Methods for exposing multiple cultures of endothelial cells to different fluid shear stresses and to cytokines, for subsequent analysis of inflammatory function.

摘要

Endothelial cells are conditioned by physicochemical environmental factors, including shear stress applied by flowing blood. However, the effects of shear conditioning on the functional responses of endothelial cells, such as ability to recruit leukocytes, remain uncertain. Here we describe a system for culturing multiple samples of endothelial cells under flow for prolonged periods, either at different shear stresses, or exposed concurrently to different concentrations of cytokines, for instance, tumour necrosis factor-alpha (TNF). The endothelial cells were cultured in glass capillaries (microslides) that could be conveniently transferred to a flow-based adhesion assay, to test the ability of the cultures to support adhesion and migration of flowing leukocytes. Paired control, 'static' samples were exposed to the identical medium and culture geometry. We found that the type of tubing used in the culture flow circuit and its maintenance at 37 degrees C were critical design factors, which could influence the response to TNF of the static controls which were exposed to recirculated medium. Endothelial cells conditioned by culture under flow showed a reduction in response to TNF, as judged by ability to induce the capture and migration of neutrophils. We found that the higher the shear stress, the weaker the ability to recruit neutrophils. This sensitivity to shear stress was greater if the cells were allowed to stabilise under static conditions for 24 h, compared to cells exposed to flow immediately after seeding. The inhibition of neutrophil recruitment was similar for cultures exposed to steady flow or flow with a pulsatile element (flow oscillation approximately 20% about the mean). Thus, we have developed a versatile culture system which allows investigations of functional modifications of endothelial cells and demonstrates the potential sensitivity of inflammatory responses to the local fluid environment.