Abstract
Primary brain microvascular endothelial cells (BMECs) are widely used in a large number of in vitro studies each year to better mimic their physiological characteristics in vivo. However, potential changes in primary endothelial cells stemming from donor variability or culture conditions may affect the reliability and reproducibility of the experiments. While working on a project regarding BMEC senescence, we noticed behavioral differences between two different batches of cells. Comparative analyses of cellular characteristics revealed that while one batch of BMECs developed a typical cobblestone morphology, the other batch displayed a spindle-shape morphology. Despite showing similar tubulogenic and barrier-forming capacities, the spindle-shaped BMECs displayed greater proliferation rates, stronger staining for CD34, a marker of stemness and higher resistance to oxidative stress-induced senescence and replicative senescence. Conversely, the spindle-shaped cells demonstrated a much weaker staining for the endothelial marker CD31. Taken together, these findings indicate that it is important to scrutinize endothelial characteristics to ensure experimental accuracy when cellular responses markedly vary between the so-called endothelial cells.