Lab on a Chip

Embryo adhesion represents the first step of implantation, yet understanding this process has been hindered by the lack of human in vitro platforms that replicate endometrial physiology. Here, we present a dual-channel microfluidic platform containing organoid-derived endometrial epithelium and primary stromal cells. Our model recapitulates important endometrial hallmarks including epithelial polarization, stromal decidualization, extracellular vesicle release, and hormone-induced receptivity. We tested its function by introducing mouse and human blastocysts and showed that embryos displayed features of initial adhesion. These included establishment of embryo-epithelial contacts initiated via the polar trophectoderm, inner cell mass repositioning, and lineage segregation. Moreover, human embryos secreted {beta}hCG indicating a functional trophoblast. Thus, this work provides a platform to study key features of embryo adhesion and endometrial receptivity, and disorders affecting embryo-endometrium interactions. TeaserEndometrium-on-a-chip shows detailed human embryo adhesion dynamics.

Extracellular vesicles (EVs) carry molecular cargo that can reflect the real-time state of parental cells, yet most in vitro EV analyses rely on bulk approaches and therefore average over pronounced heterogeneity in both cell and EV populations. Here, we present a semi-open microfluidic platform that enables multi-marker profiling of EVs released from single-cell-derived clones, allowing EV signatures to be linked to clonal progeny originating from a single parental cell. The platform integrates aligned cell and EV arrays containing 17,305 wells, assembled with a 3D-printed housing to capture released EVs in one-to-one matched wells. Captured EVs are immunolabeled for canonical tetraspanin markers (CD9, CD63, CD81) and EpCAM, imaged by high-resolution fluorescence microscopy, and quantified using an automated image-analysis pipeline. Applying the platform to single-cell-derived PC3 clones revealed substantial heterogeneity in EV marker co-expression, with hierarchical clustering identifying four distinct tetraspanin co-expression profiles. The fraction of EpCAM-positive EVs increased with PC3 cell proliferation, as assessed by endpoint cell number, whereas free (non-EV-associated) EpCAM showed no correlation. This platform enables near single-EV-level, multi-marker profiling from single-cell lineages and provides a practical approach to simultaneously dissect both cellular and EV heterogeneity.

Droplet-based microfluidics enable researchers to observe phenotypic heterogeneity within complex biological mixtures through parallel encapsulation of individual samples followed by imaging. Observing or quantifying dynamic heterogeneity remains challenging due to complexities associated with trapping and tracking many individual droplets. Current approaches for time-lapse imaging require specialized devices with droplet traps that limit accessibility and throughput. Here, using readily available materials and software, we demonstrate a simple method for stabilizing and monitoring many, individual droplets for up to 12 hours. We leveraged our method to track bacterial growth within droplets in a high-throughput manner. Our method allows tracking the changes and variation in growth rate within and across droplets, revealing heterogeneity in growth patterns hidden in batch assays. Improving the affordability and throughput of time-dependent phenotyping assays helps to advance biological discovery and biotechnology innovation.

Data-driven materials development requires large, well-characterized libraries of precisely defined formulations. While microfluidic platforms excel at generating highly controlled materials, their throughput is often limited by the challenge of efficiently interfacing device outputs with standard well plates. This bottleneck frequently necessitates manual transfer or non-microfluidic workflows, constraining both throughput and reproducibility. Here, we present LMNOP-bot (Libraries of Micro- and Nano-materials, OPen-source bot), an open-source robotic platform for the automated generation and collection of micro- and nanomaterial libraries from serial microfluidic outputs. Using synchronized, pressure-driven flow, LMNOP-bot enables continuous formulation and direct deposition into standard well plates. The system is low-cost (<$700, excluding pressure regulators), constructed from readily available or easily fabricated components, and designed for broad accessibility. LMNOP-bot collects [&ge;]30 {micro}L per formulation at a rate of one sample every four seconds, representing an approximately 50x increase in throughput over existing serial microfluidic workflows, and operates robustly for over 10,000 runs without maintenance. We demonstrate compatibility with both PDMS/glass and commercial polycarbonate devices, with seamless interfacing to 96- and 384-well plates. Repeated sampling confirms high precision and reproducibility. By removing a key bottleneck in microfluidic library generation, LMNOP-bot enables rapid, scalable, and accessible exploration of material design spaces.

