Assessing Endothelial Cell Permeability and Transport Pathways via Biotin/FITC‐Avidin Interaction in Cultured Endothelial Microchannel Networks Using Microfluidic Devices

Abstract

Microfluidic technologies have enabled in vitro studies to closely simulate the in vivo microvessel environment with sufficient complexity. Our recent studies demonstrated that endothelial cells (ECs) cultured under continuous flow within microfluidic devices form junctions closer to those observed in intact microvessels than static cell cultures. Importantly their cell signaling responses to inflammatory mediators are comparable to those derived from individually perfused intact venules. However, the commonly used impermeable polydimethysiloxane (PDMS) in microfluidic devices could not study the permeability properties of ECs. Recently the biotin‐avidin interaction has been successfully applied to static cell cultures for assessing EC permeability. The objective of this study is to apply the biotin‐avidin approach to cultured ECs in microfluidic device, and detect the spatial changes in EC permeability and differentiate transport pathways under control and stimulated conditions. Microfluidic devices were fabricated through soft lithography at different levels of channel branches. Glass coverslips were cohered as the base of devices for image acquisition. Inner channels of device were coated with biotinylated fibronectin before rat primary dermal microvascular endothelial cells were seeded and cultured along the lining of channels with a constant medium flow at 0.22 μL/min. ECs reach confluence within 3 days from seeding and successfully covered the inner surface of microchannels. Permeability was assessed by measuring integrated fluorescence intensity of region of interest after perfusion the microchannel networks with solutions containing FITC‐avidin with confocal images. The results showed that perfusion microchannels with medium alone showed no fluorescent signals. Only scattered FITC signals were observed at cellular junctions when the microchannels were perfused with BSA‐Ringer's solution containing FITC‐avidin (served as control). When PAF (10 nM) was added to the perfusate in the presence of FITC‐avidin, we observed a time dependent increases in fluorescent areas and intensity along junctions between ECs. Image analysis showed that fluorescence intensity increased 6.6 ± 0.12 and 8.94 ± 0.25 times that of the control when PAF was perfused for 4 and 10 min, respectively. The spatial distribution of FITC signal indicates paracellular pathway as the predominant mechanism of PAF‐induced permeability increase. The biotin‐avidin approach is based on high affinity ligand‐ acceptor interactions and their interaction enable the mapping of transport pathways and quantify the leaked molecules. Our study indicated that the application of biotin‐avidin approach to microfluidic devices with cultured EC networks could serve as a useful tool for assessing permeability properties of ECs that were grown under continuous flow, a cell culture condition closer to that in vivo.Support or Funding InformationSupported by HL130363 and DK097391This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.