We have developed an innovative system for ex vivo processing of patient-specific cell products to produce large numbers of T-lymphocytes in support of phase 2 adoptive immunotherapy trials for hematologic malignancies. Extensive efforts were undertaken to close the cell processing system to improve the safety profile of the process and comply with new federal regulations regarding cell and tissue processing. Our results demonstrate that apheresis products can be processed in a closed system (Cytomate) with similar yields (approximately 4 x 10(9) mononuclear cells/apheresis) and recoveries (approximately 60% of starting mononuclear cells) to manual cell processing. Cells processed with this system could be cryopreserved for up to 5 months without significant loss of recovery or viability. Additionally, we have evaluated the use of gas permeable bags and developed perfusion bioreactor protocols in which T cells can be rapidly produced in excess of 10(10) viable cells per liter of culture. Using similar methods for upfront processing, we have also developed methods for positive selection and ex vivo culture of CD4+ T cells that result in 200 to 800-fold expansion of fresh or cryopreserved samples. T cells produced in these systems were shown to retain activation-induced cytolytic capability and TH1/TH2 cytokine production as a measure of biologic potency. These new methods allow for more efficient production multiple patient-specific products by satisfying the basic tenants of safety and efficacy required for early phase clinical trials of cell products.