Sided Stimulation of Endothelial Cells Modulates Neutrophil Trafficking in an In Vitro Sepsis Model.

While the role of dysregulated polymorphonuclear leukocyte (PMN) transmigration in septic mediated tissue damage is well documented, strategies to mitigate aberrant transmigration across endothelium have yet to yield viable therapeutics. Recently, microphysiological systems (MPS) have emerged as novel in vitro mimetics that facilitate the development of human models of disease. With this advancement, aspects of endothelial physiology that are difficult to assess with other models can be directly probed. In this study, the role of endothelial cell (EC) apicobasal polarity on leukocyte trafficking response is evaluated with the µSiM-MVM (microphysiological system enabled by a silicon membrane - microvascular mimetic). Here, ECs are stimulated either apically or basally with a cytokine cocktail to model a septic-like challenge before introducing healthy donor PMNs into the device. Basally oriented stimulation generated a stronger PMN transmigratory response versus apical stimulation. Importantly, healthy PMNs are unable to migrate towards a bacterial peptide chemoattractant when ECs are apically stimulated, which mimics the attenuated PMN chemotaxis seen in sepsis. Escalating the apical inflammatory stimulus by a factor of five is necessary to elicit high PMN transmigration levels across endothelium. These results demonstrate that EC apicobasal polarity modulates PMN transmigratory behavior and provides insight into the mechanisms underlying sepsis.

Transforming Static Barrier Tissue Models into Dynamic Microphysiological Systems.

Microphysiological systems are miniaturized cell culture platforms used to mimic the structure and function of human tissues in a laboratory setting. However, these platforms have not gained widespread adoption in bioscience laboratories where open-well, membrane-based approaches serve as the gold standard for mimicking tissue barriers, despite lacking fluid flow capabilities. This issue can be primarily attributed to the incompatibility of existing microphysiological systems with standard protocols and tools developed for open-well systems. Here, we present a protocol for creating a reconfigurable membrane-based platform with an open-well structure, flow enhancement capability, and compatibility with conventional protocols. This system utilizes a magnetic assembly approach that enables reversible switching between open-well and microfluidic modes. With this approach, users have the flexibility to begin an experiment in the open-well format using standard protocols and add or remove flow capabilities as needed. To demonstrate the practical usage of this system and its compatibility with standard techniques, an endothelial cell monolayer was established in an open-well format. The system was reconfigured to introduce fluid flow and then switched to the open-well format to conduct immunostaining and RNA extraction. Due to its compatibility with conventional open-well protocols and flow enhancement capability, this reconfigurable design is expected to be adopted by both engineering and bioscience laboratories.

Use of the MicroSiM (µSiM) Barrier Tissue Platform for Modeling the Blood-Brain Barrier.

The microSiM (µSiM) is a membrane-based culture platform for modeling the blood-brain barrier (BBB). Unlike conventional membrane-based platforms, the µSiM provides experimentalists with new capabilities, including live cell imaging, unhindered paracrine signaling between 'blood' and 'brain' chambers, and the ability to directly image immunofluorescence without the need for the extraction/remounting of membranes. Here we demonstrate the basic use of the platform to establish monoculture (endothelial cells) and co-culture (endothelial cells and pericytes) models of the BBB using ultrathin nanoporous silicon-nitride membranes. We demonstrate compatibility with both primary cell cultures and human induced pluripotent stem cell (hiPSC) cultures. We provide methods for qualitative analysis of BBB models via immunofluorescence staining and demonstrate the use of the µSiM for the quantitative assessment of barrier function in a small molecule permeability assay. The methods provided should enable users to establish their barrier models on the platform, advancing the use of tissue chip technology for studying human tissues.

High levels of endothelial ICAM-1 prohibit natalizumab mediated abrogation of CD4+ T cell arrest on the inflamed BBB under flow in vitro.