Microphysiological systems (MPS) are essential for modeling tissue barriers, yet integrating electrical readouts often requires permanently sealed microfluidic architectures that limit access to open-well (direct-access) workflows used in bioscience laboratories. To resolve this issue, we present a modular approach in which functional components are added and removed from a standard MPS core using a magnetic interface. This design preserves compatibility with established open-well protocols for seeding and downstream analysis, while microfluidic perfusion or electrical sensing capabilities are added only when needed. We demonstrate this approach with an impedance-sensing module that enables continuous impedance measurements to assess barrier function. By fitting spectra to an equivalent circuit model, we quantify junctional and non-junctional electrical contributions to barrier integrity over time, alongside conventional single-frequency TEER, and complementary permeability and imaging readouts. We apply this platform across three representative use cases, including LPS-induced disruption, shear stress-mediated strengthening, and compatibility with barrier models formed above a 3D hydrogel matrix.

Co-culturing cells with mismatched densities, where one cell type adheres to surfaces while the other floats, represents a fundamental challenge in cell biology. This is particularly evident in studying macrophage-adipocyte interactions, where macrophages must engage and clear lipid-rich apoptotic adipocytes, a process critical to understanding chronic inflammation in obesity and metabolic disease. The density disparity between macrophages, which sink and adhere to culture surfaces, and adipocytes, which float due to their lipid content, has prevented conventional co-culture approaches from achieving sustained cell-cell contact. To address this challenge, we developed a microfluidic system that confines adipocytes and lipid droplets in close proximity to macrophages. This platform features recessed micro-traps within the upper surface of a microfluidic chamber that trap buoyant objects while allowing media exchange and delivery of reagents for live-cell and immunofluorescence imaging. Time lapse imaging revealed that the dynamic process of macrophages-dead corpse interactions, showing that individual macrophages cannot engulf entire corpses but instead mechanically deform them. Furthermore, the platform successfully recapitulates the formation of Crown-Like Structures (CLS), clusters of macrophages surrounding dead adipocytes that are hallmarks of adipose tissue inflammation. Long-term culture revealed that CLS effectively clear lipids compared to partial macrophage engagement, providing mechanistic insights that were previously unattainable with standard histological approaches. Beyond the macrophage-lipid interaction, this platform has potential for studying interactions between adherent cells and buoyant targets, such as microplastics, opening new avenues for research where density mismatch poses a major barrier.

Polymeric 3D printing of microfluidic devices for biosensing is an appealing fabrication alternative for rapid manufacturing of biosensing devices with complex geometry in a streamlined, repeatable and cost-effective manner without the need for expensive instrumentation such as those employed in photochemical etching and soft lithography. Hybrid 3D printed paper-based microfluidics is an emerging area which harnesses the unique properties of both, merging the construction of microfluidic structures and the inherent capillary-driven flow within paper substrates. In this work, we have fabricated hydrophobic barriers by 3D printing a single layer of machinable wax, thermoplastic polyurethane, polylactic acid and polypropylene directly on chromatography paper to create open microchannels and determine the most suitable material. Characterization of each open microchannel using the four materials revealed polypropylene as the most reliable material with high hydrophobic barrier integrity and resolution. Polypropylene achieved functional microchannels with a resolution of 621 {+/-} 33{micro}m, hydrophobic barrier integrity of (93.75 {+/-} 9.16%), wicking speed of 0.38mm/s and optimal hydrophilicity of channels (51.4 {+/-} 8.36 {degrees}) with minimal embedding during thermal curing. To demonstrate proof of principle, a fluorescence assay demonstrating the formation of a dimeric g-quadruplex structure from a g-rich sequence which significantly enhances fluorescence of thioflavin T was implemented.

Extracellular vesicles (EVs) are lipid spheres released from cells. Research utilizing EVs has met several hurdles owing to the small size of the majority of EVs and other nanoparticles (<150 nm) and the lack of detection technologies capable of providing high-throughput single particle measurements at this scale. The use of high-throughput single particle measurements is critical for the assessment of EV heterogeneity and abundance which are features often used to assess the development of isolation protocols or particle characterization. The Coulter principle, known in the field as resistive pulse sensing (RPS), has been used for several decades to size and count cells. More recently, this technology has evolved to accommodate nanoparticle analysis. In the last decade a platform utilizing microfluidic resistive pulse sensing (MRPS) has been demonstrated for nanoparticles, offering ergonomic characterization of nanoparticles along with utilizing open format data. To date, assessment of MRPS accuracy and reporting standards have not been assessed. With the aim of increasing data accuracy, ergonomics, and reporting transparency, we developed a microfluidic resistive pulse sensing post-acquisition analysis software (RPSPASS) application for automated cohort calibration, population gating, statistical output, QC plot generation, alternative data file outputs, and standardized reporting templates.

