Differentiation of human CD14+ monocytes: an experimental investigation of the optimal culture medium and evidence of a lack of differentiation along the endothelial line

ABSTRACT

The aim of this study was to determine the optimal culturing media for human CD14+ monocytes and to evaluate whether these cells are capable of differentiating into vascular endothelial cells. Human monocytes isolated from peripheral blood were cultured for 1, 3, 7, 10 or 14 days in different media containing either 10% fetal bovine serum (FBS), 10% autologous donor serum (Auto), 10% FBS with interleukin-3 and macrophage colony stimulating factor (FBS-WF) or 10% Auto and the same growth factors (AU-WF). The cells were differentiated using endothelial cell conditioning medium (EC). Viability was measured using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, and the cells were characterized by histology, immunohistochemistry and western blot analysis. Monocytes treated with Auto, FBS-WF or AU-WF medium generated a significant higher yield of vital cells after 7 days in culture compared with FBS-only medium (mean difference (MD)=0.318, P=0.01; MD=1.83, P=0.04; or MD=0.271, P=0.01 and MD=0.318, P=0.102). All tested media led to the differentiation of monocytes into macrophages, identified by CD68, especially in the FBS-WF medium (MD=+18.3% P=0.04). Differentiation into ECs caused a significant decrease in cell viability in all media. Endothelial cell markers, including CD31, CD144, VEGF, VEGF-R2 and CD34, could not be detected. Autologous serum significantly increases the yield of monocyte-derived cells with a higher effectiveness than commonly used FBS-only serum. There is no further benefit in culturing monocytes longer than 7 days. The cultivation of monocytes in the tested media leads preferentially to differentiation into macrophages. Differentiation into endothelial cells did not take place.