INTRODUCTION: The humanized anti-α4 integrin blocking antibody natalizumab (NTZ) is an effective treatment for relapsing-remitting multiple sclerosis (RRMS) that is associated with the risk of progressive multifocal leukoencephalopathy (PML). While extended interval dosing (EID) of NTZ reduces the risk for PML, the minimal dose of NTZ required to maintain its therapeutic efficacy remains unknown. OBJECTIVE: Here we aimed to identify the minimal NTZ concentration required to inhibit the arrest of human effector/memory CD4+ T cell subsets or of PBMCs to the blood-brain barrier (BBB) under physiological flow in vitro. RESULTS: Making use of three different human in vitro BBB models and in vitro live-cell imaging we observed that NTZ mediated inhibition of α4-integrins failed to abrogate T cell arrest to the inflamed BBB under physiological flow. Complete inhibition of shear resistant T cell arrest required additional inhibition of ß2-integrins, which correlated with a strong upregulation of endothelial intercellular adhesion molecule (ICAM)-1 on the respective BBB models investigated. Indeed, NTZ mediated inhibition of shear resistant T cell arrest to combinations of immobilized recombinant vascular cell adhesion molecule (VCAM)-1 and ICAM-1 was abrogated in the presence of tenfold higher molar concentrations of ICAM-1 over VCAM-1. Also, monovalent NTZ was less potent than bivalent NTZ in inhibiting T cell arrest to VCAM-1 under physiological flow. In accordance with our previous observations ICAM-1 but not VCAM-1 mediated T cell crawling against the direction of flow. CONCLUSION: Taken together, our in vitro observations show that high levels of endothelial ICAM-1 abrogate NTZ mediated inhibition of T cell interaction with the BBB. EID of NTZ in MS patients may thus require consideration of the inflammatory status of the BBB as high levels of ICAM-1 may provide an alternative molecular cue allowing for pathogenic T cell entry into the CNS in the presence of NTZ.

A computer vision approach for analyzing label free leukocyte trafficking dynamics on a microvascular mimetic.

High-content imaging techniques in conjunction with in vitro microphysiological systems (MPS) allow for novel explorations of physiological phenomena with a high degree of translational relevance due to the usage of human cell lines. MPS featuring ultrathin and nanoporous silicon nitride membranes (µSiM) have been utilized in the past to facilitate high magnification phase contrast microscopy recordings of leukocyte trafficking events in a living mimetic of the human vascular microenvironment. Notably, the imaging plane can be set directly at the endothelial interface in a µSiM device, resulting in a high-resolution capture of an endothelial cell (EC) and leukocyte coculture reacting to different stimulatory conditions. The abundance of data generated from recording observations at this interface can be used to elucidate disease mechanisms related to vascular barrier dysfunction, such as sepsis. The appearance of leukocytes in these recordings is dynamic, changing in character, location and time. Consequently, conventional image processing techniques are incapable of extracting the spatiotemporal profiles and bulk statistics of numerous leukocytes responding to a disease state, necessitating labor-intensive manual processing, a significant limitation of this approach. Here we describe a machine learning pipeline that uses a semantic segmentation algorithm and classification script that, in combination, is capable of automated and label-free leukocyte trafficking analysis in a coculture mimetic. The developed computational toolset has demonstrable parity with manually tabulated datasets when characterizing leukocyte spatiotemporal behavior, is computationally efficient and capable of managing large imaging datasets in a semi-automated manner.

Modelling a Human Blood-Brain Barrier Co-Culture Using an Ultrathin Silicon Nitride Membrane-Based Microfluidic Device.

Understanding the vesicular trafficking of receptors and receptor ligands in the brain capillary endothelium is essential for the development of the next generations of biologics targeting neurodegenerative diseases. Such complex biological questions are often approached by in vitro models in combination with various techniques. Here, we present the development of a stem cell-based human in vitro blood-brain barrier model composed of induced brain microvascular endothelial cells (iBMECs) on the modular µSiM (a microdevice featuring a silicon nitride membrane) platform. The µSiM was equipped with a 100 nm thick nanoporous silicon nitride membrane with glass-like imaging quality that allowed the use of high-resolution in situ imaging to study the intracellular trafficking. As a proof-of-concept experiment, we investigated the trafficking of two monoclonal antibodies (mAb): an anti-human transferrin receptor mAb (15G11) and an anti-basigin mAb (#52) using the µSiM-iBMEC-human astrocyte model. Our results demonstrated effective endothelial uptake of the selected antibodies; however, no significant transcytosis was observed when the barrier was tight. In contrast, when the iBMECs did not form a confluent barrier on the µSiM, the antibodies accumulated inside both the iBMECs and astrocytes, demonstrating that the cells have an active endocytic and subcellular sorting machinery and that the µSiM itself does not hinder antibody transport. In conclusion, our µSiM-iBMEC-human astrocyte model provides a tight barrier with endothelial-like cells, which can be used for high-resolution in situ imaging and for studying receptor-mediated transport and transcytosis in a physiological barrier.

