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Anticancer immunotherapy using dendritic cell (DC) based vaccines provides an adjuvant therapeutic strategy that is not cross reactive with conventional therapeutics. However, manufacturing of DC vaccines requires stringent adherence to Good Manufacturing Practice (GMP) methods and rigorous standardization. Optimally this includes a closed system for monocyte isolation, in combination with closed culture and washing systems and an effective vector transduction strategy. In this study, we used the Gambro Elutra to enrich monocytes from non-mobilized leukapheresis products collected from healthy donors. This approach enriched monocytes from an average frequency of 13.6 + 3.2% (mean + SEM), to an average frequency of 79.5 + 4.3% following enrichment with a yield of 79 to 100%. The monocytes were then cultured in a closed system using gas permeable Vuelife fluoroethylene propylene (FEP) bags and X-vivo-15 media containing 10 ng/ml granulocyte-macrophage colony-stimulation factor (GM-CSF) and 5 ng/ml Interleukin (IL) 4. The cultures were re-fed on days two and four, with a 25% media volume and cytokines. Following culture for seven days, the cells were harvested using a Cobe-2991 and concentrated using a bench centrifuge retrofitted with blocks to allow centrifugation of 72 ml bags and supernatant removed using a plasma extractor. This approach reduced the media volume to an average of 17.4 ml and an average DC concentration of 6.3 + 1.0 × 107 cells/ml, a viability of 93.8 + 2.2%, a purity of 88.9 + 3.3% and a total yield of 8.5 + 1.4 × 108 DCs. Based on the identification of DR+ cells as DCs we had an average yield of 46 + 8% using a calculation based on the number of monocytes in the apheresis product and the resulting DCs differentiated from monocytes. The use of DCs as a vaccine, required transduction with an adenovirus (Adv) vector with the tumor suppressor, p53 transgene (Adv5CMV-p53) as the antigen at a DC concentration of 9 × 106 DCs/ml at an Ad5CMV-p53: DC ratio of 20,000:1, and a 2 or 3 hour co-culture, followed by a 1:10 dilution with media and an additional 16–22 hour incubation. Following incubation, the DCs were washed twice and the supernatants removed using a plasma extractor. The average viability after infection with Ad5CMV-p53 was 87.9 ± 2.6% with an average of 20.3 + 5.4% of the DCs expressing p53. The calculated yield of DCs following Ad5CMV-p53 transduction, based on the number of monocytes in the apheresis products, averaged 12.4 + 3.8%. We conclude that it is possible to efficiently manufacture Adv transduced DCs using a functionally closed system.