A human BBB (blood brain barrier)-on-chip to assess vascular permeability

Introduction

In vitro human blood-brain barrier (BBB) models are needed to assess pathophysiological molecular transport mechanisms and enable the design of targeted therapies for neurological disorders. Several 2D culture systems have been designed but often fail to recapitulate the 3D cellular organization of brain capillaries. The generation of 3D models has allowed the production of tube-like vessels, however their geometries as well as cellular organization are still far from reflecting BBB in vivo. Here, Hajal et al., developed a human BBB-on-chip resembling the natural BBB that displays relevant gene expression profiles and vessel permeability values. They also developed quantitative measurement of perfusate molecules.

Experimental procedure

The authors provide a detailed protocol comprising the different steps to fabricate their BBB model as well as methods to quantitatively analyze molecular permeability. Briefly, PDMS microfluidic chips were molded from a silicon wafer fabricated by soft photolithography. Appropriate cell types such as human endothelial cells (ECs), pericytes (PCs) and astrocytes (ACs) were embedded in a fibrin hydrogel and loaded inside the microfluidic chip (Figure 1). As a result of cell self-organization, microvascular networks (MVNs) were formed after few days of culture within the chips.

In addition, the authors develop an approach to precisely collect interstitial fluid for direct analysis of labeled and unlabeled molecules. They used Fluigent Flow-EZ pressure controllers to perfuse their BBB model by imposing a pressure of 1kPa and further collected interstitial fluid (Figure 2). This controlled fluid perfusion and collection enable to analyze molecules present in this fluid with methods such as ELISA or mass spectrometry.

These two methods (interstitial fluid collection following vascular pressurization and fluorescence imaging by confocal microscopy) can be combined to measure effective permeability values under physiological transmural flow conditions.

This robust and flexible method could be applied to numerous applications. Analyzing the permeability of the BBB to nanoparticles for instance or the susceptibility to infectious agents such as SARS-CoV-2 would be of great interest.

Results

Compared with standard 2D assays, this model features relevant cellular organization and morphological characteristics, as well as values of molecular permeability within the range expected in vivo. After several days of culture, highly interconnected structures are formed (Figure 3) with vascular diameters in the range of 10–40 µm (slightly larger than those of human BBB capillaries in vivo). They can be perfused with solutes which make it a highly physiologically relevant model of the BBB microcirculation. Importantly, the application of physiological levels of fluid flow using a microfluidic pump was previously shown to induce lower permeabilities and resulted in prolonged model stability (Gs, O. et al. Microheart: a microfluidic pump for functional vascular culture in microphysiological systems. J. Biomech. 119, 2021).

Microvascular networks formed inside the BBB-on-chip can be collected from the gel to be analyzed. Cell types such as endothelial cells can be isolated with a cell sorter to quantify their gene and protein expression levels. The results show that under appropriate culture conditions with BBB-specific perivascular cells (pericytes and astrocytes), human ECs (ECs from induced pluripotent stem cells = iPS-ECs) adopt gene expression profiles that closely match those of human primary brain ECs. In addition, the assessment of the permeability of various molecules as well as the analyses of circulating cytokines secreted in the BBB-on-chip (via Luminex analysis of the medium perfused) provides a simple and physiologically accurate platform to study correlations between cytokine signaling and transporter gene/protein expression. Furthermore, the relevance of this protocol in designing patient-specific BBB microvascular networks may have potential applications in the clinic.