A photonic biosensor-integrated tissue chip platform for real-time sensing of lung epithelial inflammatory markers.

Tissue chip (TC) devices, also known as microphysiological systems (MPS) or organ chips (OCs or OoCs), seek to mimic human physiology on a small scale. They are intended to improve upon animal models in terms of reproducibility and human relevance, at a lower monetary and ethical cost. Virtually all TC systems are analyzed at an endpoint, leading to widespread recognition that new methods are needed to enable sensing of specific biomolecules in real time, as they are being produced by the cells. To address this need, we incorporated photonic biosensors for inflammatory cytokines into a model TC. Human bronchial epithelial cells seeded in a microfluidic device were stimulated with lipopolysaccharide, and the cytokines secreted in response sensed in real time. Sensing analyte transport through the TC in response to disruption of tissue barrier was also demonstrated. This work demonstrates the first application of photonic sensors to a human TC device, and will enable new applications in drug development and disease modeling.

A tissue chip with integrated digital immunosensors: In situ brain endothelial barrier cytokine secretion monitoring.

Organ-on-a-chip platforms have potential to offer more cost-effective, ethical, and human-resembling models than animal models for disease study and drug discovery. Particularly, the Blood-Brain-Barrier-on-a-chip (BBB-oC) has emerged as a promising tool to investigate several neurological disorders since it promises to provide a model of the multifunctional tissue working as an important node to control pathogen entry, drug delivery and neuroinflammation. A comprehensive understanding of the multiple physiological functions of the tissue model requires biosensors detecting several tissue-secreted substances in a BBB-oC system. However, current sensor-integrated BBB-oC platforms are only available for tissue membrane integrity characterization based on permeability measurement. Protein secretory pathways are closely associated with the tissue's various diseased conditions. At present, no biosensor-integrated BBB-oC platform exists that permits in situ tissue protein secretion analysis over time, which prohibits researchers from fully understanding the time-evolving pathology of a tissue barrier. Herein, the authors present a platform named "Digital Tissue-BArrier-CytoKine-counting-on-a-chip (DigiTACK)," which integrates digital immunosensors into a tissue chip system and demonstrates on-chip multiplexed, ultrasensitive, longitudinal cytokine secretion profiling of cultured brain endothelial barrier tissues. The integrated digital sensors utilize a novel beadless microwell format to perform an ultrafast "digital fingerprinting" of the analytes while achieving a low limit of detection (LoD) around 100-500 fg/mL for mouse MCP1 (CCL2), IL-6 and KC (CXCL1). The DigiTACK platform is extensively applicable to profile temporal cytokine secretion of other barrier-related organ-on-a-chip systems and can provide new insight into the secretory dynamics of the BBB by sequentially controlled experiments.

Sourcing cells for in vitro models of human vascular barriers of inflammation.

The vascular system plays a critical role in the progression and resolution of inflammation. The contributions of the vascular endothelium to these processes, however, vary with tissue and disease state. Recently, tissue chip models have emerged as promising tools to understand human disease and for the development of personalized medicine approaches. Inclusion of a vascular component within these platforms is critical for properly evaluating most diseases, but many models to date use "generic" endothelial cells, which can preclude the identification of biomedically meaningful pathways and mechanisms. As the knowledge of vascular heterogeneity and immune cell trafficking throughout the body advances, tissue chip models should also advance to incorporate tissue-specific cells where possible. Here, we discuss the known heterogeneity of leukocyte trafficking in vascular beds of some commonly modeled tissues. We comment on the availability of different tissue-specific cell sources for endothelial cells and pericytes, with a focus on stem cell sources for the full realization of personalized medicine. We discuss sources available for the immune cells needed to model inflammatory processes and the findings of tissue chip models that have used the cells to studying transmigration.

