Chun-hua Pan1, Rong-cheng Luo. 1. Department of Oncology, Nanfang Hospital, First Military Medical University, Guangzhou 510515, China.
Abstract
OBJECTIVE: To explore a new method for in vitro culture of mature dendritic cells (DCs) by utilizing the maturation-inducing effect of tumor necrosis factor-alpha(TNF-alpha) on DC precursors. METHODS: Freshly isolated DC precursors (peripheral blood-derived monocytes) were initially cultivated for 4 h in RPMI 1640 supplemented with 10% fetal bovine serum and containing TNF-alpha (200 U/ml), followed by treatment with rhGM-CSF (800 U/ml) and interleukin (IL)-4 (500 U/ml) added into the medium. After 48 and 96 h, the 3 cytokines were simultaneously added to the medium for further culture. On day 8 of cell culture, the DCs were harvested and designated as T-DCs, which were subsequently subjected to examinations of flow cytometry analysis, assay by autologous and allogeneic mixed lymphocyte reaction (MLR) for estimating their maturation, and MTT assay for their specific killing activity. RESULTS: The T-DCs expressed specific surface markers (CD83,CD1a, CD54, CD11a, CD80, CD86, HLA-DR, CD83HLA-DR) of mature DCs, and stimulated strong T-cell proliferative responses in both autologous and autogeneic MLR. It also activated CD4+ T cells, which proliferated in response to autologous tumor, and specifically lysed tumor targets. CONCLUSION: The DCs thus cultured in vitro possess typical phenotype, and can present antigens to T cells, and this method provides an alternative in culturing mature and functionally competent DCs.
OBJECTIVE: To explore a new method for in vitro culture of mature dendritic cells (DCs) by utilizing the maturation-inducing effect of tumor necrosis factor-alpha(TNF-alpha) on DC precursors. METHODS: Freshly isolated DC precursors (peripheral blood-derived monocytes) were initially cultivated for 4 h in RPMI 1640 supplemented with 10% fetal bovine serum and containing TNF-alpha (200 U/ml), followed by treatment with rhGM-CSF (800 U/ml) and interleukin (IL)-4 (500 U/ml) added into the medium. After 48 and 96 h, the 3 cytokines were simultaneously added to the medium for further culture. On day 8 of cell culture, the DCs were harvested and designated as T-DCs, which were subsequently subjected to examinations of flow cytometry analysis, assay by autologous and allogeneic mixed lymphocyte reaction (MLR) for estimating their maturation, and MTT assay for their specific killing activity. RESULTS: The T-DCs expressed specific surface markers (CD83,CD1a, CD54, CD11a, CD80, CD86, HLA-DR, CD83HLA-DR) of mature DCs, and stimulated strong T-cell proliferative responses in both autologous and autogeneic MLR. It also activated CD4+ T cells, which proliferated in response to autologous tumor, and specifically lysed tumor targets. CONCLUSION: The DCs thus cultured in vitro possess typical phenotype, and can present antigens to T cells, and this method provides an alternative in culturing mature and functionally competent DCs.