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Background: The main advantages of using CD8+ T lymphocytes for adoptive therapy of cancer is their ability to specifically target tumor cells and their long clonal life span which can be used for both immunotherapy and immunoprophylaxis.Certain issues need to be resolved for the scalable use ofCD8+ T cells, such as the low number of CD8+ T cell precursors, the difficulty in rapidly growing CD8+ T cells, and the decrease of proliferation and the loss of anti-tumor activity as expansion time increases.Currently, one of the greatest challenges in this field is the generation of large numbers of potent CD8 + T cells for the adoptive immunotherapy of cancer.Methods: Autologous tumor cells were obtained from solid tumors, malignant ascites or pleural effusions.For those patients where autologous tumor cells were not available, allogenic tumor cells (from ATCC tunaor cell lines) were used.Mononuclear cells MNCs were purified from the peripheral blood.Tumor cells and MNCs were seeded and co-cultured in an innovative three dimensional tissue culture device to rebuild tumors in vitro, and to create an environment for in vitro sensitization mimicking the in vivo cellular immunization.After proliferation and expansion, activated antitumor CD8+ T cells without residual tumor cells were harvested for use.Results: The tumors rebuilt in vitro exhibited similar biological characteristics of malignant tumor in vivo and expressed high levels of tumor associated antigens.When MNCs were co-cultured with tumor cells, early contact and interactions among MNCs and tumor cells could be observed, such as tumor antigen intake, processing and presentation by both dendritic cells and lymphocytes.There was significant proliferation and differentiation of the immune cells, including the maturation of dendritic cells.The lymphocytes were frequently forming lymph clusters containing tumor cells inside, mimicking the tumordraining lymph nodes.These sensitized immune cells surrounded tumor to prevent the growth of tumor cells and were also involved in the immune surveillance to prevent the metastasis of the tumor.More importantly, these cells directly eradicated tumor cells through cytolysis by cytotoxic T lymphocytes or phagocytosisby activated macrophages, which was associated with a significant decrease of tumor associated antigens.Conclusions: Our approach fundamentally improves the current protocol for expanding CD8+ T cells in vitro.By using an innovative three dimensional tissue culture device, an environment for in vitro sensitization that mimics the in vivo cellular immunization is created, associated with a shorter time to reach a successful initiation an expansion of large scale sensitized CD8+ T cells derived from peripheral blood.Reactivation of lymphocytes also becomes a feasible approach by the coculture of autologous or allogenie tumor cells with CD8+ T cells.