Evaluation of Barrier Integrity Using a Two-Layered Microfluidic Device Mimicking the Blood-Brain Barrier.

The blood-brain barrier (BBB) plays an essential role in maintaining the homeostasis of the brain microenvironment by controlling the influx and efflux of biological substances that are necessary to sustain the neuronal metabolic activity and functions. This barrier is established at the blood-brain interface of the brain microcapillaries by different cells. These include microvascular endothelial cells, astrocytes, and pericytes besides other components such as microglia, basal membrane, and neuronal cells forming together what is commonly referred to as the neurovascular unit; different in vivo and in vitro platforms are available to study the BBB where each system provides specific benefits and drawbacks. Recently, organ-on-a-chip platforms combine the elegance of microengineering technology with the complexity of biological systems to create near-ideal experimental models for various diseases and organs. These microfluidic devices with micron-sized channels allow the cells to be grown in a more biologically relevant environment, enabling cell to cell communications with continuous bathing in biological fluids in a tissue-like fashion. They also closely represent tissue and organ functionality by recapitulating mechanical forces as well as vascular perfusion. Here, we describe the use of humanized BBB model created with microfluidic organ-on-a-chip technology where human brain microvascular endothelial cells (BMECs) are cocultured with primary human pericytes and astrocytes. We thoroughly described the method to assess BBB integrity using a microfluidic chip and various sizes of labeled dextran as permeability markers. In addition, we provide a detailed protocol on how to microscopically investigate the tight junction proteins expression between hBMECs.