The Modular µSiM Reconfigured: Integration of Microfluidic Capabilities to Study In Vitro Barrier Tissue Models under Flow.

Microfluidic tissue barrier models have emerged to address the lack of physiological fluid flow in conventional "open-well" Transwell-like devices. However, microfluidic techniques have not achieved widespread usage in bioscience laboratories because they are not fully compatible with traditional experimental protocols. To advance barrier tissue research, there is a need for a platform that combines the key advantages of both conventional open-well and microfluidic systems. Here, a plug-and-play flow module is developed to introduce on-demand microfluidic flow capabilities to an open-well device that features a nanoporous membrane and live-cell imaging capabilities. The magnetic latching assembly of this design enables bi-directional reconfiguration and allows users to conduct an experiment in an open-well format with established protocols and then add or remove microfluidic capabilities as desired. This work also provides an experimentally-validated flow model to select flow conditions based on the experimental needs. As a proof-of-concept, flow-induced alignment of endothelial cells and the expression of shear-sensitive gene targets are demonstrated, and the different phases of neutrophil transmigration across a chemically stimulated endothelial monolayer under flow conditions are visualized. With these experimental capabilities, it is anticipated that both engineering and bioscience laboratories will adopt this reconfigurable design due to the compatibility with standard open-well protocols.

The Modular µSiM: A Mass Produced, Rapidly Assembled, and Reconfigurable Platform for the Study of Barrier Tissue Models In Vitro.

Advanced in vitro tissue chip models can reduce and replace animal experimentation and may eventually support "on-chip" clinical trials. To realize this potential, however, tissue chip platforms must be both mass-produced and reconfigurable to allow for customized design. To address these unmet needs, an extension of the µSiM (microdevice featuring a silicon-nitride membrane) platform is introduced. The modular µSiM (m-µSiM) uses mass-produced components to enable rapid assembly and reconfiguration by laboratories without knowledge of microfabrication. The utility of the m-µSiM is demonstrated by establishing an hiPSC-derived blood-brain barrier (BBB) in bioengineering and nonengineering, brain barriers focused laboratories. In situ and sampling-based assays of small molecule diffusion are developed and validated as a measure of barrier function. BBB properties show excellent interlaboratory agreement and match expectations from literature, validating the m-µSiM as a platform for barrier models and demonstrating successful dissemination of components and protocols. The ability to quickly reconfigure the m-µSiM for coculture and immune cell transmigration studies through addition of accessories and/or quick exchange of components is then demonstrated. Because the development of modified components and accessories is easily achieved, custom designs of the m-µSiM shall be accessible to any laboratory desiring a barrier-style tissue chip platform.

Molecular mechanisms underlying the heterogeneous barrier responses of two primary endothelial cell types to sphingosine-1-phosphate.

Sphingosine-1-phosphate (S1P) signals to enhance or destabilize the vascular endothelial barrier depending on the receptor engaged. Here, we investigated the differential barrier effects of S1P on two influential primary endothelial cell (EC) types, human umbilical vein endothelial cells (HUVECs) and human pulmonary microvascular endothelial cells (HPMECs). S1PR1 (barrier protective) and S1PR3 (barrier disruptive) surface and gene expression were quantified by flow cytometry and immunofluorescence, and RT-qPCR, respectively. Functional evaluation of EC monolayer permeability in response to S1P was quantified with transendothelial electrical resistance (TEER) and small molecule permeability. S1P significantly enhanced HUVEC barrier function, while promoting HPMEC barrier breakdown. Immunofluorescence and flow cytometry analysis showed select, S1PR3-high HPMECs, suggesting susceptibility to barrier destabilization following S1P exposure. Reevaluation of HPMEC barrier following S1P exposure under inflamed conditions demonstrated synergistic barrier disruptive effects of pro-inflammatory cytokine and S1P. The role of the Rho-ROCK signaling pathway under these conditions was confirmed through ROCK1/2 inhibition (Y-27632). Thus, the heterogeneous responses of ECs to S1P signaling are mediated through Rho-ROCK signaling, and potentially driven by differences in the surface expression of S1PR3.

Human Organ-on-a-Chip Microphysiological Systems to Model Musculoskeletal Pathologies and Accelerate Therapeutic Discovery.

