2 x 200 x 10e6 monocytes are transferred into culture bags by sterile welding and cultured for 5 days at a density of 2.5-5 x 10e6 cells/mL in serum free medium (CellGro, Cell Genix) in the presence of 100 ng/mL GM-CSF (Leukine, Berlex) and 20 ng/mL IL-4 (Cell Genix). After 2 days one volume fresh medium containing 2x concentrated cytokines is added.

On day 5 cells are counted and pulsed with tumor antigen derived peptides (one per culture) at a concentration of 5 µg/mL in approximately 5 mL and at a cell density of 20 x 10e6/mL for 1 h.

After peptide pulsation cells are diluted in medium containing 20 ng/mL TNF-?, plus IL-4 and GM-CSF as stated above.

On day 7 of culture 35/40 x 10e6 mature peptide pulsed DC are frozen at 5 x 10e6 cells per vial in freezing medium as above.

The frozen MoDC are subsequently used for tumor peptide specific stimulation of autologous T lymphocytes at a ratio of 1 DC per 10 T cells. Co-cultures led to antigen specific, IFN-? secreting T cells after 1 restimulation at a DC:T cell ratio of 1:20, as evaluated by IFN- ? Elispot, showing that the generated DC have good antigen presenting and co-stimulatory capacity.

After thawing cells showed good viability (>90%) and the recovery was approximately 80%.