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Introduction: Inflammatory bowel disease (IBD) is a chronic relapsing and remitting disease with a rising incidence globally. Circulating exosomes play great roles in IBD pathogenesis through exosomal cargoes, especially impacting the function of endothelial barriers. Transendothelial electrical resistance (TEER) measurement is a widely used non-invasive and label-free strategy to monitor endothelial barrier function in vitro. This study established a well-designed microfluidic device to monitor the TEER changes of endothelial cellular barrier on-chip after treated with exosome derived from IBD serum. Methods: The chip comprised two layers of microfluidic chambers with top layer for the perfusion of medium to maintain the nutrition and pressure during cell culture, and bottom layer for the extracellular matrix mimic using hydrogel, which are separated by a semipermeable membrane that permitted the formation of endothelial cell barrier. Four electrodes independent from the outlets were integrated to the chip for TEER detection. In vivo mouse models mouse models and proteome profiling were performed to finding relevant regulators. Results: With this platform, significant decrease of TEER was detected, indicating that IBD serum exosome impact the endothelial cellular barrier on-chip. In vivo mouse models, IBD serum exosome treated group showed great higher DAI scores, shorter colons, more severe histological features, and higher levers of S100A8 expression, promoting the disease progress. Proteome profiling showed that TFRC and ANXA5 have great potentials as novel regulators in IBD. Discussion: This in-house customized microfluidic chip emulates the endothelial barrier microenvironment and enables the TEER monitoring, and can be used to investigate endothelial barrier function in vitro. IBD serum exosome promote the severity of disease.

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Blood-brain-barrier (BBB) disruption has been associated with a variety of central-nervous-system diseases. In vitro BBB models enable to investigate how the barrier reacts to external injury events, commonly referred to as insults. Here, a human-cell-based BBB platform with integrated, transparent electrodes to monitor barrier tightness in real time at high resolution is presented. The BBB model includes human cerebral endothelial cells and primary pericytes and astrocytes in a 3D arrangement within a pump-free, open-microfluidic platform. With this platform, this study demonstrates that oxygen-glucose deprivation (OGD), which mimics the characteristics of an ischemic insult, induces a rapid remodeling of the cellular actin structures and subsequent morphological changes in the endothelial cells. High-resolution live imaging shows the formation of large actin stress-fiber bundles in the endothelial layer during OGD application, which ultimately leads to cell shrinkage and barrier breakage. Simultaneous electrical measurements evidence a rapid decrease of the barrier electrical resistance before the appearance of stress fibers, which indicates that the barrier function is compromised already before the appearance of drastic morphological changes. The results demonstrate that the BBB platform recapitulates the main barrier functions in vitro and can be used to investigate rapid reorganization of the BBB upon application of external stimuli.

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Over the most recent decades, the development of new biological platforms to study disease progression and drug efficacy has been of great interest due to the high increase in the rate of neurodegenerative diseases (NDDs). Therefore, blood-brain barrier (BBB) as an organ-on-a-chip (OoC) platform to mimic brain-barrier performance could offer a deeper understanding of NDDs as well as a very valuable tool for drug permeability testing for new treatments. A very attractive improvement of BBB-oC technology is the integration of detection systems to provide continuous monitoring of biomarkers in real time and a fully automated analysis of drug permeably, rendering more efficient platforms for commercialization. In this Perspective, an overview of the main BBB-oC configurations is introduced and a critical vision of the BBB-oC platforms integrating electronic read out systems is detailed, indicating the strengths and weaknesses of current devices, proposing the great potential for biosensors integration in BBB-oC. In this direction, we name potential biomarkers to monitor the evolution of NDDs related to the BBB and/or drug cytotoxicity using biosensor technology in BBB-oC.