Human Microphysiological Systems (hMPS), otherwise known as organ- and tissue-on-a-chip models, are an emerging technology with the potential to replace in vivo animal studies with in vitro models that emulate human physiology at basic levels. hMPS platforms are designed to overcome limitations of two-dimensional (2D) cell culture systems by mimicking 3D tissue organization and microenvironmental cues that are physiologically and clinically relevant. Unlike animal studies, hMPS models can be configured for high content or high throughput screening in preclinical drug development. Applications in modeling acute and chronic injuries in the musculoskeletal system are slowly developing. However, the complexity and load bearing nature of musculoskeletal tissues and joints present unique challenges related to our limited understanding of disease mechanisms and the lack of consensus biomarkers to guide biological therapy development. With emphasis on examples of modeling musculoskeletal tissues, joints on chips, and organoids, this review highlights current trends of microphysiological systems technology. The review surveys state-of-the-art design and fabrication considerations inspired by lessons from bioreactors and biological variables emphasizing the role of induced pluripotent stem cells and genetic engineering in creating isogenic, patient-specific multicellular hMPS. The major challenges in modeling musculoskeletal tissues using hMPS chips are identified, including incorporating biological barriers, simulating joint compartments and heterogenous tissue interfaces, simulating immune interactions and inflammatory factors, simulating effects of in vivo loading, recording nociceptors responses as surrogates for pain outcomes, modeling the dynamic injury and healing responses by monitoring secreted proteins in real time, and creating arrayed formats for robotic high throughput screens. Overcoming these barriers will revolutionize musculoskeletal research by enabling physiologically relevant, predictive models of human tissues and joint diseases to accelerate and de-risk therapeutic discovery and translation to the clinic.

Real time imaging of single extracellular vesicle pH regulation in a microfluidic cross-flow filtration platform.

Extracellular vesicles (EVs) are cell-derived membranous structures carrying transmembrane proteins and luminal cargo. Their complex cargo requires pH stability in EVs while traversing diverse body fluids. We used a filtration-based platform to capture and stabilize EVs based on their size and studied their pH regulation at the single EV level. Dead-end filtration facilitated EV capture in the pores of an ultrathin (100 nm thick) and nanoporous silicon nitride (NPN) membrane within a custom microfluidic device. Immobilized EVs were rapidly exposed to test solution changes driven across the backside of the membrane using tangential flow without exposing the EVs to fluid shear forces. The epithelial sodium-hydrogen exchanger, NHE1, is a ubiquitous plasma membrane protein tasked with the maintenance of cytoplasmic pH at neutrality. We show that NHE1 identified on the membrane of EVs is functional in the maintenance of pH neutrality within single vesicles. This is the first mechanistic description of EV function on the single vesicle level.

Rapid and specific detection of intact viral particles using functionalized microslit silicon membranes as a fouling-based sensor.

The COVID-19 pandemic demonstrated the public health benefits of reliable and accessible point-of-care (POC) diagnostic tests for viral infections. Despite the rapid development of gold-standard reverse transcription polymerase chain reaction (RT-PCR) assays for SARS-CoV-2 only weeks into the pandemic, global demand created logistical challenges that delayed access to testing for months and helped fuel the spread of COVID-19. Additionally, the extreme sensitivity of RT-PCR had a costly downside as the tests could not differentiate between patients with active infection and those who were no longer infectious but still shedding viral genomes. To address these issues for the future, we propose a novel membrane-based sensor that only detects intact virions. The sensor combines affinity and size based detection on a membrane-based sensor and does not require external power to operate or read. Specifically, the presence of intact virions, but not viral debris, fouls the membrane and triggers a macroscopically visible hydraulic switch after injection of a 40 µL sample with a pipette. The device, which we call the µSiM-DX (microfluidic device featuring a silicon membrane for diagnostics), features a biotin-coated microslit membrane with pores ∼2-3× larger than the intact virus. Streptavidin-conjugated antibody recognizing viral surface proteins are incubated with the sample for ∼1 hour prior to injection into the device, and positive/negative results are obtained within ten seconds of sample injection. Proof-of-principle tests have been performed using preparations of vaccinia virus. After optimizing slit pore sizes and porous membrane area, the fouling-based sensor exhibits 100% specificity and 97% sensitivity for vaccinia virus (n = 62). Moreover, the dynamic range of the sensor extends at least from 105.9 virions per mL to 1010.4 virions per mL covering the range of mean viral loads in symptomatic COVID-19 patients (105.6-107 RNA copies per mL). Forthcoming work will test the ability of our sensor to perform similarly in biological fluids and with SARS-CoV-2, to fully test the potential of a membrane fouling-based sensor to serve as a PCR-free alternative for POC containment efforts in the spread of infectious disease.

