Generation of Monocyte-Derived Dendritic Cells

Leukapheresis was performed using a Cobe Spectra separator (Cobe BCT, Lakewood, CO, USA). All of the following operations were performed under Good Manufacturing Practice (GMP) conditions in the GMP facility of University Hospital Motol using the protocol for DC generation that was approved by the State Institute for Drug Control, as previously described [25 (link), 26 (link)]. The leukapheretic product was diluted in PBS + 1 mM EDTA (Lonza, Verviers, Belgium), and mononuclear cells were separated by Ficoll-Paque Premium (GE Healthcare, Waukesha, WI, USA) gradient centrifugation. Collected mononuclear cells (PBMC) were washed in PBS + 1 mM EDTA (Lonza), resuspended in CellGro medium (CellGenix, Freiburg, Germany) and plated in triple flasks (Thermo Scientific, Waltham, MA, USA) at 1 × 10⁶ cells per cm² of surface area. After 2 h, non-adherent cells were washed with PBS (Lonza). Adherent monocytes were cultured for 6 days in CellGro (CellGenix) medium with 20 ng/ml of IL-4 (Gentaur, Kampenhout, Belgium) and 500 U/ml of GM-CSF (Gentaur); fresh cytokines were added on day 3. Immature DCs were harvested on day 6, washed in PBS (Lonza) and resuspended in CellGro (CellGenix).