition to supporting proliferation of normal lymphoid cells, CITTL also sustains growth of human and murine hybridomas, as well as a variety of transformed lymphoid cell lines. The ability of a minimum essential medium supplemented with 5 or 6 factors to support growth of normal and malignant lymphoid cells suggests that only a limited number of extracellular signals are required for mitogenesis of these cells.