Effect of differentiation on endocytic profiles of endothelial and epithelial cell culture models

Understanding mechanisms of endocytosis and trafficking of nanoparticles through endothelial and epithelial barriers leads potentially to improved efficacy of nanoparticulate drug delivery systems. Detailed characterizations of cell models with respect to endocytic pathway expression and activity (endocytic profiling) should facilitate data interpretation. We performed endocytic profiling of CaCo-2 and hCMEC/D3 cell lines, widely used as human intestinal and blood–brain barrier permeability models, respectively, during cell differentiation. Furthermore, we compared endocytic profiles of cell lines with those of primary cells. Expression of genes involved in specific endocytic pathways was analyzed at mRNA levels by quantitative real time PCR. Where possible, the respective protein levels were analyzed by Western blotting, and endocytic activities of cells were analyzed by flow cytometry. We showed that differentiated CaCo-2 cells formed tight, well polarized monolayers with reduced endocytic activity accompanied by reduced mRNA expression of most of the endocytosis-related genes. In contrast, hCMEC/D3 cells formed a leaky, less polarized barrier, and in vitro differentiation had little effect on either the expression of endocytosis-related genes or endocytic activity of these cells. Endocytic profiling of in vitro models and comparison with primary cells is an important measure to avoid misleading conclusions in nanoparticle permeation studies. [Display omitted] •We study the impact of cell differentiation on endocytosis.•We examine changes in endocytic marker expression/activity during differentiation.•We perform endocytic profiling of CaCo-2 and hCMEC/D3 cells.•Differentiated CaCo-2 cells show reduced endocytic marker expression and activity.•Poorly differentiated hCMEC/D3 cells show little changes in endocytic activity.