Autologous Tumor Lysate-Pulsed Dendritic Cell Vaccine

Fresh tumors were sent to the cell therapy laboratory. Tumor single-cell suspensions were obtained by mechanical disaggregation and then frozen and stored. Tumor lysate was obtained through four cycles of thawing and freezing, and then irradiated and stored at −20 °C. Seven days after dexamethasone termination, peripheral blood mononuclear cells were collected by leukapheresis. CD14+ cells were selected by immunomagnetic separation using a CliniMacs™ (Miltenyi Biotec, Bergisch Gladbach, Germany) following manufacturer’s instructions. These cells were cultured at 2 × 106 cells/mL in AIM-V (Gibco, Grand Island NY 14072,USA) supplemented with antibiotics, 1000 UI/mL of IL-4 (R&D Systems, Minneapolis, MN, USA) and 1000 UI/mL GM-CSF (Leukine, Genzyme Corporation, Bayer Healthcare, Seattle, WA, USA) in culture bags (Cellgenix, Gaithersburg, MD 20877, USA) at 37 °C in a humidified incubator. IL-4 (500 UI/mL) and GM-CSF (500 UI/mL) were further added to the medium on the 4th day, and cultured cells were harvested on the 7th day. These immature dendritic cells were adjusted at 107 cells/mL and pulsed with autologous tumor lysate (median 69.82 μg/mL, rank 27.9–75 μg/mL) for 2 h at 37 °C and 5% CO₂. At that time, to induce dendritic cells maturation, 50 ng/mL of TNF-α (Beromun, Boehringer Ingelheim, Barcelona, España), 1000 UI/mL of IFN-α (Intron A, Schering Corporation, Kenilworth, NJ, USA) and 20 ng/mL Poli I:C (Amersham, GE Healthcare, Madrid, España) were added to the medium and cells were placed in culture bags at 2 × 106 cells/mL. Mature dendritic cells were harvested on the 8th day and frozen in aliquots following standard procedures until use. Briefly, the cells were resuspended in RPMI-1640 complete medium (500 mL RPMI-1640 (GIBCO, Life Technology, Madrid, España) + 50 mL of 10% FCS + 5 mL of l-Glutamine 200 mM (GIBCO, Life Technology, Madrid, España) + 5 mL Pen/Strep solution (solution with 10,000 U/mL Pen, 10 mg/mL Strep, GIBCO, Life Technology) at twice the desired cryopreservation concentration. The cryopreservation solution was prepared, containing 40% complete RPMI-1640, 40% FCS and 20% DMSO. The cryopreservation vials were placed in the cryopreservation box (5100 Crio 1° Freezing Container, Nalgene) and 500 microliters of the cell suspension was added to each vial; then, 500 μL of the cryopreservation solution was added and the final suspension was carefully mixed. The cryopreservation box was brought to a −80 °C freezer, and after 24 h, the cell vials were stored in a liquid nitrogen tank. Ten million cells was considered the optimal dose for each administration. The viability of cells was determined before and after freezing [22 (link)].