Here we report on the development of a breakthrough microfluidic human in vitro cerebrovascular barrier (CVB) model featuring stem cell-derived brain-like endothelial cells (BLECs) and nanoporous silicon nitride (NPN) membranes (µSiM-CVB). The nanoscale thinness of NPN membranes combined with their high permeability and optical transparency makes them an ideal scaffold for the assembly of an in vitro microfluidic model of the blood–brain barrier (BBB) featuring cellular elements of the neurovascular unit (NVU). Dual-chamber devices divided by NPN membranes yield tight barrier properties in BLECs and allow an abluminal pericyte-co-culture to be replaced with pericyte-conditioned media. With the benefit of physiological flow and superior imaging quality, the µSiM-CVB platform captures each phase of the multi-step T-cell migration across the BBB in live cell imaging. The small volume of <100 µL of the µSiM-CVB will enable in vitro investigations of rare patient-derived immune cells with the human BBB. The µSiM-CVB is a breakthrough in vitro human BBB model to enable live and high-quality imaging of human immune cell interactions with the BBB under physiological flow. We expect it to become a valuable new tool for the study of cerebrovascular pathologies ranging from neuroinflammation to metastatic cancer.

The extraordinary permeability and manufacturability of ultrathin silicon-based membranes are enabling devices with improved performance and smaller sizes in such important areas as molecular filtration and sensing, cell culture, electroosmotic pumping, and hemodialysis. Because of the robust chemical and mechanical properties of silicon nitride (SiN), several laboratories have developed techniques for patterning nanopores in SiN using reactive ion etching (RIE) through a template structure. These methods however, have failed to produce pores small enough for ultrafiltration (<100 nm) in SiN and involve templates that are prone to microporous defects. Here we present a facile, wafer-scale method to produce nanoporous silicon nitride (NPN) membranes using porous nanocrystalline silicon (pnc-Si) as a self-assembling, defect free, RIE masking layer. By modifying the mask layer morphology and the RIE etch conditions, the pore sizes of NPN can be adjusted between 40 nm and 80 nm with porosities reaching 40%. The resulting NPN membranes exhibit higher burst pressures than pnc-Si membranes while having 5× greater permeability. NPN membranes also demonstrate the capacity for high resolution separations (<10 nm) seen previously with pnc-Si membranes. We further demonstrate that human endothelial cells can be grown on NPN membranes, verifying the biocompatibility of NPN and demonstrating the potential of this material for cell culture applications.

Selective cellular transmigration across the microvascular endothelium regulates innate and adaptive immune responses, stem cell localization, and cancer cell metastasis. Integration of traditional microporous membranes into microfluidic vascular models permits the rapid assay of transmigration events but suffers from poor reproduction of the cell permeable basement membrane. Current microporous membranes in these systems have large nonporous regions between micropores that inhibit cell communication and nutrient exchange on the basolateral surface reducing their physiological relevance. Here, the use of 100 nm thick continuously nanoporous silicon nitride membranes as a base substrate for lithographic fabrication of 3 µm pores is presented, resulting in a highly porous (≈30%), dual‐scale nano‐ and microporous membrane for use in an improved vascular transmigration model. Ultrathin membranes are patterned using a precision laser writer for cost‐effective, rapid micropore design iterations. The optically transparent dual‐scale membranes enable complete observation of leukocyte egress across a variety of pore densities. A maximal density of ≈14 micropores per cell is discovered beyond which cell–substrate interactions are compromised giving rise to endothelial cell losses under flow. Addition of a subluminal extracellular matrix rescues cell adhesion, allowing for the creation of shear‐primed endothelial barrier models on nearly 30% continuously porous substrates.

Silk consists primarily of two proteins-a fibrous core protein, fibroin, and a glue protein, sericin, which envelops the fibroin fibers with sticky layers thus helping in the formation of cocoon, achieved by cementing the silk fibers together. Sericin, a water soluble protein, has traditionally been discarded in silk processing, despite great potential for use as a biomaterial. Here we show that this glue protein, sericin, has the ability to form self-assembled nano and microstructures with hierarchical self-similarity across length scales in the form of a diffusion-limited, fractal assembly. Sericin obtained from two silkworms, a domesticated mulberry Bombyx mori and a wild non-mulberry Antheraea mylitta, were studied, with particular insight into its structure and morphology as it dried on a surface. High-resolution atomic force microscopy was used to image the self-assembled protein, with investigations on the various factors that influenced this process. We describe the self-assembly patterns formed by the sericin protein and analyze the images based on the theory of diffusion limited aggregation to explain the fractal nature of these architectures observed. The unique physical behavior and fractal nature of this glue protein may represent a key step in understanding its biological relevance as well as the role it plays in the assembly and formation of silks.

Microfluidic systems are powerful tools for cell biology studies because they enable the precise addition and removal of solutes in small volumes. However, the fluid forces inherent in the use of microfluidics for cell cultures are sometimes undesirable. An important example is chemotaxis systems where fluid flow creates well-defined and steady chemotactic gradients but also pushes cells downstream. Here we demonstrate a chemotaxis system in which two chambers are separated by a molecularly thin (15 nm), transparent, and nanoporous silicon membrane. One chamber is a microfluidic channel that carries a flow-generated gradient while the other chamber is a shear-free environment for cell observation. The molecularly thin membranes provide effectively no resistance to molecular diffusion between the two chambers, making them ideal elements for creating flow-free chambers in microfluidic systems. Analytical and computational flow models that account for membrane and chamber geometry, predict shear reduction of more than five orders of magnitude. This prediction is confirmed by observing the pure diffusion of nanoparticles in the cell-hosting chamber despite high input flow (Q = 10 µL min−1; vavg ~45 mm min−1) in the flow chamber only 15 nm away. Using total internal reflection fluorescence (TIRF) microscopy, we show that a flow-generated molecular gradient will pass through the membrane into the quiescent cell chamber. Finally we demonstrate that our device allows us to expose migrating neutrophils to a chemotactic gradient or fluorescent label without any influence from flow.