Over the past decade, the integration of microgel-based granular hydrogels in biomedical technologies has experienced substantial growth due to the numerous benefits microgels offer. However, the inability to easily adopt uniform microgel fabrication workflows at scale constitutes a major bottleneck, or in some cases, a barrier-to-entry that stunts further growth of the field. The gold-standard technique for emulsion-based microgel production is through microfluidic droplet-generating devices that produce liquid gel precursor droplets that gel post-production. However, traditional microfluidic workflows often require multiple independent flows and controlled pressure sources, along with a steep learning curve in using microfluidics to achieve uniform droplet sizes reproducibly and repeatedly. This difficulty in adopting microgel fabrication is further compounded by low throughput and the extensive flow rate calibration required when switching to new formulations (e.g., material type, droplet size). In this work, we present a step-emulsion system that bridges the gap by providing a robust and simple setup. We experimentally characterize and evaluate how flow and outlet channel dimension contribute to the generation of uniform droplet populations at specific sizes. With our large dataset consisting of various outlet channel dimensions, we evaluated outlet channel geometrical impacts (height, width, cross-sectional area, aspect-ratio, etc.) on gel precursor droplet size and generation throughput. We demonstrate robust, highly compatible, and repeatably uniform droplet generation from various gel precursor polymer backbones, users with varying microfluidics experience, and a wide viscosity range, including alginate solutions with 650 times the viscosity of water. Furthermore, we confirmed consistent gel precursor droplet generation outcomes driven by a constant flow source (syringe pump) and by direct manual injection as a simple and highly adoptable option for the generation of gel precursor droplets. This platform is ideal for researchers seeking rapid and easy microgel fabrication, regardless of microfluidics experience.

Paper-based diagnostics such as lateral flow assays (LFAs) and microfluidic paper-based analytical devices ({micro}PADs) have attracted considerable attention because of their low cost, portability, and ease of use. Currently, to enable fabrication of {micro}PADs and improve LFA performance, hydrophobic blocks are patterned on paper substrates. However, fabrication of high-resolution hydrophobic barriers remains a major challenge. In this work, we developed a novel silicone extrudable ink for the fabrication of hydrophobic features on paper substrates. The ink was formulated using a vinyl-terminated polydimethylsiloxane (vPDMS) and polymethylhydrosiloxane (PMHS) system crosslinked through platinum-catalyzed hydrosilylation, and its rheological properties were tailored by incorporating silica fillers, obtaining a shear-thinning gel suitable for extrusion. The resulting formulation provided tunable properties, controlled deposition, and stable feature formation, enabling simple, low-cost, rapid, and robust fabrication of high-resolution hydrophobic barriers. Using this approach, we demonstrated improved fluid confinement and pattern fidelity on paper substrates, fabricated high-resolution paper microfluidic devices down to 150 {micro}m channel width, and enhanced the sensitivity of an LFA for a malaria diagnostic test. These results highlight the potential of this silicone ink platform as a practical and scalable strategy for advancing high-performance paper-based diagnostic technologies.

1.Adipocyte size is an independent predictor of several metabolic disorders, including type 2 diabetes, liver and cardiovascular diseases. However, technical limitations due to the fragile nature of mature adipocytes have restricted the functional analyses of size-separated adipocytes using conventional methods. Therefore, we have developed a microfluidic device, based on deterministic lateral displacement, for sorting intact, mature adipocytes. Cell-size distribution was determined from time-lapse recordings inside the device, in separate outlets, and by Coulter counter analysis of the collected cell fractions. This approach allowed size-separation with minimal size-overlap with mean diameters of (small fraction) 47 {micro}m and (large fraction) 82 {micro}m based on Coulter counter measurements. Viability of the separated cells was verified by insulin stimulation and western blotting of key insulin signaling proteins. The sample recovery, comparing input versus output material, was relatively high, 42% for the large fraction with a purity of 93%. We demonstrate that microfluidics is a suitable approach to overcome the limitations of sorting mature adipocytes according to size. Together, the high recovery rate, high throughput capacity, accurate separation and the fact that the cells maintained hormonal response after sorting provides compelling evidence of the strength and usability of the microfluidic approach for exploring adipocyte function in relation to size.

