In vitro induction of cell-mediated immunity to murine leukemia cells. I. Optimization of tissue culture conditions for the generation of cytotoxic lymphocytes

Cytotoxic lymphocytes (CTL) to allogeneic and syngeneic murine leukemia cells were generated in vitro in 'one way' mixed lymphocyte-tumor cell cultures carried out under a variety of conditions. In an attempt to define the optimal culture conditions for sensitization, the following parameters were analyzed: culture vessel, culture volume, responder: stimulator (R : S) cell ratio, lymphoid cell density, source of lymphoid cells, duration of culture, fetal calf serum (FCS) concentration, 2-mercaptoethanol (2-ME) concentration, concentration of amino acids, and buffering system. Of the many variables examined, of particular importance were the R : S cell ratio, the cell density, the FCS concentration, the presence of 2-ME, and the time of harvesting. These factors exerted various effects, both quantitatively and qualitatively, on the magnitude and kinetics of the responses induced in microcultures (5 × 10⁶ lymphocytes) and macrocultures (25 × 10⁶ lymphocytes). Moreover, optimal sensitization in syngeneic cultures required different conditions than those for allogeneic cultures. Cytotoxic activity was assessed in vitro by a quantitative ⁵¹Cr-release assay. The sensitized lymphocytes demonstrated the characteristics of T lymphocytes and reacted specifically with the sensitizing leukemia cells.