Microfluidic Prototyping by Xurography to Engineer Fully‐lumenized Microvessels In Vitro

Abstract

Vascular permeability (VP) is a measure of the barrier function (BF) of an intact endothelial cell (EC) monolayer. This parameter is especially important in physiology, as deregulation of permeability leads to leaky blood vessels with a transvascular imbalance of chemical gradients, fluidic pressure, and in some instances excessive trafficking of blood‐borne cells into the surrounding tissue. Regulation of VP is a highly coordinated process. However, monitoring VP in vivo can be challenging as it typically requires invasive and labor intensive intravital microscopy. Here we present an in vitro alternative by developing a microfluidic model of a blood vessel analog that permits characterization of endothelial BF under the influence of different extracellular matrix (ECM) and perivascular cellular constituents. Our model relies on the manufacturing process known as xurography, an inexpensive and rapid print‐and‐peel prototyping method for assembling multiple layers of polymers. This method has been recently applied to bypass the need for high‐end fabrication facilities that are not readily available at all research institutions. However, to date most experimental platforms manufactured with xurography have focused on biochemical studies with few cell culture‐based applications. Briefly, after using a cutting plotter to form a channel (.8 mm x 8 mm) on a PDMS layer (~ 400 um thick), the device is constructed through a series of bindings to other PDMS layers (3 x ~ 250 um) and biopsy punching that form two regions. The inlet and outlet ports for cell seeding (2mm and 4mm) are located at the extremes of the model. An area for ECM hydrogels is allocated in the middle, with a PDMS rod placed in the center (Fig 1). After the construction, collagen type I at 6mg/ml is inserted into the central region. Once polymerized the PDMS rod is pulled to leave a lumen, permitting the addition of ECs into the formed ductal structure. In order to assess BF, the vessels were stained and imaged with confocal microscopy for the intercellular adherent junction molecule, VE‐cadherin as well as cytoskeletal proteins. To quantify VP, a fluorescent tracer molecule was infused in the vessel, and this parameter was determined by measuring changes of intensity as the dye permeated from the vessel to the surrounding hydrogel. Importantly, our results indicated the vessel analog demonstrated VP on the same order of magnitude as similar studies with ECs growing in static conditions (Fig 2). Notably, the microdevice allows the addition of cells that can support EC function. These findings will be fundamental as we explore the crosstalk between cells that serve in regulating blood vessel BF. Moreover, the inexpensive fabrication process can be adapted seamlessly by research institutions across the nation to probe further into vascular biology.Support or Funding InformationFunding was provided by the National Heart, Lung, and Blood Institute (R01HL141941) and The Mark Foundation for Cancer Research ASPIRE Award. MC‐M acknowledges funding from a NIH Diversity Supplement (R01HL141941).Microfluidic model design and Confocal Imaging ‐ A) Assembly of microfluidic vessel analogue through binding of layers after fabrication. B) Staining for cytoskeletal protein, cell nuclei, and VE‐cadherin.Figure 1Apparent Permeability: Quantification of Vascular Permeability. Images of fluorescent dye permeating through the endothelial cell wall (time point ‐ 0 to 5 min.)Figure 2