The endothelium lines the inner surface of all blood vessels representing an important tissue with vital functions to mediate tissue homeostasis. Tissue-tissue interfaces play a critical role throughout the human body where endothelial cells (ECs) contribute by creating vital barriers with tight and adherens junctions to regulate permeability of macromolecules and fluids while protecting and nourishing adjacent tissue. They are the most prominent cell type to experience physical forces of shear, stretch and strain through the laminar pulsatile nature of the bloodstream. Alterations in physiological flow profiles have a strong impact on EC pathology contributing to diseases like atherosclerosis and coronary heart disease as well as inflammatory conditions. Further, they are among the first cell types to interact with xenobiotics. Endothelial endocytosis and barrier regulation have profound impact on drug-tissue interactions. There is the need for technological innovations and improvements to study EC biology, EC-epithelial and EC-nanocarrier interactions in more complex settings that take physiological biophysical and biochemical cues into account. For this purpose, the Multi-Organ-Tissue-Flow (MOTiF) biochip has been invented and its design has been finalised during the beginning of this thesis. The objective was to develop handling and cellular seeding protocols for the biochip to establish more in vivo-like and more complex in vitro EC culture approaches. Within the scope of this thesis, the biochip has been characterised for perfused EC culture. Complex co-cultures with tissue resident macrophages and further with murine cortical spheroids present in liver sinusoidal structures and at the blood-brain barrier (BBB) have been established, respectively. Additionally, first applications under physiological parameters of shear stress have been made. The focus was on nanocarrier uptake profiles and microvascular endothelial barrier interaction.