Purification Of Human Peripheral Blood Monocytes On Gelatin-coated Surface And Differentiated Into Dendritic Cells

Objective:To investigate the efficacy of purification of human peripheral blood monocytes on gelatin-coated surfaces, observe the morphology and analyze the phenotype of dendritic cells (DC) generated by these monocytes, and make a comparison with conventional plastic adhesion method.Methods:Human peripheral blood mononuclear cells (PBMC) were harvested by Ficoll-Hypaque gradient centrifugation. Gelatin group and common plastic group were designated according to coating flasks with or without gelatin. Equally divided harvested PBMC, isolated monocytes using different flasks in each group. Dendritic cells were derived by culturing the monocytes in the presence of rhIL-4and rhGM-CSF followed by the maturation by the cocktail rhIL-1β, rhIL-6, rhTNF-α and PGE2. Count the number of isolated monocytes of each group. Observed and compared the contamination of platelet in each group. CD14, CD3, CD19positive rate of monocytes were determined by flow cytometry, represented the purity of monocytes, T and B lymphocytes contamination rate respectively. Cell viability was determined by trypan blue staining. After monocytes differentiated into dendritic cells, observed the morphology of dendritic cells of the two groups. And the expression of CD la, CD83on immature and mature dendritic cells were determined by flow cytometry.Results:Average30ml peripheral blood of each group, harvested (12.3±3.56)and(15.8±3.05)×106monocytes of common plastic group and gelatin-coated group respectively,p<0.05,there were significant difference between the two groups. It means that the number of monocytes in the gelatin group was higher than that in the common plastic group. Observed the platelet contamination of the common plastic group was higher than that in the gelatin-coated group. CD14, CD3, CD19positive rate of the monocytes were (67.25±5.77)%and (81.56±5.83)%,(4.68±1.01)%and (2.89±0.81)%,(10.89±1.45)%and (7.68±1.54)%respectively, P<0.05, there were significant difference between the two groups, it means that the purity of monocytes in gelatin-coated group was higher than that in common plastic group, contaminated rate of T, B lymphcytes in gelatin-coated group were lower than that in common plastic group. Cell viability of each group were (94.6±1.58)%and (95.2±1.64)%, P>0.05, there were no significant difference between the two groups, it means that gelatin has no obvious adverse effects on cell viability. After monocytes differentiated into dendritic cells, dendritic cells in both of the two groups matured with typical morphological features. There was no significant difference of the expression of CD la, CD83on immature and mature dendritic cells, regards that monocytes isolated by gelatin-coated surface can successfully stimulate into dendritic cells without adversely affecting the function of dendritic cells.Conclusion:Gelatin-coated surfaces can effectively and simply isolate monocytes and successfully stimulate them into mature dendritic cells with typical morphological features and phenotypes.