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.

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.

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.

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.

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.

Development of isoporous microslit silicon nitride membranes for sterile filtration applications.

The widely used 0.2/0.22 µm polymer sterile filters were developed for small molecule and protein sterile filtration but are not well-suited for the production of large nonprotein biological therapeutics, resulting in significant yield loss and production cost increases. Here, we report on the development of membranes with isoporous sub-0.2 µm rectangular prism pores using silicon micromachining to produce microslit silicon nitride (MSN) membranes. The very high porosity (~33%) and ultrathin (200 nm) nature of the 0.2 µm MSN membranes results in a dramatically different structure than the traditional 0.2/0.22 µm polymer sterile filter, which yielded comparable performance properties (including gas and hydraulic permeance, maximum differential pressure tolerance, nanoparticle sieving/fouling behavior). The results from bacteria retention tests, conducted according to the guidance of regulatory agencies, demonstrated that the 0.2 µm MSN membranes can be effectively used as sterile filters. It is anticipated that the results and technologies presented in this study will find future utility in the production of non-protein biological therapeutics and in other biological and biomedical applications.

Ultrathin Dual-Scale Nano- and Microporous Membranes for Vascular Transmigration Models.

Selective cellular transmigration across the microvascular endothelium regulates innate and adaptive immune responses, stem cell localization, and cancer cell metastasis. Integration of traditional microporous membranes into microfluidic vascular models permits the rapid assay of transmigration events but suffers from poor reproduction of the cell permeable basement membrane. Current microporous membranes in these systems have large nonporous regions between micropores that inhibit cell communication and nutrient exchange on the basolateral surface reducing their physiological relevance. Here, the use of 100 nm thick continuously nanoporous silicon nitride membranes as a base substrate for lithographic fabrication of 3 µm pores is presented, resulting in a highly porous (≈30%), dual-scale nano- and microporous membrane for use in an improved vascular transmigration model. Ultrathin membranes are patterned using a precision laser writer for cost-effective, rapid micropore design iterations. The optically transparent dual-scale membranes enable complete observation of leukocyte egress across a variety of pore densities. A maximal density of ≈14 micropores per cell is discovered beyond which cell-substrate interactions are compromised giving rise to endothelial cell losses under flow. Addition of a subluminal extracellular matrix rescues cell adhesion, allowing for the creation of shear-primed endothelial barrier models on nearly 30% continuously porous substrates.

An in vitro platform for elucidating the molecular genetics of S. aureus invasion of the osteocyte lacuno-canalicular network during chronic osteomyelitis.

Staphylococcus aureus osteomyelitis is a devasting disease that often leads to amputation. Recent findings have shown that S. aureus is capable of invading the osteocyte lacuno-canalicular network (OLCN) of cortical bone during chronic osteomyelitis. Normally a 1 µm non-motile cocci, S. aureus deforms smaller than 0.5 µm in the sub-micron channels of the OLCN. Here we present the µSiM-CA (Microfluidic - Silicon Membrane - Canalicular Array) as an in vitro screening platform for the genetic mechanisms of S. aureus invasion. The µSiM-CA platform features an ultrathin silicon membrane with defined pores that mimic the openings of canaliculi. While we anticipated that S. aureus lacking the accessory gene regulator (agr) quorum-sensing system would not be capable of invading the OLCN, we found no differences in propagation compared to wild type in the µSiM-CA. However the µSiM-CA proved predictive as we also found that the agr mutant strain invaded the OLCN of murine tibiae.

DNA Translocations through Nanopores under Nanoscale Preconfinement.

