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Scale up of dendritic cell production is a critical challenge that is infeasible with current static culture systems such as well plates, T-flasks, and bags. We have developed a fully enclosed, sterile cell culture system, called EDEN, that allows for continuous perfusion of fresh differentiation medium into the cell culture cartridge and simultaneous removal of depleted medium. EDEN generated ca. 25 million immature dendritic cells (iDCs) per run with a yield, relative to seeded monocytes, of 20-30%. Immunophenotyping showed that EDEN generated iDCs were phenotypically similar to 6-well plate generated iDCs. Maturation of EDEN iDCs using a standard maturation cocktail was successful with upregulation of CD80/83/86 and downregulation of CD209. Computational fluid dynamics simulations aided the EDEN cartridge design to ensure proper differentiation medium perfusion. These results indicate that EDEN successfully generates clinically relevant numbers of iDCs in a single cell culture cartridge with fewer manual interventions compared to standard culture techniques.