BACKGROUND AND AIMS: The aim of the present study was to compare several in vitro anti-CD3/CD28-coated magnetic bead expansion methods as a means of improving T cell recovery and to evaluate the impact of the respective methods on T cell viability, differentiation and function. The effect of the respective expansion protocols on cytokine production and cytotoxicity was also characterized. METHODS: Peripheral blood mononuclear cells were isolated from healthy donors, expanded for 7 days by different methods in vitro and then counted. T cell viability, phenotype and differentiation status were determined by flow cytometry. The cytotoxicity of collected cells was evaluated by a non-radioactive assay. RESULTS: An equal bead-to-cell ratio in the presence of IL-2, IL-7 and IL-15 generated the highest T cell yields (median 4.75-fold increase [range 4.10-6.25], P=0.043) with a median of 79.20% viable cells (range 70.00%-80.30%). By contrast, a high bead-to-cell ratio (3:1) favored the selection of central memory T cells (CD4: 0.44 [0.40-1.69]; CD8: 0.77 [0.42-1.19], P=0.043) with increased interferon-gamma (IFN-γ) production following re-stimulation (48.5% [42.1%-68.2%], P=0.043) and also a trend towards enhanced cytotoxicity against target cells (10:1 ratio: 61.80% [40.80%-80.00%], P=0.068). CONCLUSIONS: An equal bead-to-cell ratio is optimal in keeping the balance between promoting proliferations and preserving cellular vitality and the combination of homeostatic cytokines further improved cell output, whereas a high bead-to-cell ratio favored the production of bioactive cells. Crown
BACKGROUND AND AIMS: The aim of the present study was to compare several in vitro anti-CD3/CD28-coated magnetic bead expansion methods as a means of improving T cell recovery and to evaluate the impact of the respective methods on T cell viability, differentiation and function. The effect of the respective expansion protocols on cytokine production and cytotoxicity was also characterized. METHODS: Peripheral blood mononuclear cells were isolated from healthy donors, expanded for 7 days by different methods in vitro and then counted. T cell viability, phenotype and differentiation status were determined by flow cytometry. The cytotoxicity of collected cells was evaluated by a non-radioactive assay. RESULTS: An equal bead-to-cell ratio in the presence of IL-2, IL-7 and IL-15 generated the highest T cell yields (median 4.75-fold increase [range 4.10-6.25], P=0.043) with a median of 79.20% viable cells (range 70.00%-80.30%). By contrast, a high bead-to-cell ratio (3:1) favored the selection of central memory T cells (CD4: 0.44 [0.40-1.69]; CD8: 0.77 [0.42-1.19], P=0.043) with increased interferon-gamma (IFN-γ) production following re-stimulation (48.5% [42.1%-68.2%], P=0.043) and also a trend towards enhanced cytotoxicity against target cells (10:1 ratio: 61.80% [40.80%-80.00%], P=0.068). CONCLUSIONS: An equal bead-to-cell ratio is optimal in keeping the balance between promoting proliferations and preserving cellular vitality and the combination of homeostatic cytokines further improved cell output, whereas a high bead-to-cell ratio favored the production of bioactive cells. Crown