Co-culture of Lymphocytes and Tumor Cells

One day before co-culture, lymphocytes were thawed and cultured in lymphocyte medium supplemented with 200 IU/ml IL-2 overnight at 37℃. Lymphocyte medium consisted of RPMI-1640 (Wako)/AIM V(Gibco) supplemented with 12.5 mM HEPES, 2-Mercapto-ethanol and 5% human AB serum. Tumor cells were stimulated overnight with 200 IU/ml of human recombinant IFNγ to facilitate HLA expression. 96-well U-bottom plates were coated with 5 μg/ml anti-CD28 (clone CD28.2) and kept overnight at 4 ℃. On the next day, tumor cells were dissociated to single cells with TrypLE (Gibco) and resuspended in lymphocytes medium. Lymphocytes were seeded at a density of 10⁵ cells/well (total 1 × 10⁶ cells) and co-cultured with tumor cells at a 20:1 ratio (lymphocytes: cancer cells, respectively). Co-culture was kept in the presence of 200 IU/ml IL-2 and 10 μg/ml anti-PD-1 antibody (clone EH12.2H7, Biolegend). Half of the medium was replaced two to three times per week. 1 week after the co-culture, lymphocytes were collected, counted, and replated at the concentration of 10⁵ cells/well with fresh tumor cells [24 (link)].