Leveraging avidin-biotin interaction to quantify permeability property of microvessels-on-a-chip networks

Microvessels-on-a-chip have enabled in vitro studies to closely simulate in vivo microvessel environment. However, assessing microvessel permeability, a functional measure of microvascular exchange, has not been attainable in nonpermeable microfluidic platforms. This study developed a new approach that enables permeability coefficients (P_s) to be quantified in microvessels developed in nonpermeable chip platforms by integrating avidin-biotin technology. Microvessels were developed on biotinylated fibronectin-coated microfluidic channels. Solute transport was assessed by perfusing microvessels with fluorescence-labeled avidin. Avidin molecules that crossed endothelium were captured by substrate biotin and recorded with real-time confocal images. The P_s was derived from the rate of avidin-biotin accumulation at the substrate relative to solute concentration difference across microvessel wall. Avidin tracers with different physiochemical properties were used to characterize the barrier properties of the microvessel wall. The measured baseline P_s and inflammatory mediator-induced increases in P_s and endothelial cell (EC) [Ca²⁺]_i resembled those observed in intact microvessels. Importantly, the spatial accumulation of avidin-biotin at substrate defines the transport pathways. Glycocalyx layer is well formed on endothelium and its degradation increased transcellular transport without affecting EC junctions. This study demonstrated that in vitro microvessels developed in this simply designed microfluidics structurally possess in vivo-like glycocalyx layer and EC junctions and functionally recapitulate basal barrier properties and stimuli-induced responses observed in intact microvessels. This new approach overcomes the limitations of nonpermeable microfluidics and provides an easily executed highly reproducible in vitro microvessel model with in vivo microvessel functionality, suitable for a wide range of applications in blood and vascular research and drug development.NEW & NOTEWORTHY Our study developed a novel method that allows permeability coefficient to be measured in microvessels developed in nonpermeable microfluidic platforms using avidin-biotin technology. It overcomes the major limitation of nonpermeable microfluidic system and provides a simply designed easily executed and highly reproducible in vitro microvessel model with permeability accessibility. This model with in vivo-like endothelial junctions, glycocalyx, and permeability properties advances microfluidics in microvascular research, suitable for a wide range of biomedical and clinical applications.