Dual Channel Microfluidics for Mimicking the Blood–Brain Barrier

Abstract

High-resolution imaging is essential for analysis of the steps and way stations of cargo transport in in vitro models of the endothelium. In this study, we demonstrate a microfluidic system consisting of two channels horizontally separated by a cell-growth-promoting membrane. Its design allows for high-resolution (down to single-molecule level) imaging using a high numerical aperture objective with a short working distance. To reduce optical aberrations and enable single-molecule-sensitive imaging, an observation window was constructed in the membrane via laser cutting with subsequent structuring using 3D multiphoton lithography for improved cell growth. The upper channel was loaded with endothelial cells under flow conditions, which showed polarization and junction formation. A coculture of human vascular endothelial cells with pericytes was developed that mimics the blood–brain barrier. Finally, this dual channel microfluidics system enabled 3D localization microscopy of the cytoskeleton and 3D single-molecule-sensitive tracing of lipoprotein particles.

CRISPR/Cas9 Genome Editing vs. Over-Expression for Fluorescent Extracellular Vesicle-Labeling: A Quantitative Analysis

“…Considering the high dilution of GFP, each detected fluorescence spot with high probability represents a single GFP molecule ( Figure 3 a). Each detected spot was parametrized by 2D Gaussian fitting [ 54 , 55 , 56 , 57 ]. By measuring a large number of spots ( n = 247) one can obtain the distribution of fluorescence intensities ( Figure 2 c and Figure 3 a).…”

“…Afterwards, the cells were fixed and imaged at the single-molecule level. For the visualization of the EV distribution inside a cell, 3D reconstruction of astigmatism distorted single-molecule emitter point spread functions was used [ 54 , 56 , 57 , 59 , 60 ]. A high signal–noise ratio (on average >30) enabled the 3D localization of the subdiffractional EVs and aggregates inside the cells.…”

Microfluidic Tissue Engineering and Bio‐Actuation

“…This prevascularization approach can be achieved by patterning microfluidic materials to create microfluidic networks. [208,[263][264][265] Engineering micro and meso-vasculature systems within hydrogels relies on three main types of techniques, being: Micromolding, 3D printing, and microfluidic spinning. [266] Various types of hydrogels can be produced and shaped via micromolding, microfluidic spinning, 3D printing, photopatterning, and other methods, to form controllable and immediately perfusable networks.…”

Modeling the blood-brain barrier for treatment of central nervous system (CNS) diseases

“…To achieve this, in vitro modeling is required to mimic the BBB and then analyze the transport across the BBB. 72 One study created a two-channel microfluidic device, with human vascular endothelial cells cultured in one channel and pericytes. The outer layer of the chip was composed of acrylic glass, which increased the mechanical soundness of the chip.…”