Staphylococcus aureus Cell Wall Biosynthesis Modulates Bone Invasion and Osteomyelitis Pathogenesis.

Staphylococcus aureus invasion of the osteocyte lacuno-canalicular network (OLCN) is a novel mechanism of bacterial persistence and immune evasion in chronic osteomyelitis. Previous work highlighted S. aureus cell wall transpeptidase, penicillin binding protein 4 (PBP4), and surface adhesin, S. aureus surface protein C (SasC), as critical factors for bacterial deformation and propagation through nanopores in vitro, representative of the confined canaliculi in vivo. Given these findings, we hypothesized that cell wall synthesis machinery and surface adhesins enable durotaxis- and haptotaxis-guided invasion of the OLCN, respectively. Here, we investigated select S. aureus cell wall synthesis mutants (Δpbp3, Δatl, and ΔmreC) and surface adhesin mutants (ΔclfA and ΔsasC) for nanopore propagation in vitro and osteomyelitis pathogenesis in vivo. In vitro evaluation in the microfluidic silicon membrane-canalicular array (µSiM-CA) showed pbp3, atl, clfA, and sasC deletion reduced nanopore propagation. Using a murine model for implant-associated osteomyelitis, S. aureus cell wall synthesis proteins were found to be key modulators of S. aureus osteomyelitis pathogenesis, while surface adhesins had minimal effects. Specifically, deletion of pbp3 and atl decreased septic implant loosening and S. aureus abscess formation in the medullary cavity, while deletion of surface adhesins showed no significant differences. Further, peri-implant osteolysis, osteoclast activity, and receptor activator of nuclear factor kappa-B ligand (RANKL) production were decreased following pbp3 deletion. Most notably, transmission electron microscopy (TEM) imaging of infected bone showed that pbp3 was the only gene herein associated with decreased submicron invasion of canaliculi in vivo. Together, these results demonstrate that S. aureus cell wall synthesis enzymes are critical for OLCN invasion and osteomyelitis pathogenesis in vivo.

Brain endothelial tricellular junctions as novel sites for T cell diapedesis across the blood-brain barrier.

The migration of activated T cells across the blood-brain barrier (BBB) is a critical step in central nervous system (CNS) immune surveillance and inflammation. Whereas T cell diapedesis across the intact BBB seems to occur preferentially through the BBB cellular junctions, impaired BBB integrity during neuroinflammation is accompanied by increased transcellular T cell diapedesis. The underlying mechanisms directing T cells to paracellular versus transcellular sites of diapedesis across the BBB remain to be explored. By combining in vitro live-cell imaging of T cell migration across primary mouse brain microvascular endothelial cells (pMBMECs) under physiological flow with serial block-face scanning electron microscopy (SBF-SEM), we have identified BBB tricellular junctions as novel sites for T cell diapedesis across the BBB. Downregulated expression of tricellular junctional proteins or protein-based targeting of their interactions in pMBMEC monolayers correlated with enhanced transcellular T cell diapedesis, and abluminal presence of chemokines increased T cell diapedesis through tricellular junctions. Our observations assign an entirely novel role to BBB tricellular junctions in regulating T cell entry into the CNS. This article has an associated First Person interview with the first author of the paper.

Endothelial cell apicobasal polarity coordinates distinct responses to luminally versus abluminally delivered TNF-α in a microvascular mimetic.