To reduce unwanted variation in the passage speed of DNA through solid-state nanopores, we demonstrate nanoscale preconfinement of translocating molecules using an ultrathin nanoporous silicon nitride membrane separated from a single sensing nanopore by a nanoscale cavity. We present comprehensive experimental and simulation results demonstrating that the presence of an integrated nanofilter within nanoscale distances of the sensing pore eliminates the dependence of molecular passage time distributions on pore size, revealing a global minimum in the coefficient of variation of the passage time. These results provide experimental verification that the inter- and intramolecular passage time variation depends on the conformational entropy of each molecule prior to translocation. Furthermore, we show that the observed consistently narrower passage time distributions enables a more reliable DNA length separation independent of pore size and stability. We also demonstrate that the composite nanofilter/nanopore devices can be configured to suppress the frequency of folded translocations, ensuring single-file passage of captured DNA molecules. By greatly increasing the rate at which usable data can be collected, these unique attributes will offer significant practical advantages to many solid-state nanopore-based sensing schemes, including sequencing, genomic mapping, and barcoded target detection.

Finite element modeling to analyze TEER values across silicon nanomembranes.

Silicon nanomembranes are ultrathin, highly permeable, optically transparent and biocompatible substrates for the construction of barrier tissue models. Trans-epithelial/endothelial electrical resistance (TEER) is often used as a non-invasive, sensitive and quantitative technique to assess barrier function. The current study characterizes the electrical behavior of devices featuring silicon nanomembranes to facilitate their application in TEER studies. In conventional practice with commercial systems, raw resistance values are multiplied by the area of the membrane supporting cell growth to normalize TEER measurements. We demonstrate that under most circumstances, this multiplication does not 'normalize' TEER values as is assumed, and that the assumption is worse if applied to nanomembrane chips with a limited active area. To compare the TEER values from nanomembrane devices to those obtained from conventional polymer track-etched (TE) membranes, we develop finite element models (FEM) of the electrical behavior of the two membrane systems. Using FEM and parallel cell-culture experiments on both types of membranes, we successfully model the evolution of resistance values during the growth of endothelial monolayers. Further, by exploring the relationship between the models we develop a 'correction' function, which when applied to nanomembrane TEER, maps to experiments on conventional TE membranes. In summary, our work advances the the utility of silicon nanomembranes as substrates for barrier tissue models by developing an interpretation of TEER values compatible with conventional systems.

Ultrathin nanoporous membranes for insulator-based dielectrophoresis.

Insulator-based dielectrophoresis (iDEP) is a simple, scalable mechanism that can be used for directly manipulating particle trajectories in pore-based filtration and separation processes. However, iDEP manipulation of nanoparticles presents unique challenges as the dielectrophoretic force [Formula: see text] exerted on the nanoparticles can easily be overshadowed by opposing kinetic forces. In this study, a molecularly thin, SiN-based nanoporous membrane (NPN) is explored as a breakthrough technology that enhances [Formula: see text] By numerically assessing the gradient of the electric field square [Formula: see text]-a common measure for [Formula: see text] magnitude-it was found that the unique geometrical features of NPN (pore tapering, sharp pore corner and ultrathin thickness) act in favor of intensifying the overall [Formula: see text] A comparative study indicated that [Formula: see text] generated in NPN are four orders of magnitude larger than track-etched polycarbonate membranes with comparable pore size. The stronger [Formula: see text] suggests that iDEP can be conducted under lower voltage bias with NPN: reducing joule heating concerns and enabling solutions to have higher ionic strength. Enabling higher ionic strength solutions may also extend the opportunities of iDEP applications under physiologically relevant conditions. This study also highlights the effects of [Formula: see text] induced by the ion accumulation along charged surfaces (electric-double layer (EDL)). EDL-based [Formula: see text] exists along the entire charged surface, including locations where geometry-based iDEP is negligible. The high surface-to-volume ratio of NPN offers a unique platform for exploiting such EDL-based DEP systems. The EDL-based [Formula: see text] was also found to offset the geometry-based [Formula: see text] but this effect was easily circumvented by reducing the EDL thickness (e.g. increasing the ionic strength from 0.1 to 100 mM). The results from this study imply the potential application of iDEP as a direct, in-operando antifouling mechanism for ultrafiltration technology, and also as an active tuning mechanism to control the cut-off size limit for continuous selectivity of nanomembrane-based separations.

High-performance, low-voltage electroosmotic pumps with molecularly thin silicon nanomembranes.

