Abstract
Microfluidic cellular models, commonly referred to as “organs‐on‐chips,” continue to advance the field of bioengineering via the development of accurate and higher throughput models, captivating the essence of living human organs. This class of models can mimic key in vivo features, including shear stresses and cellular architectures, in ways that cannot be realized by traditional two‐dimensional in vitro models. Despite such progress, current organ‐on‐a‐chip models are often overly complex, require highly specialized setups and equipment, and lack the ability to easily ascertain temporal and spatial differences in the transport kinetics of compounds translocating across cellular barriers. To address this challenge, we report the development of a three‐dimensional human blood brain barrier (BBB) microfluidic model (μHuB) using human cerebral microvascular endothelial cells (hCMEC/D3) and primary human astrocytes within a commercially available microfluidic platform. Within μHuB, hCMEC/D3 monolayers withstood physiologically relevant shear stresses (2.73 dyn/cm2) over a period of 24 hr and formed a complete inner lumen, resembling in vivo blood capillaries. Monolayers within μHuB expressed phenotypical tight junction markers (Claudin‐5 and ZO‐1), which increased expression after the presence of hemodynamic‐like shear stress. Negligible cell injury was observed when the monolayers were cultured statically, conditioned to shear stress, and subjected to nonfluorescent dextran (70 kDa) transport studies. μHuB experienced size‐selective permeability of 10 and 70 kDa dextrans similar to other BBB models. However, with the ability to probe temporal and spatial evolution of solute distribution, μHuBs possess the ability to capture the true variability in permeability across a cellular monolayer over time and allow for evaluation of the full breadth of permeabilities that would otherwise be lost using traditional end‐point sampling techniques. Overall, the μHuB platform provides a simplified, easy‐to‐use model to further investigate the complexities of the human BBB in real‐time and can be readily adapted to incorporate additional cell types of the neurovascular unit and beyond.
Highlights
The blood–brain barrier (BBB) is the prominent barrier at the interface of the blood stream and the central nervous system (CNS) and is primarily responsible for maintaining brain homeostasis and protecting the CNS from harmful foreign entities.[1]
We further demonstrate that μHuB is modular and can be readily adapted for more complex, coculture experiments to further bridge the gap between existing tools for investigating the human blood brain barrier (BBB) and underlying biology
ΜHuB can be expanded to incorporate additional components of interest, including the use of differentiation factors (e.g., 8-CPTcAMP and Ro 20–1,724),[79] primary human brain endothelial cells instead of the immortalized line, modification of cell type ratios to represent different regions of the brain,[80] and modulation of the applied shear stress, to create a holistic model of a healthy BBB. μHuB can be readily modified to further investigate how transport is affected in a diseased state, such as when there is inflammation caused by a traumatic brain injury or as the result of an invasive glioblastoma
Summary
The blood–brain barrier (BBB) is the prominent barrier at the interface of the blood stream and the central nervous system (CNS) and is primarily responsible for maintaining brain homeostasis and protecting the CNS from harmful foreign entities.[1]. Higher than physiologically relevant (40 dyn/cm2) and pulsatile shear stresses resulted in downregulation of ZO-1, Claudin[5], and P-gp; tight junction marker expression recovered when physiological shear was reestablished,[33] further suggesting the importance of maintaining hemodynamic shear stress among in vitro systems to more accurately represent the BBB microenvironment Recent developments in this field have resulted in a diversity of three-dimensional cell culture models and several dynamic systems with the ability to incorporate hemodynamic shear.[34,35] Still, simultaneous visualization of the BBB and the associated transport through the barrier in real-time remains a challenge.[36,37,38,39] Direct visualization at a cellular level provides real-time monitoring of the cellular morphology and can be used as a proxy for cell behavior. We further demonstrate that μHuB is modular and can be readily adapted for more complex, coculture experiments to further bridge the gap between existing tools for investigating the human BBB and underlying biology
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