Endothelial cells (ECs) are an active component of the immune system and interact directly with inflammatory cytokines. While ECs are known to be polarized cells, the potential role of apicobasal polarity in response to inflammatory mediators has been scarcely studied. Acute inflammation is vital in maintaining healthy tissue in response to infection; however, chronic inflammation can lead to the production of systemic inflammatory cytokines and deregulated leukocyte trafficking, even in the absence of a local infection. Elevated levels of cytokines in circulation underlie the pathogenesis of sepsis, the leading cause of intensive care death. Because ECs constitute a key barrier between circulation (luminal interface) and tissue (abluminal interface), we hypothesize that ECs respond differentially to inflammatory challenge originating in the tissue versus circulation as in local and systemic inflammation, respectively. To begin this investigation, we stimulated ECs abluminally and luminally with the inflammatory cytokine tumor necrosis factor alpha (TNF-α) to mimic a key feature of local and systemic inflammation, respectively, in a microvascular mimetic (µSiM-MVM). Polarized IL-8 secretion and polymorphonuclear neutrophil (PMN) transmigration were quantified to characterize the EC response to luminal versus abluminal TNF-α. We observed that ECs uniformly secrete IL-8 in response to abluminal TNF-α and is followed by PMN transmigration. The response to abluminal treatment was coupled with the formation of ICAM-1-rich membrane ruffles on the apical surface of ECs. In contrast, luminally stimulated ECs secreted five times more IL-8 into the luminal compartment than the abluminal compartment and sequestered PMNs on the apical EC surface. Our results identify clear differences in the response of ECs to TNF-α originating from the abluminal versus luminal side of a monolayer for the first time and may provide novel insight into future inflammatory disease intervention strategies.

Identification of Penicillin Binding Protein 4 (PBP4) as a critical factor for Staphylococcus aureus bone invasion during osteomyelitis in mice.

Staphylococcus aureus infection of bone is challenging to treat because it colonizes the osteocyte lacuno-canalicular network (OLCN) of cortical bone. To elucidate factors involved in OLCN invasion and identify novel drug targets, we completed a hypothesis-driven screen of 24 S. aureus transposon insertion mutant strains for their ability to propagate through 0.5 µm-sized pores in the Microfluidic Silicon Membrane Canalicular Arrays (µSiM-CA), developed to model S. aureus invasion of the OLCN. This screen identified the uncanonical S. aureus transpeptidase, penicillin binding protein 4 (PBP4), as a necessary gene for S. aureus deformation and propagation through nanopores. In vivo studies revealed that Δpbp4 infected tibiae treated with vancomycin showed a significant 12-fold reduction in bacterial load compared to WT infected tibiae treated with vancomycin (p<0.05). Additionally, Δpbp4 infected tibiae displayed a remarkable decrease in pathogenic bone-loss at the implant site with and without vancomycin therapy. Most importantly, Δpbp4 S. aureus failed to invade and colonize the OLCN despite high bacterial loads on the implant and in adjacent tissues. Together, these results demonstrate that PBP4 is required for S. aureus colonization of the OLCN and suggest that inhibitors may be synergistic with standard of care antibiotics ineffective against bacteria within the OLCN.

Free Standing, Large-Area Silicon Nitride Membranes for High Toxin Clearance in Blood Surrogate for Small-Format Hemodialysis.

Developing highly-efficient membranes for toxin clearance in small-format hemodialysis presents a fabrication challenge. The miniaturization of fluidics and controls has been the focus of current work on hemodialysis (HD) devices. This approach has not addressed the membrane efficiency needed for toxin clearance in small-format hemodialysis devices. Dr. Willem Kolff built the first dialyzer in 1943 and many changes have been made to HD technology since then. However, conventional HD still uses large instruments with bulky dialysis cartridges made of ~2 m2 of 10 micron thick, tortuous-path membrane material. Portable, wearable, and implantable HD systems may improve clinical outcomes for patients with end-stage renal disease by increasing the frequency of dialysis. The ability of ultrathin silicon-based sheet membranes to clear toxins is tested along with an analytical model predicting long-term multi-pass experiments from single-pass clearance experiments. Advanced fabrication methods are introduced that produce a new type of nanoporous silicon nitride sheet membrane that features the pore sizes needed for middle-weight toxin removal. Benchtop clearance results with sheet membranes (~3 cm2) match a theoretical model and indicate that sheet membranes can reduce (by orders of magnitude) the amount of membrane material required for hemodialysis. This provides the performance needed for small-format hemodialysis.