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Rationale: The blood-brain barrier (BBB) while functioning as a gatekeeper of the brain, impedes cerebral drug delivery. An emerging technology to overcome this limitation is focused ultrasound (FUS). When FUS interacts with intravenously injected microbubbles (FUS+MB), the BBB opens, transiently allowing the access of therapeutic agents into the brain. However, the ultrasound parameters need to be tightly tuned: when the acoustic pressure is too low there is no opening, and when it is too high, tissue damage can occur. We therefore asked whether barrier permeability can be increased by combining FUS+MB with a second modality such that in a clinical setting lower acoustic pressures could be used. Methods: Given that FUS+MB achieves BBB opening in part by disruption of tight junction (TJ) proteins such as claudin-5 of brain endothelial cells, we generated a stable MDCK (Madin-Darby Canine Kidney) II cell line (eGFP-hCldn5-MDCK II) that expresses fluorescently tagged human claudin-5. Two claudin-5 binders, the peptide mC5C2 and cCPEm (truncated form of an enterotoxin), reported previously to weaken the barrier, were synthesized and assessed for their abilities to enhance the permeability of cellular monolayers. We then performed a comparative analysis of single and combination treatments, measuring transendothelial electrical resistance (TEER) and cargo leakage, combined with confocal image analysis. Results: We successfully generated a novel cell line that formed functional monolayers as validated by an increased TEER reading and a low (< 0.2%) permeability to sodium fluorescein (376 Da). We found that the binders exerted a time- and concentration-dependent effect on barrier opening when incubated over an extended period, whereas FUS+MB caused a rapid opening followed by recovery after 12 hours within the tested pressure range. Importantly, preincubation with cCPEm prior to FUS+MB treatment resulted in greater barrier opening compared to either FUS+MB or cCPEm alone as measured by reduced TEER values and an increased permeability to fluorescently labelled 40 kDa dextran (FD40). Conclusion: The data suggest that pre incubation with clinically suitable binders to TJ proteins may be a general strategy to facilitate safer and more effective ultrasound-mediated BBB opening in cellular and animal systems and potentially also for the treatment of human diseases of the brain.

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The blood-brain barrier (BBB) regulates the interaction between the highly vulnerable central nervous system (CNS) and the peripheral parts of the body. Disruption of the BBB has been associated with multiple neurological disorders, in which immune pathways in microglia are suggested to play a key role. Currently, many in vitro BBB model systems lack a physiologically relevant microglia component in order to address questions related to the mechanism of BBB integrity or the transport of molecules between the periphery and the CNS. To bridge this gap, we redefined a serum-free medium in order to allow for the successful co-culturing of human inducible pluripotent stem cell (hiPSC)-derived microglia and hiPSC-derived brain microvascular endothelial-like cells (BMECs) without influencing barrier properties as assessed by electrical resistance. We demonstrate that hiPSC-derived microglia exposed to lipopolysaccharide (LPS) weaken the barrier integrity, which is associated with the secretion of several cytokines relevant in neuroinflammation. Consequently, here we provide a simplistic humanised BBB model of neuroinflammation that can be further extended (e.g., by addition of other cell types in a more complex 3D architecture) and applied for mechanistic studies and therapeutic compound profiling.

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Assessing the ability of pharmaceutics to cross biological barriers and reach the site-of-action requires faithful representation of these barriers in vitro. Difficulties have arisen in replicating in vivo resistance in vitro. This paper investigated serum starvation as a method to increase Caco-2 barrier stability and resistance. The effect of serum starvation on tight junction production was examined using transwell models; specifically, transendothelial electrical resistance (TEER), and the expression and localization of tight junction proteins, occludin and zonula occludens-1 (ZO-1), were studied using western blotting and immunofluorescence. Changing cells to serum-free media 2 days post-seeding resulted in TEER readings of nearly 5000 Ω cm2 but the TEER rapidly declined subsequently. Meanwhile, exchanging cells to serum-free media 4-6 days post-seeding produced barriers with resistance readings between 3000 and 4000 Ω cm2, which could be maintained for 18 days. This corresponded to an increase in occludin levels. Serum starvation as a means of barrier formation is simple, reproducible, and cost-effective. It could feasibly be implemented in a variety of pre-clinical pharmaceutical assessments of drug permeability across various biological barriers with the view to improving the clinical translation of novel therapeutics.

