The blood-brain barrier (BBB) is a critical structure that serves as the gatekeeper between the central nervous system and the rest of the body. It is the responsibility of the BBB to facilitate the entry of required nutrients into the brain and to exclude potentially harmful compounds; however, this complex structure has remained difficult to model faithfully in vitro. Accurate in vitro models are necessary for understanding how the BBB forms and functions, as well as for evaluating drug and toxin penetration across the barrier. Many previous models have failed to support all the cell types involved in the BBB formation and/or lacked the flow-created shear forces needed for mature tight junction formation. To address these issues and to help establish a more faithful in vitro model of the BBB, we have designed and fabricated a microfluidic device that is comprised of both a vascular chamber and a brain chamber separated by a porous membrane. This design allows for cell-to-cell communication between endothelial cells, astrocytes, and pericytes and independent perfusion of both compartments separated by the membrane. This NeuroVascular Unit (NVU) represents approximately one-millionth of the human brain, and hence, has sufficient cell mass to support a breadth of analytical measurements. The NVU has been validated with both fluorescein isothiocyanate (FITC)-dextran diffusion and transendothelial electrical resistance. The NVU has enabled in vitro modeling of the BBB using all human cell types and sampling effluent from both sides of the barrier.

The sophistication and success of recently reported microfabricated organs-on-chips and human organ constructs have made it possible to design scaled and interconnected organ systems that may significantly augment the current drug development pipeline and lead to advances in systems biology. Physiologically realistic live microHuman (µHu) and milliHuman (mHu) systems operating for weeks to months present exciting and important engineering challenges such as determining the appropriate size for each organ to ensure appropriate relative organ functional activity, achieving appropriate cell density, providing the requisite universal perfusion media, sensing the breadth of physiological responses, and maintaining stable control of the entire system, while maintaining fluid scaling that consists of ~5 mL for the mHu and ~5 µL for the µHu. We believe that successful mHu and µHu systems for drug development and systems biology will require low-volume microdevices that support chemical signaling, micro-fabricated pumps, valves and microformulators, automated optical microscopy, electrochemical sensors for rapid metabolic assessment, ion mobility-mass spectrometry for real-time molecular analysis, advanced bioinformatics, and machine learning algorithms for automated model inference and integrated electronic control. Toward this goal, we are building functional prototype components and are working toward top-down system integration.

BackgroundUnderstanding blood-brain barrier responses to inflammatory stimulation (such as lipopolysaccharide mimicking a systemic infection or a cytokine cocktail that could be the result of local or systemic inflammation) is essential to understanding the effect of inflammatory stimulation on the brain. It is through the filter of the blood-brain barrier that the brain responds to outside influences, and the blood-brain barrier is a critical point of failure in neuroinflammation. It is important to note that this interaction is not a static response, but one that evolves over time. While current models have provided invaluable information regarding the interaction between cytokine stimulation, the blood-brain barrier, and the brain, these approaches—whether in vivo or in vitro—have often been only snapshots of this complex web of interactions.MethodsWe utilize new advances in microfluidics, organs-on-chips, and metabolomics to examine the complex relationship of inflammation and its effects on blood-brain barrier function ex vivo and the metabolic consequences of these responses and repair mechanisms. In this study, we pair a novel dual-chamber, organ-on-chip microfluidic device, the NeuroVascular Unit, with small-volume cytokine detection and mass spectrometry analysis to investigate how the blood-brain barrier responds to two different but overlapping drivers of neuroinflammation, lipopolysaccharide and a cytokine cocktail of IL-1β, TNF-α, and MCP1,2.ResultsIn this study, we show that (1) during initial exposure to lipopolysaccharide, the blood-brain barrier is compromised as expected, with increased diffusion and reduced presence of tight junctions, but that over time, the barrier is capable of at least partial recovery; (2) a cytokine cocktail also contributes to a loss of barrier function; (3) from this time-dependent cytokine activation, metabolic signature profiles can be obtained for both the brain and vascular sides of the blood-brain barrier model; and (4) collectively, we can use metabolite analysis to identify critical pathways in inflammatory response.ConclusionsTaken together, these findings present new data that allow us to study the initial effects of inflammatory stimulation on blood-brain barrier disruption, cytokine activation, and metabolic pathway changes that drive the response and recovery of the barrier during continued inflammatory exposure.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0760-y) contains supplementary material, which is available to authorized users.

Engineered 3D cardiac tissue constructs (ECTCs) can replicate complex cardiac physiology under normal and pathological conditions. Currently, most measurements of ECTC contractility are either made isometrically, with fixed length and without control of the applied force, or auxotonically against a variable force, with the length changing during the contraction. The “I-Wire” platform addresses the unmet need to control the force applied to ECTCs while interrogating their passive and active mechanical and electrical characteristics. A six-well plate with inserted PDMS casting molds containing neonatal rat cardiomyocytes cultured with fibrin for 13–15 days is mounted on the motorized mechanical stage of an inverted microscope equipped with a fast sCMOS camera. A calibrated flexible probe provides strain load of the ECTC via lateral displacement, and the microscope detects the deflections of both the probe and the ECTC. The ECTCs exhibited longitudinally aligned cardiomyocytes with well-developed sarcomeric structure, recapitulated the Frank-Starling force-tension relationship, and demonstrated expected transmembrane action potentials, electrical and mechanical restitutions, and responses to both β-adrenergic stimulation and blebbistatin. The I-Wire platform enables creation and mechanical and electrical characterization of ECTCs, and hence can be valuable in the study of cardiac diseases, drug screening, drug development, and the qualification of cells for tissue-engineered regenerative medicine.

Nuclear Factor (erythroid-derived 2)-like 2 (Nrf2) expression is deregulated in many cancers. Genetic and biochemical approaches coupled with functional assays in cultured cells were used to explore the consequence of Nrf2 repression. Nrf2 suppression by Keap1-directed ubiquitylation or expression of independent shRNA/siRNA sequences enhanced cellular ROS, Smad-dependent tumor cell motility, and growth in soft agar. Loss of Nrf2 was accompanied by concomitant Smad linker region/C-terminus phosphorylation, induction of the E-Cadherin transcriptional repressor Slug, and suppression of the cell-cell adhesion protein E-Cadherin. Ectopic expression of wildtype Nrf2, but not dominant negative Nrf2, suppressed the activity of a synthetic TGF-β1 responsive CAGA-directed luciferase reporter. shRNA knock-down of Nrf2 enhanced the activity of the synthetic CAGA-reporter, as well as the expression of the endogenous Smad target gene plasminogen activator inhibitor-1. Finally, we found that Nrf2/Smad3/Smad4 formed an immunoprecipitable nuclear complex. Thus, loss of Nrf2 increased R-Smad phosphorylation and R-Smad signaling, supporting the hypothesis that loss of Nrf2 in an oncogenic context-dependent manner can enhance cellular plasticity and motility, in part by using TGF-β/Smad signaling.