Stereolithography (SLA) 3D printing has become increasingly popular for fabricating microfluidic devices, with applications including hydrogel patterning and tissue modeling. In open-channel systems with surface tension-driven flow, 3D-printer-induced discrepancies in channel surface texture can significantly impact fluid flow and device performance. While previous work has focused on comparing different 3D printing methods for microchannel fabrication, the effect of device orientation during SLA printing on microchannel morphology and capillary-driven flow has not been systematically evaluated. Furthermore, there is minimal research elucidating the influence of channel surface texture on the flow of biologically relevant hydrogel precursors commonly used in organ-on-a-chip applications. Herein, we investigated the impact of print orientation on channel morphology, fluid wetting behavior, and fluid flow by comparing laser SLA-based parts where the length of the channel was tilted at 0{degrees}, 15{degrees}, 45{degrees}, or 90{degrees} during printing. We demonstrated that channel floor surface texture is greatly affected by print orientation: the highest axial surface roughness was measured in 15{degrees} printed channels, and the highest axial surface tortuosity-which describes the real length along the surface-was measured in 45{degrees} printed channels. Print angles of 15{degrees} and 45{degrees} also resulted in asymmetric roughness of the channel floor, which caused asymmetric wetting of glycerol solution. Surface tension-driven flow of glycerol solution, agarose precursor solution, and collagen precursor solution was affected by print orientation, in which the 45{degrees} printed flow devices had slowest flow for all test fluids. Root mean square roughness was not a reliable predictor of slower flow; instead, surface tortuosity should be considered. Potential alternatives to better theoretically model how print angle-induced surface texture affects open-channel flow are discussed as well. These findings provide a framework of fabrication considerations for laser SLA printing of open microchannels that can also be applied to other layer-by-layer, vat photopolymerization-based 3D printing technologies.

Organ chips offer a disruptive innovation to study human diseases with tissue-specific resolution within a predictable and tunable in vitro environment. However, these platform technologies have for the most part failed to translate to broad use in the private sector due to a lack of high-throughput, user-friendly platforms. Here we present an automated high-throughput organ chip seeded with iPSC-derived cardiomyocytes transduced with GCaMP6f and interface with translational technologies to bridge the current academia-industry gap. Cardiomyocytes were seeded on-chip fully hands-free using an entry-level fluid handling robot to significantly reduce user handling requirements. Pipette interfaces were paramount to facilitating seeding and feeding through improved tolerances for establishing a functional connection to dispense and collect small fluidic volumes. Following successful seeding, GCaMP6f activity on-chip was monitored with our automated, non-invasive fiber-optic sensing platform. We show a significant decrease in cardiomyocyte beat rate in response to decreased ambient culture temperature using data collected with our optical sensing platform. This study provides a potential translational blueprint for academia-industry partnership toward broad adoption of organ chip technology in drug development and disease modeling.

In vitro fertilization (IVF) is essential for many couples facing infertility, e.g. in cases of low sperm count (oligospermia), where natural fertilization is unlikely. Medical microrobotics, making use of microscopic devices designed to perform targeted tasks inside the body under imaging guidance and controlled actuation, represents a promising strategy to guide sperm cells toward the oocyte. This approach may significantly reduce the time, invasiveness, and patient burden of conventional IVF, with long-term potential for in vivo assisted reproduction. Here, we report the first successful in vitro fertilization (IVF) using magnetically functionalized spermatozoa, termed magnetotactic sperm cells (MSCs), as a step toward in vivo microrobotic guidance of sperm cells for targeted artificial insemination. We present a protocol for the preparation of MSCs for their use in IVF, resulting in samples largely free of non-functionalized sperm cells (99.69% purity). We systematically evaluate the effect of particle functionalization on sperm health, including acrosome integrity, DNA fragmentation, mitochondrial membrane potential, oxidative stress, and epithelial interactions, and observe no adverse effects. Notably, MSCs showed improved mitochondrial membrane integrity compared to the control samples after two hours of incubation. Using MSCs, we successfully performed complete IVF cycles that resulted in embryos developing to the blastocyst stage at a comparable rate as non-functionalized sperm cells of the same concentration. Lower concentrations of non-functionalized sperm cells (comparable to those remaining in the MSC sample after purification) did not result in any development of embryos to blastocysts. To facilitate manipulation and translation, we implemented automated image-based recognition, magnetic manipulation, and pre-clustering routines that increased guidance efficiency and are compatible with standard IVF workflows. Together, these results demonstrate that magnetic functionalization can be applied without compromising key sperm quality metrics and can enable directed sperm guidance for assisted oocyte fertilization. This work provides a practical framework for integrating microrobotic sperm manipulation into assisted-reproduction workflows and supports further development toward automated in vitro and eventual in vivo applications.

