Monocyte-Derived Dendritic Cell Preparation

Monocyte-derived DCs were established from adherent peripheral blood mononuclear cells (PBMC) obtained via leukapheresis performed at the UCLA Hemapheresis Unit, as we have published previously^{3 (link),6 (link),52 (link)}. All ex vivo DC preparations were performed in the UCLA-Jonsson Cancer Center GMP facility under sterile and monitored conditions. In brief, dendritic cells were prepared by culturing adherent cells from peripheral blood in RPMI-1640 (Gibco) and supplemented with 10% autologous serum, 500 U/mL GM-CSF (Leukine®, Amgen, Thousand Oaks, CA) and 500 U/mL of IL-4 (CellGenix), using techniques described previously^{2 (link)}. Following culture, DCs were collected by vigorous rinsing and washed with sterile 0.9% NaCl solution. The purity and phenotype of each DC lot was also determined by flow cytometry (FACScan flow cytometer; BD Biosciences, San Jose, CA). Cells were stained with FITC-conjugated CD83, PE-conjugated CD86 and PerCP-conjugated HLA-DR mAb’s (BD Biosciences). Release criteria were >70% viable by trypan blue exclusion, and >30% of the large cell gate being CD86⁺ and HLA-DR⁺. One day before each vaccination, DC were pulsed (co-cultured) with tumor lysate overnight, washed, and the final product was tested for sterility by Gram stain, mycoplasma, and endotoxin testing prior to injection.