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Sepsis represents a life-threatening event often mediated by the host's response to pathogens such as gram-negative organisms, which release the proinflammatory lipopolysaccharide (LPS). Within the endothelium, the mitogen-activated protein kinase (MAPK) pathway is an important driver of endothelial injury during sepsis, of which oxidant-sensitive apoptosis signal-regulating kinase 1 (ASK1) is postulated to be a critical upstream regulator. We hypothesized that ASK1 would play a key role in endothelial inflammation during bacterial challenge. Utilizing RNA sequencing data from patients and cultured human microvascular endothelial cells (HMVECs), ASK1 expression was increased in sepsis and after LPS challenge. Two ASK1 inhibitors, GS444217 and MSC2023964A, reduced cytokine production in HMVECs following LPS stimulation, but had no effect on permeability as measured by transendothelial electrical resistance and intercellular space. MAPKs are known to interact with endothelial nitric oxide synthase (eNOS) and ASK1 expression levels correlated with eNOS expression in patients with septic shock. In addition, eNOS physically interacted with ASK1, though this interaction was not altered by ASK1 inhibition, nor did inhibition alter MAPK p38 activity. Instead, among MAPKs, ASK1 inhibition only impaired LPS-induced JNK phosphorylation. The reduction in JNK activation caused by ASK1 inhibition impaired JNK-mediated cytokine production without affecting permeability. Thus, LPS triggers JNK-dependent cytokine production that requires ASK1 activation, but both its effects on permeability and activation of p38 are ASK1-independent. These data demonstrate how distinct MAPK signaling pathways regulate endothelial inflammatory outputs during acute infectious challenge.

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The blood-brain barrier (BBB) is a structure located in brain capillaries that protects the brain from toxic substances in blood due to its high barrier function. The brain capillaries form a layered structure with pericytes, neurons, glial cells, and extracellular matrix proteins that is called neurovascular unit, and the structure is important to express the high barrier function of BBB. Here, we propose a method to construct a three-dimensional BBB tissue using three human BBB-composing cells, including brain endothelial cells, pericytes, and astrocytes, that mimics the in vivo BBB-like layered structure. Primary human brain endothelial cells were plated on the back side (outside) of the collagen vitrigel membrane of a culture insert, pericytes were plated on the upper side (inside), and astrocytes mixed in Matrigel were plated on the pericyte layer. The layered structure was maintained for at least 2 wk. The BBB tissue-loaded collagen vitrigel membrane can be detached from the insert frame using acetone with the tissue fixed intact and used for vertical cryosectioning to analyze the tissue interior. We also measured transendothelial electrical resistance (TEER) in the three-dimensional BBB co-culture to investigate barrier function of the brain endothelial cells. We believe that our co-culture method is useful to study engineered BBB tissues and develop reliable in vitro human BBB models in the future.

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Tight junctions are important structures that form the barrier of cells and tissues, and they play key roles in maintaining homeostasis of our body. The backbone of the tight junction proteins are claudins, which composed more than twenty members. The tight junction protein 1 (TJP1), also called ZO-1 (Zonula Occludens-1), is one of the tight junction related proteins, and it is widely used in literature to label tight junctions. Here we showed that TJP1 (ZO-1) is highly expressed in cancerous HeLa cells, fibroblast cells, HUVEC as well as MDCK cells, while claudin-1 is highly expressed in HUVEC and MDCK cells, but not expressed in HeLa and fibroblast cells. We aimed to investigate whether tight junction is present in HeLa and fibroblast cells. We used transepithelial/transendothelial electrical resistance (TEER) to measure tight junction dynamics in these cells. The results showed that there is no TEERs in HeLa and fibroblast cells, while there is relatively high TEER in HUVEC and MDCK cells. Importantly, the TEER in MDCK cells is dramatically reduced after knockdown of TJP1 (ZO-1). These results suggest that TJP1 (ZO-1) cannot be used as a marker of tight junctions in a variety of cells, while TJP1 (ZO-1) may play an important role in regulation of tight junctions in MDCK cells.

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In this study, we fabricated a hybrid elastomer-plastic microdevice using the silicone elastomer poly(dimethylsiloxane) (PDMS) and the plastic polycarbonate (PC), to mimic the human blood-brain barrier (BBB) in vitro. Specifically, the microchannel-imprinted elastomer was first coated with 3-aminopropyltriethoxysilane to produce amine-terminated PDMS. Then, simply by conformal contact at room temperature, the amine-functionalized PDMS was bonded to pristine PC through the formation of urethane linkages. Aside from realizing device bonding, the amine functionalization also assisted in subsequent dopamine coating to form polydopamine and provide a stable surface for culturing human endothelial cells and central nervous system-related cells (e.g., astrocytes) inside the microchannels. Successful mimicking of the BBB-like microenvironment was assessed by 3D co-culturing of human endothelial cells and astrocytes, where the microdevice was verified as an acceptable in vitro BBB model according to the following four criteria: the formation of tight junctions at the cell-cell boundaries of the endothelial cells, evaluated by the expression of the tight junction marker ZO-1; the formation of actin filaments, evaluated using rhodamine phalloidin dye; low permeability, tested using the fluorescent tracer 40-kDa FITC-dextran; and good transendothelial electrical resistance (a measure of the tight junction integrity formed between the endothelial cells). The fabricated PDMS-PC microfluidic device ensured simple yet stable device sealing, and simultaneously enhanced BBB-mimicking cell attachment, thus fulfilling all major criteria for its application as a convenient in vitro BBB model.