A systematic optimization of throughput and operational stability in Sorting by Interfacial Tension (SIFT) is presented. Reducing droplet size and enabling a broader distribution of droplet trajectories increased the number of droplets processed per sorting element, resulting in about a four fold improvement in throughput from 30 to 125 droplets per second. Throughput was further enhanced through device parallelization, with devices incorporating two and four independent sorting regions demonstrated. These configurations distributed droplets evenly across sorting elements that exhibited comparable pH sorting thresholds, indicating similar flow conditions and drag forces within each region. Among the designs evaluated, the two-element configuration provided the optimal balance of throughput, robustness, and simplicity, achieving maximum throughputs of about 250 droplets per second. Throughput and pH sorting thresholds were preserved throughout two hours of continuous sorting. The improved platform was applied to examine the relationship between cellular glycolysis and iron homeostasis at the single-cell level for Jurkat cells, revealing a subpopulation of highly glycolytic cells with significantly elevated iron uptake, consistent with prior reports linking iron regulation and T cell metabolism. Collectively, these advances expand the scale, stability, and biological applicability of SIFT, enabling large-scale functional studies while facilitating the capture of rare and metabolically distinct cell populations.

Cardiac rhythm is a critical clinical indicator for cardiac arrhythmias and adverse events during drug toxicity studies. In vivo, cardiomyocyte responses to pharmacological agents occur within minutes and are strongly influenced by dynamic drug delivery through blood flow. However, conventional 2D and 3D static culture systems fail to replicate these fluid flow kinetics, limiting their physiological relevance for assessing beat rate responses. Here, we present Mera, an advanced microphysiological system (MPS) developed by Hooke Bio, designed for high-throughput, long-term culture and functional analysis of 3D cardiac spheroids composed of human induced pluripotent stem cell-derived cardiomyocytes and cardiac fibroblasts. Mera enables dynamic perfusion, allowing investigation of cardiomyocyte beat rates under physiologically relevant flow conditions. The platform supports up to 640 spheroids per run and integrates automated imaging, fluid handling, and user-friendly software, operating under controlled physiological conditions (37{degrees}C, 5% CO2). Flow rates are tunable between 0 and 12.5 mL/min to mimic in vivo environments. Pharmacological testing with verapamil, isoproterenol, calcium chloride, and propranolol demonstrated real-time, reversible modulation of beat rate under flow, including recovery following drug-induced suppression. System variability was comparable to a temperature-controlled reference platform, supporting robust statistical analysis. Dose-response studies yielded IC values consistent with literature, confirming physiological relevance. Collectively, these results demonstrate that Mera provides a reproducible, scalable, and human-relevant platform for cardiac drug testing. By enabling dynamic drug exposure and automated analysis, Mera represents a powerful new approach methodology (NAM) for improving the predictive assessment of cardiac safety and beat-rate modulation drug responses.

Background and ObjectivesCardiovascular cells differentiated from human induced pluripotent stem cells (iPSCs), including cardiomyocytes, are valuable for evaluating human cardiac pharmacology and toxicity. Early assessment of cardiotoxicity, especially for novel drugs like anticancer agents, is essential for improving drug development efficiency and reducing costs. This study aimed to develop a highly sensitive bioassay system capable of evaluating the physiological function of human cardiac tissue in vitro. MethodsHuman iPSCs were differentiated into cardiovascular cell types (cardiomyocytes, vascular endothelial cells, and vascular mural cells) and assembled into a cardiac tissue model on aligned fiber device. This tissue was cultured dynamically to induce the formation of vascular network-like structure. By combining the fiber device with our previously developed heart-on-a-chip microdevice (HMD), we created a new model of HMD (Aligned Fiber-based HMD; AF-HMD) with improved throughput and stability. Pulsatile force changes induced by drug exposure were quantified by tracking the displacement of fluorescent microbeads within the microchannels. ResultsAF-HMD demonstrated functional responses to known cardiac agonists and toxicants, such as doxorubicin. The device also replicated clinically relevant cardiotoxic events, including the synergistic effects of trastuzumab and doxorubicin, showing marked reductions in contractile force and beat rate, mirroring clinical observations. ConclusionsThe AF-HMD system provides a sensitive and reproducible platform for evaluating cardiotoxicity in drug development. It offers a promising tool for preclinical screening, with potential applications in personalized medicine and predicting cardiotoxic risk in cancer therapy.

