Effective clinical-scale production of dendritic cell vaccines by monocyte elutriation directly in medium, subsequent culture in bags and final antigen loading using peptides or RNA transfection.

摘要: Dendritic cell (DC) vaccination approaches are advancing fast into the clinic. The major obstacle for further improvement is current lack of a simple functionally "closed" system to generate standardized monocyte-derived (mo) DC vaccines. Here, we significantly optimized use Elutra counterflow elutriation enrich monocytic precursors by (1) developing an algorithm avoid red blood debulking and associated monocyte loss before elutriation, (2) directly in culture medium rather than phosphate-buffered saline. Upon bags containing collected monocytes simply transferred incubator progeny as final "open" washing step no longer required. Elutriation resulted more (> or = 2-fold) purer standard gradient centrifugation/adherence-based enrichment, whereas morphology, maturation markers, viability, migratory capacity, T stimulatory capacity were identical. Subsequently, compared RNA transfection, this increasingly used approach load with antigen. Elutra-derived adherence-derived could be electroporated similar, high efficiency (on average >85% green fluorescence protein positive), appeared also equal antigen expression kinetics. Both DC, when loaded MelanA peptide RNA, showed stimulation that is, priming MelanA-specific CD8+ cells. Our Elutra-based procedure straightforward, clearly superior centrifugation/plastic adherence protocol, now allows generation large numbers peptide-loaded RNA-transfected closed system.