Endothelial growth medium mv

Human aortic endothelial cells (HAEC) from male and female donors were obtained from Promocell and cultured on fibronectin-coated plasticware in Promocell Endothelial Growth Medium MV with Supplement Mix. Culture medium was replaced every 48-72h and cells were sub-cultured using Trypsin-EDTA solution (0.25%). For flow experiments, cells were seeded (at passage 6-7) in 6-well plates coated with fibronectin (10µg.ml -1 ) and cultured for 24-48h until confluent. For immunostaining experiments, cells were seeded in glass-bottomed 6-well plates (Cellvis). Once confluent, 1.902 ml fresh medium was added to each well (equivalent to 2mm medium height). Plates were placed on an orbital shaker housed inside the incubator (Grant Instruments; 150rpm with 5mm orbital radius) and exposed to flow, induced by the swirling of the media across the base of the well [14] [15] [16] . Computational fluid dynamics, using StarCCM+ analysis software, was used to determine various steady-state flow metrics at the base of the well (analysis published elsewhere) 17 . Briefly, EC at the centre of the well were exposed to low magnitude multidirectional flow (LMMF), whereas EC at the edge of the well were exposed to higher magnitude unidirectional flow (HMUF) 13, 17 . Cells were exposed to flow for 72h unless stated otherwise.