We have developed electroosmotic pumps (EOPs) fabricated from 15-nm-thick porous nanocrystalline silicon (pnc-Si) membranes. Ultrathin pnc-Si membranes enable high electroosmotic flow per unit voltage. We demonstrate that electroosmosis theory compares well with the observed pnc-Si flow rates. We attribute the high flow rates to high electrical fields present across the 15-nm span of the membrane. Surface modifications, such as plasma oxidation or silanization, can influence the electroosmotic flow rates through pnc-Si membranes by alteration of the zeta potential of the material. A prototype EOP that uses pnc-Si membranes and Ag/AgCl electrodes was shown to pump microliter per minute-range flow through a 0.5-mm-diameter capillary tubing with as low as 250 mV of applied voltage. This silicon-based platform enables straightforward integration of low-voltage, on-chip EOPs into portable microfluidic devices with low back pressures.

The electric field strength in orifice-like nanopores of ultrathin membranes.

Here we show that the electric field inside an ultrathin membrane is weaker than conventional theory would predict, and that the reduced field is predictive of measured electroosmotic flow rates. Our theoretical analysis shows that the electric field inside a charged nanopore is affected by end effects and dependent on the Dukhin number Du when the pore length-to-diameter aspect ratio λ is less than 80 for Du â‰ª 1 or 300 for Du â‰« 1. The electric field follows an unconventional scaling law; it no longer scales uniformly with the thickness of membrane, but with the local value of λ for each nanopore.

Influence of silicon dioxide capping layers on pore characteristics in nanocrystalline silicon membranes.

Porous nanocrystalline silicon (pnc-Si) membranes are a new class of membrane material with promising applications in biological separations. Pores are formed in a silicon film sandwiched between nm thick silicon dioxide layers during rapid thermal annealing. Controlling pore size is critical in the size-dependent separation applications. In this work, we systematically studied the influence of the silicon dioxide capping layers on pnc-Si membranes. Even a single nm thick top oxide layer is enough to switch from agglomeration to pore formation after annealing. Both the pore size and porosity increase with the thickness of the top oxide, but quickly reach a plateau after 10 nm of oxide. The bottom oxide layer acts as a barrier layer to prevent the a-Si film from undergoing homo-epitaxial growth during annealing. Both the pore size and porosity decrease as the thickness of the bottom oxide layer increases to 100 nm. The decrease of the pore size and porosity is correlated with the increased roughness of the bottom oxide layer, which hinders nanocrystal nucleation and nanopore formation.

Opposing roles for RhoH GTPase during T-cell migration and activation.

T cells spend the majority of their time perusing lymphoid organs in search of cognate antigen presented by antigen presenting cells (APCs) and then quickly recirculate through the bloodstream to another lymph node. Therefore, regulation of a T-cell response is dependent upon the ability of cells to arrive in the correct location following chemokine gradients ("go" signal) as well as to receive appropriate T-cell receptor (TCR) activation signals upon cognate antigen recognition ("stop" signal). However, the mechanisms by which T cells regulate these go and stop signals remain unclear. We found that overexpression of the hematopoietic-specific RhoH protein in the presence of chemokine signals resulted in decreased Rap1-GTP and LFA-1 adhesiveness to ICAM-1, thus impairing T-cell chemotaxis; while in the presence of TCR signals, there were enhanced and sustained Rap1-GTP and LFA-1 activation as well as prolonged T:APC conjugates. RT-PCR analyses of activated CD4(+) T cells and live images of T-cell migration and immunological synapse (IS) formation revealed that functions of RhoH took place primarily at the levels of transcription and intracellular distribution. Thus, we conclude that RhoH expression provides a key molecular determinant that allows T cells to switch between sensing chemokine-mediated go signals and TCR-dependent stop signals.

Highly porous silicon membranes fabricated from silicon nitride/silicon stacks.

