We describe here the design and implementation of an in vitro microvascular open model system using human brain microvascular endothelial cells. The design has several advantages over other traditional closed microfluidic platforms: (1) it enables controlled unidirectional flow of media at physiological rates to support vascular function, (2) it allows for very small volumes which makes the device ideal for studies involving biotherapeutics, (3) it is amenable for multiple high resolution imaging modalities such as transmission electron microscopy (TEM), 3D live fluorescence imaging using traditional spinning disk confocal microscopy, and advanced lattice light sheet microscopy (LLSM). Importantly, we miniaturized the design, so it can fit within the physical constraints of LLSM, with the objective to study physiology in live cells at subcellular level. We validated barrier function of our brain microvessel-on-a-chip by measuring permeability of fluorescent dextran and a human monoclonal antibody. One potential application is to investigate mechanisms of transcytosis across the brain microvessel-like barrier of fluorescently-tagged biologics, viruses or nanoparticles.

Bidirectional cargo transport along microtubules is carried out by opposing teams of kinesin and dynein motors. Despite considerable study, the factors that determine whether these competing teams achieve net anterograde or retrograde transport in cells remain unclear. The goal of this work is to use stochastic simulations of bidirectional transport to determine the motor properties that most strongly determine overall cargo velocity and directionality. Simulations were carried out based on published optical tweezer characterization of kinesin-1 and kinesin-2, and for available data for cytoplasmic dynein and the dynein-dynactin-BicD2 (DDB) complex. By varying dynein parameters and analyzing cargo trajectories, we find that net cargo transport is predicted to depend minimally on the dynein stall force, but strongly on dynein load-dependent detachment kinetics. In simulations, dynein is dominated by kinesin-1, but DDB and kinesin-1 are evenly matched, recapitulating recent experimental work. Kinesin-2 competes less well against dynein and DDB, and overall, load-dependent motor detachment is the property that most determines a motor's ability to compete in bidirectional transport. It follows that the most effective intracellular regulators of bidirectional transport are predicted to be those that alter motor detachment kinetics rather than motor velocity or stall force. K E Y W O R D S axon, dynein, Kinesin, microtubule, motor protein, neuron, optical tweezer, single-molecule, transport, vesicle

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ABSTRACTWe describe here the design and implementation of an in-vitro BBB-on-a-chip open model system capable of reconstituting the microenvironment of the blood brain barrier. This system allows controlled unidirectional flow of nutrients and biologicals on the lumen of the artificial microvessel. This BBB-on-a-chip is suitable for high resolution electron microscopy and it is amenable for quantitative 3D live fluorescence imaging using spinning confocal disk or lattice light sheet microscopy (LLSM) to follow, for example the transcytosis across the BBB-like barrier of fluorescently-tagged biological, viruses or nanoparticles.