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Immunotherapy of malignant diseases based on dendritic cells (DCs) pulsed with tumor antigens is a promising approach. Therefore, there is a demand for large-scale, clinical-grade ex vivo generation of DCs. Here, a procedure is presented that combines monocyte selection and tissue culture in closed systems under current good manufacturing practice conditions. Leukocytes from three patients with urologic cancers were collected by leukapheresis and subjected to immunomagnetic enrichment. From leukapheresis products containing 1.6 ± 0.2 × 1010 (mean ± SEM) leukocytes with a frequency of CD14+ monocytes of 18.7 ± 2.3%, monocytes were enriched to 94.3 ± 2.2%. CD14+ cell recovery was 67.0 ± 4.7%. After 6 days of culture in Teflon bags in X-Vivo 15 medium supplemented with autologous plasma, GM-CSF, and IL-4, cells showed an immature DC phenotype and efficient antigen uptake. Following an additional 3 days of culture in the presence of GM-CSF, IL-4, IL-1β, IL-6, TNFα, and PGE2, cells (82.0 ± 5.8% CD83+) displayed a mature DC morphology and phenotype, including expression of CD11b, CD11c, CD18, CD25, CD40, CD54, CD58, CD80, CD86, HLA class I, and HLA-DR as well as expression of CCR7 but not CCR5. The mature DC phenotype remained stable for at least 5 days in the absence of cytokines. Yield of DC was 14.0 ± 4.7% and viability was 91.9 ± 3.5%. Mature DCs effectively clustered with naive T cells and potently induced allogeneic T-cell proliferation and IL-2 and IFNγ but not IL-4 production. Thus, this procedure allows large-scale generation of stably mature, Th1 responses inducing DCs under cGMP conditions in a closed system from cancer patients and is therefore well suited for immunotherapy.