Nanopore formation in silicon films has previously been demonstrated using rapid thermal crystallization of ultrathin (15 nm) amorphous Si films sandwiched between nm-thick SiO2 layers. In this work, the silicon dioxide barrier layers are replaced with silicon nitride, resulting in nanoporous silicon films with unprecedented pore density and novel morphology. Four different thin film stack systems including silicon nitride/silicon/silicon nitride (NSN), silicon dioxide/silicon/silicon nitride (OSN), silicon nitride/silicon/silicon dioxide (NSO), and silicon dioxide/silicon/silicon dioxide (OSO) are tested under different annealing temperatures. Generally the pore size, pore density, and porosity positively correlate with the annealing temperature for all four systems. The NSN system yields substantially higher porosity and pore density than the OSO system, with the OSN and NSO stack characteristics fallings between these extremes. The higher porosity of the Si membrane in the NSN stack is primarily due to the pore formation enhancement in the Si film. It is hypothesized that this could result from the interfacial energy difference between the silicon/silicon nitride and silicon/silicon dioxide, which influences the Si crystallization process.

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.

Ingestion of polyethylene terephthalate microplastic water contaminants by Xenopus laevis tadpoles negatively affects their resistance to ranavirus infection and antiviral immunity.

Small plastic debris (0.1 µm-5 mm) or microplastics (MPs) have become major pollutants of aquatic ecosystems worldwide and studies suggest that MPs exposure can pose serious threats to human and wildlife health. However, to date the potential biological impacts of MPs accumulating in low amount in tissues during early life remains unclear. Here, for a more realistic assessment, we have used environmentally representative, mildly weathered, polyethylene terephthalate microplastics (PET MPs), cryomilled (1-100 µm) and fluorescently labelled. We leveraged the amphibian Xenopus laevis tadpoles as an animal model to define the biodistribution of PET MPs and determine whether exposure to PET MPs induce perturbations of antiviral immunity. Exposure to PET MPs for 1-14 days resulted in detectable PET MPs biodistribution in intestine, gills, liver, and kidney as determined by fluorescence microscopy on whole mount tissues. PET MPs accumulation rate in tissues was further evaluated via a novel in situ enzymatic digestion and subsequent filtration using silicon nanomembranes, which shows that PET MPs rapidly accumulate in tadpole intestine, liver and kidneys and persist over a week. Longer exposure (1 month) of tadpoles to relatively low concentration of PET MPs (25 µg/ml) significantly increased susceptibility to viral infection and altered innate antiviral immunity without inducing overt inflammation. This study provides evidence that exposure to MPs negatively impact immune defenses of aquatic vertebrates.

Rapid Assessment of Biomarkers on Single Extracellular Vesicles Using 'Catch and Display' on Ultrathin Nanoporous Silicon Nitride Membranes.

Extracellular vesicles (EVs) are particles secreted by all cells that carry bioactive cargo and facilitate intercellular communication with roles in normal physiology and disease pathogenesis. EVs have tremendous diagnostic and therapeutic potential and accordingly, the EV field has grown exponentially in recent years. Bulk assays lack the sensitivity to detect rare EV subsets relevant to disease, and while single EV analysis techniques remedy this, they are undermined by complicated detection schemes often coupled with prohibitive instrumentation. To address these issues, we propose a microfluidic technique for EV characterization called 'catch and display for liquid biopsy (CAD-LB)'. CAD-LB rapidly captures fluorescently labeled EVs in the similarly-sized pores of an ultrathin silicon nitride membrane. Minimally processed sample is introduced via pipette injection into a simple microfluidic device which is directly imaged using fluorescence microscopy for a rapid assessment of EV number and biomarker colocalization. In this work, nanoparticles were first used to define the accuracy and dynamic range for counting and colocalization by CAD-LB. Following this, the same assessments were made for purified EVs and for unpurified EVs in plasma. Biomarker detection was validated using CD9 in which Western blot analysis confirmed that CAD-LB faithfully recapitulated differing expression levels among samples. We further verified that CAD-LB captured the known increase in EV-associated ICAM-1 following the cytokine stimulation of endothelial cells. Finally, to demonstrate CAD-LB's clinical potential, we show that EV biomarkers indicative of immunotherapy responsiveness are successfully detected in the plasma of bladder cancer patients undergoing immune checkpoint blockade.

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.