The cardiac autonomic nervous system is a key driver of various cardiac disorders and arrhythmias. However, investigating neuronal regulation of the human heart has proven difficult due to immitted and reliable experimental models. Here, we present a novel microphysiological system utilizing a compartmentalized microfluidic device (MFD) to integrate co-cultured human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hiPSC-CMs) and sympathetic neurons (hiPSC-SNs). MFD is composed of two wide-open chambers separated by microfluidic microchannels. hiPSC-SNs were characterized by confocal imaging and RT-qPCR for the expression of peripherin, tyrosine hydroxylase, and {beta}-tubulin III, as well as high levels of dopamine {beta}-hydroxylase and nicotinic acetylcholine receptors. Furthermore, patch-clamp techniques confirmed their functional maturity, showing spontaneous action potentials and positive responses to nicotine (1{micro}M). Co-culturing hiPSC-CMs and hiPSC-SNs within the MFD facilitated axonal projection into the cardiomyocyte chamber, establishing a physical connection between the two cell types. After 10 days of co-culture, functional integration was confirmed by a significant increase in the action potential frequency and beating rate of hiPSC-CMs, as recorded by patch-clamp and video motion tracking, respectively. Notably, nicotine application in the neuronal chamber accelerated these rates in hiPSC-CMs chamber, whereas the administration of the {beta}-blocker, propranolol (5{micro}M), effectively decreased the beating rates. Collectively, these data demonstrate the feasibility of differentiating hiPSCs into functional sympathetic neurons and establishing a robust neuro-cardiac interface. This microphysiological system represents a powerful platform for investigating disorders characterized by impaired neuro-cardiac interactions, offering a valuable tool for both disease modeling and pharmacological screening.

Three-dimensional stem cell-derived neural organoids provide a promising platform for investigating early brain development and interregional circuit formation. Although co-culture of region-specific organoids into assembloids has enabled the study of cortical and subcortical interactions, these models lack directional specificity and spatial control, limiting their ability to recapitulate canonical circuit architecture. Here, we present a microfluidic platform for constructing directional and tunable interregional circuits while preserving anatomical distinction. This system, which we term "directoids" incorporates micropatterned polydimethylsiloxane (PDMS) microstructures to control uni- and bidirectional axonal growth between cortical and thalamic organoids. We observed a 70.4% success rate of axons traversing the full channel length in the permissive direction and reaching the opposing organoid, whereas no neurites successfully crossed the probative direction. These results demonstrate robust directionally bias in axon outgrowth and establish a scalable, reproducible strategy for controlling asymmetric connectivity between anatomically distinct neural organoids. Using high-density CMOS microelectrode arrays, we further validated directional tuning of extracellular action potential propagation within directoid microchannels, a feature not observed in straight-channel connectoid controls. Directoids also exhibited significant asymmetry in firing rates between channel entry and exit sites, consistent with engineered bias in signal flow. This provides an experimental paradigm for dissecting how anatomical connectivity and functional activity converge to shape neuronal networks. Together, these findings establish a microfluidic platform for investigating the mechanisms underlying hierarchical circuit formation, regional specification, and functional integration in developing human neural organoid models at cellular resolution not possible in vivo.

Metastasis is the leading cause of death in breast cancer patients, yet there are no drugs specifically designed to block cancer cell intravasation, an early step of the metastatic cascade that originates circulating tumour cells (CTCs). A major challenge in developing anti-intravasation drugs is the scarcity of relevant in vitro platforms suitable for predictable drug discovery. Intravasation is a fundamental step of metastasis and involves the crossing of cancer cells through an endothelial barrier to enter the blood circulation. Here we developed an intravasation-on-a-chip model with controlled extracellular matrix composition, fluid flow and shear stress, which mimics the dynamic tumour-endothelium interface. The systems allows real-time imaging of intravasation and the isolation and quantification of intravasated cancer cells. As a proof-of-concept for drug testing, we show that perfusion with the PI3K/mTOR inhibitor Dactolisib, significantly reduced intravasation without compromising endothelial cell viability. The system also provides the capability to evaluate inhibitor on-target activity via imaging analysis. This intravasation-on-a-chip model offers a powerful, scalable, and imaging-compatible platform for discovering and evaluating anti-intravasation compounds.