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The human blood-nerve barrier (BNB) formed by endoneurial microvascular endothelial cells, serves to maintain the internal microenvironment in peripheral nerves required for normal axonal signal transduction to and from the central nervous system. The mechanisms of human BNB formation in health and disease are not fully elucidated. Prior work established a sufficient role for glial-derived neurotrophic factor (GDNF) in enhancing human BNB biophysical properties following serum withdrawal in vitro via RET-tyrosine kinase-dependent cytoskeletal remodeling. The objective of the study was to ascertain the downstream signaling pathway involved in this process and more comprehensively determine the molecular changes that may occur at human BNB intercellular junctions under the influence of GDNF. Proteomic studies suggested expression of several mitogen-activated protein kinases (MAPKs) in confluent GDNF-treated endoneurial endothelial cells following serum withdrawal. Using electric cell-substrate impedance sensing to continuously measure transendothelial electrical resistance and static transwell solute permeability assays with fluoresceinated small and large molecules to evaluate BNB biophysical function, we determined MAPK signaling was essential for GDNF-mediated BNB TEER increase following serum withdrawal downstream of RET-tyrosine kinase signaling that persisted for up to 48 hours in vitro. This increase was associated with reduced solute permeability to fluoresceinated sodium and high molecular weight dextran. Specific GDNF-mediated alterations were detected in cytoskeletal and intercellular junctional complex molecular transcripts and proteins relative to basal conditions without exogenous GDNF. This work provides novel insights into the molecular determinants and mechanisms responsible for specialized restrictive human BNB formation in health and disease.

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Human blood-brain barrier (BBB) in vitro models pose a promising tool in drug development and understanding of mechanistic regulations during health and disease. Human-induced pluripotent stem cells (hiPS cells) represent an unlimited cell source to generate functional cells of the neurovascular unit (NVU), independent of variations or limitations during isolation and in vitro cultivation. This unit describes the standardized 2-D differentiation of adherent hiPS cells into BBB endothelial cells and neuronal stem cells (NSCs). Both cell types are combined with primary astrocytes and pericytes to develop complex, physiological BBB in vitro models. The endothelial cells in the apical compartment of the transwell models are separated from the basolateral seeded co-culture mixture by a synthetic membrane, simplifying analyses. The barrier integrity and functionality of the endothelium is improved by the specific mixture of NVU niche cells, determined here by decrease in the paracellular permeability of sodium-fluorescein and transendothelial electrical resistance (TEER) measurement. © 2018 by John Wiley & Sons, Inc.

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High extracellular S100A4 level proves a specific characteristic of some cancer cases, including malignant melanoma. Concerning the latter, extracellular S100A4 in an autocrine manner was shown to promote prometastatic activation of A375 cells by interaction with the receptor for advanced glycation endproducts (RAGE). We hypothesized that interaction of extracellular S100A4 with RAGE in a paracrine manner will affect endothelial cell (EC) integrity thus further promoting melanoma metastasis. We investigated the influence of recombinant and cell (A375)-derived S100A4 on junction protein expression and EC (hCMEC/D3) integrity by measuring transendothelial electrical resistance (TEER). Decrease of TEER and diminished expression of both occludin and VE-cadherin revealed the loss of EC integrity. Transmigration of transgenic A375 cells (A375-hS100A4/A375-hRAGE) through the EC monolayer was significantly higher compared to wild-type A375 cells, and was substantially decreased by sRAGE. A pilot study in mice, intracardially injected with A375-hS100A4 or A375-hRAGE cells, showed lower survival rates and a higher incidence of metastases compared to wild-type A375 cells. Tumor development was mostly located in the brain, bones, and ovaries. These findings provide further evidence on extracellular S100A4 as paracrine mediator of prometastatic endothelial dysfunction involving its interaction with RAGE.

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