Optimizing Dendritic Cell Preparation for Fusion with Melanoma Cells

Abstract

<span><b>Background</b>: </span> <span><span>Fusion of dendritic cells (DCs) with melanoma cells could reinforce the</span></span> antigenicity of tumors as a strategy for the treatment of malignant melanoma. However, the insufficient quantity of DCs and the low fusion efficiency limits the development of such approach. <span><br/><b>Objective</b>: </span><span>To define the dosage of the stimulating factors as well as the</span> induction condition for the optimal DCs preparation and cell fusion. <span>Methods</span><span>: DCs</span> were generated from murine bone marrow cells, and cultured with four different concentrations of the granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). DCs were confirmed to be mature by detecting the expression of MHC-II, CD11c, CD80, and CD83 by flowcytometry. DCs-melanoma fusion cells were generated using polyethylene glycols (PEG) with different molecular weights and the fusion efficiency was detected by fluorescence-activated cell sorter (FACS). <span><br/><b>Results</b>:</span> The largest quantity of DCs was found when cells were cultured with 1000 U/ ml of GM-CSF and 500 U/ml of IL-4 (1.69 ± 0.04 ×10 <span style="font-family: TimesNewRomanPSMT; font-size: xx-small;"><span style="font-family: TimesNewRomanPSMT; font-size: xx-small;">6 </span></span><span>ml</span><span style="font-family: TimesNewRomanPSMT; font-size: xx-small;"><span style="font-family: TimesNewRomanPSMT; font-size: xx-small;">-1</span></span><span>, p<0.001 when compared with</span> the other three groups). The expression levels of MHC-II and CD83 on day 7 after incubation were significantly lower than those on day 3 (MHC-II: p<0.001; CD83: p<0.001). The efficiency of cell fusion under induction of PEG-3000 was significantly higher than that of PEG-4000 (15.4 ± 0.56% vs. 11.1 ± 0.45%, p<0.001). <span><br/><b>Conclusions</b>:</span> The largest quantity for mature DCs was stimulated with 1000 U/ml of GM-CSF and 500 U/ml of IL-4 and the highest fusion efficiency was under induction of PEG-3000.